Micro-RNA-based methods and compositions for the diagnosis and treatment of colon related diseases

ES 2 425 387 T3

DESCRIPTION

Mir-106a to diagnose colon adenocarcinoma with poor survival prognosis

Background of the invention

Colon adenocarcinoma is a leading cause of cancer mortality worldwide¹. Colorectal cancer is the third most common and the second leading cause of cancer death in the United States². Sporadic colon adenocarcinomas begin as adenomas and progress through a progression of molecular, cellular, and histological changes³. Although 5-year mortality rates have declined modestly over the past 3 decades⁴, there is still a need to identify new prognostic biomarkers and therapeutic targets for this disease. Currently, chemotherapy has significant therapeutic value but surgery is the only curative form of treatment.⁵.

Ideal therapeutic targets should be causally associated with the disease and be amenable to the design of therapeutic interventions; whereas ideal biomarkers should be easy to measure and have strong associations with clinical outcomes. MicroRNAs could meet both criteria^6-8.

MicroRNAs are non-coding RNA molecules of 18-25 nucleotides, which regulate the translation of many genes.⁹. Since its discovery^10,11, have been found to regulate various cellular processes including apoptosis¹²⁴, differentiation^{10, 11, 15} and cell proliferation¹⁶. MicroRNAs may also have a causal role in carcinogenesis^6,17. MicroRNA expression levels are altered in most tumor types^18,19, including colon tumors^19-22. The miR-15 and miR-16a microRNAs are suppressed or down-regulated in most chronic lymphocytic leukemias²³. Experimental manipulation of specific microRNAs modulates tumor development in mouse model systems^16,24-26. The prognostic potential of microRNAs has also been demonstrated for chronic lymphocytic leukemia⁷, lung cancer⁸ and neuroblastomas²⁴.

Aberrant microRNA expression can cause carcinogenesis, inhibition of specific microRNAs can have therapeutic implications. Modified antisense oligonucleotides can be designed to specifically inhibit microRNA function²⁸. Antagomirs are a type of antisense oligonucleotide that has been shown to be effective in inhibiting microRNA function in vivo in mice.²⁹. The ease of designing specific inhibitors of microRNA function makes them candidates for therapeutic targets.

Summary of the invention

In a general aspect, provided herein is a method of diagnosing whether a subject has, is at risk of developing, or has a reduced survival prognosis for a colon cancer-related disease. The method includes measuring the level of at least one miR gene product in a test sample from the subject, wherein an alteration in the level of the miR gene product in the test sample, relative to the level of a gene product of corresponding miR in a control sample, is indicative that the subject has, or is at risk of developing, colon cancer-related disease. In a particular aspect, the at least one miR gene product is selected from the group consisting of miR20a, miR21, miR-106a, miR-181b, miR-203, and combinations thereof. In one embodiment, the miR gene product is miR-21.

In another general aspect, provided herein is an assay method for detecting at least an onset of, predisposition to, or reduced survival prognosis for a colon cancer-related disease response, comprising:

(1) determining an expression level of at least one marker in a sample from a test subject; the at least one marker including at least one miR gene product selected from the group consisting of miR20a, miR21, miR-106a, miR-181b, miR-203, and combinations thereof;

(2) comparing the expression level determined in step (1) with a marker control expression level in a sample from a healthy subject; and (3) consider that the subject has a colon cancer-related disease when the result of the comparison in step (2) indicates that: i) the level of expression of the at least one marker in the test subject is greater than in the control or ii) the expression level of the at least one marker in the test subject is lower than in the control.

The sample can comprise one or more of tissue, blood, plasma, serum, urine, and feces. Furthermore, all process steps can be performed in vitro.

In another general aspect, provided herein is a method of diagnosing whether a subject has, is at risk of developing, or has a reduced survival prognosis for a colon cancer-related disease, comprising:

(1) reverse transcribing RNA from a test sample obtained from the subject to provide a set of

ES 2 425 387 T3 target oligodeoxynucleotides;

(2) hybridizing the target oligodeoxynucleotides to a microarray comprising miRNA-specific probe oligonucleotides to provide a hybridization profile for the test sample; and (3) comparing the hybridization profile of the test sample with a hybridization profile generated from a control sample, in which an alteration in the signal of at least one miRNA is indicative that the subject has, is at risk to develop or have a reduced survival prognosis for a disease related to colon cancer.

In a particular aspect, the signal of at least one miRNA, relative to the signal generated from the control sample, is up or down regulated. In addition, the microarray may comprise miRNA-specific probe oligonucleotides for one or more miRNAs selected from the group consisting of miR20a, miR-21, miR-106a, miR-181b, miR-203, and combinations thereof.

In another general aspect, provided herein is a method of inhibiting tumorigenesis in a subject who has, or is suspected of having, a colon cancer-related disease in which at least one miR gene product selected from the group consisting in miR20a, miR-21, miR-106a, miR-181b, miR-203 and combinations thereof, it is down-regulated or up-regulated in cancer cells of the subject, relative to control cells, comprising:

(1) when the at least one miR gene product is down-regulated in cancer cells, administering to the subject an effective amount of at least one isolated miR gene product selected from the group consisting of miR20a, miR-21, miR-106a , miR-181b, miR-203, and combinations thereof, such that tumorigenesis is inhibited in the subject; or (2) when the at least one miR gene product is upregulated in cancer cells, administering to the subject an effective amount of at least one compound to inhibit the expression of the at least one miR gene product selected from the group consisting of miR20a, miR-21, miR-106a, miR-181b, miR-203, and combinations thereof, so as to inhibit tumorigenesis in the subject.

In a particular aspect, at least one miR gene product isolated in step (1) and / or in step (2) is miR-21 or an isolated variant or biologically active fragment or functional equivalent thereof, or an antibody that joins them.

In another general aspect, provided herein is a method of inhibiting tumorigenesis in a subject having colon cancer, comprising:

(1) determining the amount of at least one miR gene product in cancer cells of the subject, relative to control cells; and (2) alter the amount of miR gene product expressed in cancer cells:

(i) administering to the subject an effective amount of at least one isolated miR gene product selected from the group consisting of miR20a, miR-21, miR-106a, miR-181b, miR-203, and combinations thereof, if the amount of the miR gene product expressed in the cancer cells is less than the amount of the miR gene product expressed in the control cells; or (ii) administering to the subject an effective amount of at least one compound to inhibit the expression of the at least one miR gene product, if the amount of miR gene product expressed in cancer cells is greater than the amount of the miR gene product. miR expressed in control cells so as to inhibit tumorigenesis in the subject.

In a particular aspect, the at least one miR gene product isolated in step (i) is miR-21 or an isolated variant or biologically active fragment thereof. Furthermore, in certain embodiments, the at least one miR gene product in step (ii) is selected from the group consisting of miR20a, miR-21, miR-106a, miR-181b, miR203, and combinations thereof, or a isolated variant or biologically active fragment thereof.

In another general aspect, provided herein is a method of identifying an inhibitor of tumorigenesis, which comprises providing a test agent to a cell and measuring the level of at least one miR gene product associated with an altered expression level in a colon cancer-related disease, in which an increase or decrease in the level of the miR gene product in the cell, relative to a suitable control cell, it is indicative that the test agent is an inhibitor of tumorigenesis.

In another general aspect, provided herein is a method of identifying an inhibitor of tumorigenesis, comprising providing a test agent to a cell and measuring the level of at least one miR gene product associated with an altered expression level in a colon cancer-related disease, in which a reduction in the level of the miR gene product in the cell, relative to a cell of

ES 2 425 387 T3 suitable control is indicative that the test agent is an inhibitor of tumorigenesis.

In another general aspect, a marker is provided herein to assess one or more metabolic pathways that contribute to at least one of onset, progression, severity, pathology, aggressiveness, grade, activity, disability, mortality, morbidity, sub-classification. disease or other underlying pathogenic or pathological feature of at least one colon cancer-related disease, the marker comprising one or more miR gene products selected from the group consisting of miR20a, miR-21, miR-106a, miR181b, miR-203, and combinations thereof.

In another general aspect, a composition comprising one or more of the markers described herein is provided herein.

In another general aspect, provided herein is a method for identifying a potential for the initiation or development of at least one colon cancer-related disease in a subject, the method providing to measure one or more of the markers described herein. document. In certain embodiments, one or more markers are present in an isolated sample and all steps of the procedure are performed in vitro.

In another general aspect, provided herein is a reagent for assays for detecting a colon cancer-related disease, the reagent comprising a polynucleotide comprising the nucleotide sequence of at least one marker described herein or a sequence of nucleotides complementary to the nucleotide sequence of the marker.

In another general aspect, provided herein is a reagent for assays for detecting a colon cancer-related disease, the reagent comprising an antibody that recognizes a protein encoded by at least one marker described herein.

In another general aspect, provided herein is a DNA microplate for assays for colon cancer-related disease, in which a probe for assaying at least one marker described herein has been immobilized.

In another general aspect, provided herein is a method for evaluating the efficacy of a therapy to prevent, diagnose, and / or treat at least one colon cancer-related disease comprising:

1) subjecting an animal to a therapy whose efficacy is being evaluated, and

2) determining the level of efficacy of the treatment being tested in treating or preventing colon cancer-related disease by evaluating at least one marker described herein.

In certain embodiments, the candidate therapeutic agent comprises one or more of: pharmaceutical compositions, nutraceutical compositions, and homeopathic compositions. Furthermore, the therapy being evaluated may be for use in a human subject. In certain embodiments, the procedure is not a procedure for treating the human or animal body by surgery or therapy.

In another general aspect, provided herein is a method for evaluating the potential of at least one material with respect to an ability to initiate a colon cancer-related disease response in an animal model, the method providing:

1) measuring one or more of the positively or negatively regulated markers described herein after exposure of the animal to one or more materials in amounts sufficient to initiate a colon cancer-related disease response in the animal; Y

2) determine if at least one of the positively or negatively regulated markers has the ability to initiate a disease response related to colon cancer.

In another general aspect, provided herein is a pharmaceutical composition for treating a colon cancer-related disease, comprising: at least one miR gene product selected from the group consisting of miR20a, miR-21, miR-106a, miR-181b, miR -203, and combinations thereof; and a pharmaceutically acceptable carrier.

In another general aspect, provided herein is a pharmaceutical composition for treating colon cancer, comprising at least one miR expression inhibiting compound and a pharmaceutically acceptable carrier, wherein the at least one inhibiting compound of miR expression is specific for a miR gene product selected from the group consisting of miR20a, miR-21, miR-106a, miR181b, miR-203, and combinations thereof.

In another general aspect, provided herein is an article of manufacture comprising: at least one capture reagent that binds to a marker for a colon cancer-related disease

ES 2 425 387 T3 selected from at least one of the markers described herein.

In another general aspect, provided herein is a kit to screen for a candidate compound for a therapeutic agent to treat a colon cancer-related disease, the kit comprising: one or more reagents for at least one marker described in this docu , and a cell that expresses at least one marker. In certain embodiments, the presence of the marker is detected using a reagent comprising an antibody or an antibody fragment that specifically binds with at least one marker. Furthermore, in certain embodiments, the reagent is labeled, radiolabeled, or labeled with biotin and / or the antibody or antibody fragment is radiolabeled, chromophore labeled, fluorophore labeled, or enzyme labeled. In a particular embodiment, the kit also includes a container comprising at least one of the markers. In addition, the reagent may comprise one or more of: an antibody, a probe to which the reagent binds or may bind, and an immobilized metal chelate.

In another general aspect, provided herein is a screening assay regarding a colon cancer-related disease comprising:

contacting one or more of the markers of claim 20 with a substrate for said marker and with a test agent and determining whether the test agent modulates the activity of the marker.

In certain embodiments, all steps of the process can be performed in vitro.

In another general aspect, provided herein is a microarray for predicting the presence of a colon cancer-related disease in a subject comprising an antibody directed to at least one marker of claim 20.

In another general aspect, methods, compositions and the like are provided herein, wherein an expression level of the marker is assessed by detecting the presence of a transcribed polynucleotide or part thereof, the transcribed polynucleotide comprising a coding region of the marker. In addition, the sample can be a body fluid or tissue associated with colon cancer. In a particular embodiment, the sample comprises cells obtained from the patient.

In another general aspect, provided herein is a method of treating, preventing, reversing, or limiting the severity of a colon cancer-related disease complication in an individual in need thereof, comprising:

administering to the individual an agent that interferes with at least one colon cancer-related disease response signaling pathway, in an amount sufficient to interfere with said signaling, wherein the agent comprises at least one miR gene product selected from the group consisting of miR20a, miR-21, miR-106a, miR-181b, miR-203, and combinations thereof.

In another general aspect, provided herein is the use of an agent that interferes with at least one colon cancer-related disease response signaling pathway, for the preparation of a medicament to treat, prevent, reverse, or limit the severity of a colon cancer-related disease complication in an individual, wherein the agent comprises at least one miR gene product selected from the group consisting of miR20a, miR-21, miR-106a, miR-181b, miR-203, and combinations thereof.

In another general aspect, provided herein is a method of treating, preventing, reversing, or limiting the severity of a colon cancer-related disease complication in an individual in need thereof, comprising administering to the individual an agent that interferes with at least one colon cancer-related disease response cascade, wherein the agent comprises at least one miR gene product selected from the group consisting of miR20a, miR-21, miR-106a, miR-181b, miR-203, and combinations thereof.

In another general aspect, provided herein is the use of an agent that interferes with at least one colon cancer-related disease response cascade for the preparation of a medicament to treat, prevent, reverse, or limit the severity of a disease. complication of colon cancer-related disease in an individual, wherein the agent comprises at least one miR gene product selected from the group consisting of miR20a, miR-21, miR-106a, miR-181b, miR-203 and combinations thereof.

In another general aspect, provided herein is a computer-readable medium comprising a database having a plurality of digitally encoded reference profiles, wherein at least a first reference profile represents a level of at least one first marker in one or more samples from one or more subjects showing an indication of a colon cancer-related disease response, wherein the marker comprises one or more miR gene products selected from the group consisting of miR20a, miR-21, miR-106a, miR-181b, miR-203, and combinations thereof.

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In certain embodiments, the computer-readable medium includes at least one second reference profile that represents a level of at least one second marker in one or more samples from one or more subjects that show indications of a colon cancer-related disease response. ; or subjects who have a colon cancer-related disease.

In another general aspect, provided herein is a computer system for determining whether a subject has, is predisposed to, or has a poor survival prognosis for a colon cancer-related disease comprising the database described herein. document and a server comprising a computer executable code to make the computer receive a profile of a subject, identifying from the database a matching reference profile that is diagnostically relevant to the subject's profile; and generating an indication of whether the subject has, or is predisposed to have, a colon cancer-related disease.

In another general aspect, provided herein is a computer-assisted method for evaluating the presence, absence, nature, or extent of a colon cancer-related disease in a subject, comprising:

1) providing a computer comprising a model or algorithm for classifying data from a sample obtained from the subject, wherein the classification includes analyzing the data with respect to the presence, absence, or amount of at least one marker, wherein the marker comprises one or more miR gene products selected from the group consisting of miR20a, miR-21, miR-106a, miR-181b, miR-203, and combinations thereof;

2) Enter data of the biological sample obtained from the subject; Y

3) classify the biological sample to indicate the presence, absence, nature or extent of a disease related to colon cancer.

In another general aspect, at least one miR gene product and combinations thereof include isolated variants or biologically active fragments or functional equivalents thereof, or antibodies that bind thereto.

In another general aspect, an animal model for colon cancer is provided herein in which at least one of the following biological or chemical processes occurs in the animal model. Up or down regulation of one or more miR gene products is selected from the group consisting of miR20a, miR21, miR-106a, miR-181b, miR-203, and combinations thereof. In certain embodiments, the animal model is a non-human vertebrate. In particular embodiments, the animal model is a mouse, rat, rabbit, or primate.

Various objects and advantages of the present invention will be apparent to those skilled in the art from the following detailed description of the preferred embodiment, when read in light of the accompanying drawings.

Brief description of the drawings

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the office upon request and upon payment of the necessary fee.

Figures 1a - 1g: miR-21 is expressed at higher levels in colon adenocarcinomas with increasing expression in more advanced tumors.

(Figure 1a) In situ hybridization for miR-21 was optimized to distinguish high and low expressions of miR-21. Colonic epithelial cells in human tumor (T) express higher levels of miR-21 compared to adjacent non-tumor tissue (N). (Figure 1c) The nuclei and cytoplasm of colonic epithelial cells in tumor tissue express significant amounts of miR-21 in tumor tissue, at high power. (Figure 1e) Non-tumor tissue does not show significant expression of miR-21 at the same magnification.

(Figure 1b, Figure 1d, Figure 1f) The mixed control probe shows no significant staining at low or high magnification in serial sections of tumor and non-tumor tissue, as expected. Scale bars (Figures 1 cf) indicate that miR-21 500 pM (g) is expressed at higher levels in more advanced tumors. Dot plots represent relative miR-21 (from quantitative RT-PCR) Ct values ​​for adenoma and tumor expression levels that have been normalized to paired non-adenoma or non-tumor tissues, respectively. Tissue types have been ordered from adenoma to stage I-IV tumors. The bars indicate the median of the value. There is a significant tendency for more advanced tumors to have higher expression of miR-21 (non-parametric test for trend between ordered groups).

Figure 2: miR-21 is expressed at higher levels in more advanced tumors. MicroRNA microarrays were used to measure miR-21 expression levels. Dot plots represent log2 (tumor / non-tumor ratios) of miR-21 as calculated from original cohort microRNA microarrays. Probe

ES 2 425 387 T3 hsa-miR-21-prec 17 No. 1 microarray was used to measure miR-21 expression. Tissues with undetectable miR-21 expression were excluded based on the microarray data. Tissue types of TNM tumors from stage I to stage IV have been ordered. The bars indicate the median of the value. There is a significant tendency for more advanced tumors to have higher expression of miR-21 (p = 0.04; non-parametric test with respect to the trend between ordered groups).

Figures 3a and 3b: High miR-21 expression in tumors predicts poor survival in subjects with typical adenocarcinoma histology in both independent cohorts. This analysis excludes subjects with histology of mucinous adenocarcinoma or adenosquamous carcinoma.

(Figure 3a) MicroRNA microarrays were used in the Maryland test cohort to measure microRNA expression levels from tumors and non-tumor tissues. Tissues with undetectable expression of miR-21 based on microarray data were excluded. High miR-21 expression was ranked based on the highest tertile. The red lines indicate individuals with high expression while the green lines correspond to low expression. For non-tumor tissue, 24/69 tissues were classified as high while 26/72 tumors were classified as high. High expression of miR-21 in tumors (right) is associated with poor survival while it is not associated in non-tumor tissue.

(Figure 3b) Validation of the association with high miR-21 expression in tumors and negative prognosis in an independent cohort. The expression levels of miR-21 were measured by quantitative RT-PCR. The high expression is based on the highest tertile. 35/103 non-tumor tissues were classified as high and 34/103 tumor tissues were classified as high. The p-values ​​are log-rank p-values ​​from Kaplan-Meier analysis. The Xs in all lines indicate when an individual was censored.

Figures 4a and 4b: the high expression of miR-21 in tumors predicts poor survival in both independent cohorts. This analysis includes all subjects regardless of adenocarcinoma histology.

(Figure 4a) microRNA microarrays were used in the Maryland test cohort to measure microRNA expression levels from tumors and non-tumor tissues. Tissues with undetectable miR-21 expression were excluded based on microarray data. The high expression of miR21 was ranked based on the highest tertile. The red lines indicate individuals with high expression while the green lines correspond to low expression. For non-tumor tissues, 26/74 tissues were classified as high while 28/79 tumors were classified as high. High expression of miR-21 in tumors (right) is associated with poor survival while it is not associated in non-tumor tissue.

(Figure 4b) Validation of the association with high expression of miR-21 in tumors and poor prognosis in an independent cohort. The miR-21 expression levels were measured by quantitative RT-PCR. High expression is based on the highest tertile, 37/111 non-tumor tissues were classified as high and 37/111 tumor tissues were classified as high. All p values ​​are p log rank values ​​from Kaplan-Meier analysis. The Xs in all lines indicate when an individual was censored.

Figures 5a, 5b and 5c: high miR-21 expression is associated with poor response to adjuvant chemotherapy for cases with conventional adenocarcinoma histology. This analysis includes subjects from the validation cohort, excluding subjects with mucinous adenocarcinoma or adenosquamous carcinoma histologies.

(Figure 5a) comparison of survival rates for TNM stage II / III subjects with conventional adenocarcinoma histology using miR-21 expression levels and receipt of adjuvant chemotherapy. For the 77 stage II / III subjects, 25 were classified as low miR-21 therapy recipients, 28 as low miR-21 and non-therapy recipients, 11 as high therapy miR-21 recipients, and 13 as high miR-21 and not recipients of therapy. For stage II / III subjects receiving adjuvant chemotherapy, high expression of miR-21 in tumors is associated with poor survival (p = 0.03).

(Figure 5b) Comparison of stage II TNM subjects with conventional adenocarcinoma histology. For the 33 stage II subjects, 8 were classified as low miR-21 recipients of therapy, 15 as low miR-21 and not therapy recipients, 3 as high miR-21 therapy recipients, and 7 as high miR-21 and not. therapy recipients. All stage II subjects who received chemotherapy survived during the course of this study.

(Figure 5c) Comparison of stage III TNM subjects with conventional adenocarcinoma histology. For the 44 stage III subjects, 17 were classified as low miR-21 therapy recipients, 13 as low miR-21 and non-therapy recipients, 8 as high miR-21 than therapy recipients, and 6 as high miR-21 and not recipients of therapy. For stage III subjects who received adjuvant chemotherapy, high expression of miR-21 in tumors is associated with poor survival (p = 0.02). The Xs in all lines indicate when an individual was censored.

Figures 6a, 6b, and 6c: Combined analysis of the Maryland trial cohort and the Hong Kong validation cohort examining associations between miR-21 expression in tumors and the receipt of adjuvant chemotherapy with prognosis. This analysis includes all TNM stage II / III subjects from both cohorts. Individuals with histologies of mucinous adenocarcinoma or adenosquamous carcinoma were excluded. The left column includes

ES 2 425 387 T3 Kaplan-Meier representations analyzing the association between receipt of adjuvant therapy and prognosis. The middle column includes analysis of the association between high expression of miR-21 in tumors and prognosis, and the right column subdivides individuals based on both chemotherapy and miR-21 expression status.

(Figure 6a) All TNM stage II / III subjects. For the 119 stage II / III subjects, 40 were classified as low miR-21 therapy recipients, 41 as low miR-21 and non-therapy recipients, 16 as high therapy miR-21 recipients, and 22 as high miR-21 and not recipients of therapy. High miR-21 expression is associated with poor survival for those receiving chemotherapy (p = 0.003) as well as those receiving no therapy (p = 0.04).

(Figure 6b) All stage II TNM subjects. For the 52 stage II / III subjects, 10 were classified as low miR21 therapy recipients, 25 as low miR-21 and non-therapy recipients, 4 as high therapy MIR-21 recipients, and 13 as high miR-21 and not. therapy recipients. Associations between high miR-21 expression and prognosis were not statistically significant in individuals who received chemotherapy (p = 0.11) or those who did not receive chemotherapy (p = 0.06).

(Figure 6c) All stage III TNM subjects. For the 67 stage III subjects, 30 were classified as low miR21 who received therapy, 16 as low miR-21 who received no therapy, 12 as high miR-21 who received therapy, and 9 as high miR-21 who received no therapy. therapy. High miR-21 expression is significantly associated with poor survival in stage III subjects who received chemotherapy (P = 0.007), but not in subjects who did not receive chemotherapy (P = 0.30). The Xs in all lines indicate when an individual was censored.

Figures 7a - 7c. Global miRNA profiles are associated with clinical TNM stage classification and survival prognosis. Hierarchical clustering of miRNA TIN relationships resulted in the formation of two clusters arbitrarily named cluster A and cluster B. The resulting HEAT map and cluster assignments are shown in Figure 7a. These two groups were composed of individuals with significantly different survival prognoses for TNM stages, with individuals in group B being more likely to be diagnosed as stage III or IV compared to individuals in group A (Figure 7b). The KaplanMeier analysis shows that individuals in group B also have a worse survival prognosis (Figure 7c).

Figures 8a - 8i. The TIN ratios of individual miRNAs are predictive of survival prognosis. Presented herein are graphs showing TIN relationships by TNM staging (left) and Kaplan-Meier analysis (right) for each of these 9 miRNAs. The Y axis (graphs of the ratio of TIN by TNM stage) indicates the ratio of TIN transformed in log (2) for each individual, while the Y axis groups individuals by TNM stages (I, II, III or IV) . The significance values ​​shown are the result of a non-parametric test with respect to the trend of the mean TIN ratio values ​​between individuals grouped by stage. The Kaplan-Meier plots include all individuals with TIN relationship data for that particular miRNA. The inventors found that TIN relationships were associated with both clinical stage and survival prognosis.

Figures 9a and 9b. A miRNA identification of 9 miRNAs predicts risk of death from colon cancer. Each of the TIN ratios of miR-21, miR-106a, miR181b, miR-16b, miR-203, let-7 g, miR-29a, miR103-2 and miR-10a was predictive of prognosis of colon cancer. Hierarchical grouping of TIN relationships from these 9 miRNAs resulted in dividing individuals into two groups (1A) with significantly different survival prognoses (1B). Individuals in group B had a significantly higher risk of dying from colon cancer than those in group A. Individuals were excluded from this analysis if they lacked more than 2 of the 9 TIN relationships that made up the miRNA identification.

Detailed description of the preferred embodiment

In a general aspect, provided herein is the identification of particular microRNAs whose expression is altered in cancer cells associated with different colon cancers, relative to normal control cells.

As used interchangeably herein, a "miR gene product", "microRNA", "miR", or "miRNA" refers to the unprocessed (eg, precursor) or processed (eg. , mature) of a miR gene. Since miR gene products are not translated into protein, the term "miR gene products" does not include proteins. The unprocessed miR gene transcript is also referred to as a "miR precursor" or "miR prec" and typically comprises an RNA transcript approximately 70-100 nucleotides in length. The miR precursor can be processed by digestion with an RNase (eg Dicer, Argonaut or RNase III (eg RNase III from E. coli)) into an active 19-25 nucleotide RNA molecule. This active 19-25 nucleotide RNA molecule is also referred to as the "processed" miRNA gene transcript or "mature" miRNA.

The active 19-25 nucleotide RNA molecule can be obtained from the miR precursor by natural processing routes (for example, using intact cells or cell lysates) or by synthetic processing routes (for example, using isolated processing enzymes, such as Dicer, Argonaut or

ES 2 425 387 T3

Isolated RNase III). It is understood that the active 19-25 nucleotide RNA molecule can also be produced directly by biological or chemical synthesis, without having been processed from the miR precursor. When a microRNA is referred to herein by name, the name corresponds to both the precursor and mature forms, unless otherwise indicated.

In one aspect, provided herein are methods for diagnosing whether a subject has, or is at risk of developing, colon cancer, which comprise measuring the level of at least one miR gene product in a test sample from the subject. and comparing the level of the miR gene product in the test sample with the level of a corresponding miR gene product in a control sample. As used herein, a "subject" can be any mammal that has, or is suspected of having, a solid cancer. In a preferred embodiment, the subject is a human who has, or is suspected of having, colon cancer.

In one embodiment, the at least one miR gene product measured in the test sample is selected from the group consisting of miR20a, miR-21, miR-106a, miR-181b, miR-203, and combinations thereof. In a preferred embodiment, the miR gene product is miR-21.

Colon cancer related disease can be any disorder or cancer arising from the colon tissues. Such cancers are normally associated with the formation and / or presence of tumor masses and can be, for example, adenocarcinomas.

In one embodiment, the colon is an adenocarcinoma and the at least one miR gene product measured in the test sample is selected from the group consisting of miR20a, miR-21, miR-106a, miR-181b, miR-203, and combinations thereof.

In a further embodiment, the at least one miR gene product measured in the test sample is miR-21.

The level of at least one miR gene product can be measured in a biological sample (eg, cells, tissues) obtained from the subject. For example, a tissue sample (eg, from a tumor) can be removed from a subject suspected of having colon cancer-related disease by standard biopsy techniques. In another embodiment, a blood sample can be withdrawn from the subject, and blood cells (eg, white blood cells) can be isolated for DNA extraction by conventional techniques. The blood or tissue sample is preferably obtained from the subject prior to the initiation of radiotherapy, chemotherapy, or other therapeutic treatment. A corresponding control tissue or blood sample can be obtained from unaffected tissues from the subject, from a normal human individual or a population of normal individuals, or from cultured cells corresponding to the majority of cells in the subject sample. The control blood or tissue sample is then processed along with the subject sample, so that the levels of the miR gene product produced from a given miR gene in cells from the subject sample can be compared with the levels of the gene product. of corresponding miRs from cells of the control sample. A reference miR expression pattern for the biological sample can also be used as a control.

An alteration (eg, an increase or decrease) in the level of a miR gene product in the sample obtained from the subject, relative to the level of a corresponding miR gene product in a control sample, is indicative of the presence of a colon cancer-related disease in the subject.

In one embodiment, the level of the at least one miR gene product in the test sample is greater than the level of the corresponding miR gene product in the control sample (i.e., the expression of the miR gene product is "up-regulated ”). As used herein, the expression of a miR gene product is "up-regulated" when the amount of miR gene product in a cell or tissue sample from a subject is greater than the amount of the same gene product in a sample. cell or tissue control.

In another embodiment, the level of the at least one miR gene product in the test sample is less than the level of the corresponding miR gene product in the control sample (i.e., the expression of the miR gene product is "down-regulated ”). As used herein, the expression of a miR gene is "down-regulated" when the amount of the miR gene product produced from that gene in a tissue cell sample from a subject is less than the amount produced from it. gene in a control cell or tissue sample.

Relative miR gene expression in control and normal samples can be determined relative to one or more RNA expression patterns. The standards may comprise, for example, a zero miR gene expression level, the miR gene expression level in a conventional cell line, the miR gene expression level in unaffected tissues of the subject, or the mean expression level. miR gene previously obtained for a population of normal human controls.

The level of a miR gene product in a sample can be measured using any technique that is suitable for detecting RNA expression levels in a biological sample. Suitable techniques (eg, Northern blot analysis, RT-PCR, in situ hybridization) for determining RNA expression levels in a biological sample (eg, cells, tissues) are well known to those of skill in the art. In a particular embodiment, the level of at least one miR gene product is detected using transfer analysis of

ES 2 425 387 T3

Northern. For example, total cellular RNA can be purified from cells by homogenization in the presence of nucleic acid extraction buffer, followed by centrifugation. Nucleic acids are precipitated and DNA is removed by DNase treatment and precipitation. The RNA molecules are then separated by gel electrophoresis on agarose gels according to conventional techniques, and transferred to nitrocellulose filters. The RNA is then immobilized on the filters by heating. Detection and quantification of specific RNA is achieved using appropriately labeled RNA or DNA probes complementary to the RNA in question. See, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook et al, eds, 2^to edition, Cold Spring Harbor Laboratory Press, 1989, Chapter 7.

Suitable probes for Northern blot hybridization of a given miR gene product can be produced from the nucleic acid sequences and include, but are not limited to, probes that are at least about 70%, 75%, 80%, 85% , 90%, 95%, 98%, 99% or complete complementarity with a miR gene product of interest. Procedures for the preparation of labeled DNA and RNA probes and the conditions for hybridizing them to target nucleotide sequences are described in Molecular Cloning: A Laboratory Manual, J. Sambrook et al, eds, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, chapters 10 and 11.

In a non-limiting example, the nucleic acid probe can be labeled with, for example, a radionuclide, such as or qo 'q'í ¹ 14 or ¹ iai

H, P, P, C or S; a heavy metal; a ligand capable of acting as a specific binding pair member for a labeled ligand (eg, biotin, avidin, or an antibody.); a fluorescent molecule; a chemiluminescent molecule, an enzyme, or the like.

Probes can be labeled for high specific activity by the notch translation procedure of Rigby et al. (1977). J. Mol. Biol. 113: 237-251 or by the random primer procedure of Fienberg et al. (1983), Anal. Biochem. 132: 6-13. The latter is the procedure chosen to synthesize probes labeled with³²P with high specific activity of single-stranded DNA or RNA templates. For example, by replacing pre-existing nucleotides with highly radioactive nucleotides according to the notch translation procedure, it is possible to prepare nucleic acid probes labeled with³²P with a specific activity well above 10⁸cpm / microgram. Autoradiographic detection of hybridization can then be performed by exposing hybridized filters to photographic film. Densitometric scanning of the photographic films exposed by the hybridized filters provides an accurate measurement of miR gene transcript levels. Using another approach, miR gene transcript levels can be quantified by computer imaging systems, such as the Molecular Dynamics 400-B 2D Phosphorimager available from Amersham Biosciences, Piscataway, NJ.

When radionuclide labeling of DNA or RNA probes is not practical, the random primer procedure can be used to incorporate an analog, for example, the dTTP analog 5- (N- (N-biotinyl-apsilonaminocaproyl) -3-aminoallyl ) deoxyuridine triphosphate, in the probe molecule. The biotinylated probe oligonucleotide can be detected by reaction with biotin-binding proteins, such as avidin, streptavidin, and antibodies (eg, anti-biotin antibodies) coupled to fluorescent dyes or enzymes that produce color reactions.

In addition to Northern and other RNA hybridization techniques, it is possible to determine the levels of RNA transcripts using the in situ hybridization technique. This technique requires fewer cells than the Northern blotting technique, and involves depositing whole cells on a microscope cover slip and scanning the nucleic acid content of the cell with a solution containing nucleic acid probes (eg, cDNA or RNA). radioactive or otherwise marked. This technique is particularly well suited to analyzing tissue biopsy samples from subjects. The practice of the in situ hybridization technique is described in more detail in US Patent No. 5,427,916.

In a non-limiting example, suitable probes for in situ hybridization of a given miR gene product can be produced from the nucleic acid sequences, and include, but are not limited to, probes that are at least about 70%, 75% , 80%, 85%, 90%, 95%, 98%, 99%, or complete complementarity with a miR gene product of interest, as described above.

The relative number of miR gene transcripts in cells can also be determined by reverse transcription of miR gene transcripts, followed by amplification of the reverse transcripts by polymerase chain reaction (RT-PCR). The levels of miR gene transcripts can be quantified in comparison to an internal standard, eg, the level of mRNA of a "housekeeping" gene present in the same sample. A suitable "housekeeping" gene for use as an internal standard includes, for example, myosin or glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Procedures for performing quantitative and semi-quantitative RT-PCR, and variations thereof, are well known to those skilled in the art.

In some cases, it may be desirable to simultaneously determine the level of expression of a plurality of different miR gene products in a sample. In other cases, it may be desirable to determine the level of expression of the transcripts of all known miR genes correlated with cancer. Assessing the cancer-specific expression levels of hundreds of miR genes or gene products is time consuming and requires a large amount of total RNA (for example, at least 20 gg for each Northern blot)

ES 2 425 387 T3 and autoradiographic techniques requiring radioactive isotopes.

To overcome these limitations, an oligo library can be constructed, in microplate format (ie, a microarray), containing a set of oligonucleotide probes (eg, oligodeoxynucleotides) that are specific for a set of miR genes. Using such a microarray, the level of expression of multiple microRNAs in a biological sample can be determined by reverse transcription of the RNAs to generate a set of target oligodeoxynucleotides, and hybridize them to screen the oligonucleotides on the microarray to generate a hybridization, or expression profile. The hybridization profile of the test sample can then be compared to that of a control sample to determine which microRNAs have an altered expression level in solid cancer cells.

As used herein, "probe oligonucleotide" or "probe oligodeoxynucleotide" refers to an oligonucleotide that is capable of hybridizing to a target oligonucleotide. "Target oligonucleotide" or "target oligodeoxynucleotide" refers to a molecule to detect (eg, by hybridization). By "miR-specific probe oligonucleotide" or "miR-specific probe oligonucleotide" is meant a probe oligonucleotide having a sequence selected to hybridize to a specific miR gene product, or to a reverse transcript of the specific miR gene product.

An "expression profile" or "hybridization profile" of a particular sample is essentially an identification of the state of the sample; While two states can have any particular gene similarly expressed, the evaluation of several genes simultaneously allows the generation of a gene expression profile that is unique to the state of the cell. That is, normal tissue can be distinguished from cancerous tissue (eg, tumor), and within cancerous tissue, different prognostic states (eg, good or bad long-term survival prospects) can be determined. By comparing the expression profiles of colon cancer tissue in different states, information is obtained regarding which genes are important (including both positive and negative regulation of genes) in each of these states. The identification of sequences that are differentially expressed in colon cancer tissue, as well as differential expression that results in different prognostic results, allows the use of this information in a number of ways.

In a non-limiting example, a particular treatment regimen can be evaluated (eg, to determine whether a chemotherapeutic drug works to improve the long-term prognosis in a particular patient). Similarly, the diagnosis can be made or confirmed by comparing patient samples with known expression profiles. Furthermore, these expression profiles (or individual genes) allow the exploration of pharmacological candidates that suppress the expression profile of colon cancer or convert a negative prognostic profile into a better prognostic profile.

Accordingly, also provided herein are methods of diagnosing whether a subject has, or is at risk of developing, colon cancer, which comprise reverse transcribing RNA from a test sample obtained from the subject to provide a set of target oligodeoxynucleotides, hybridize target oligodeoxynucleotides to a microarray comprising miRNA-specific probe oligonucleotides to provide a hybridization profile for the test sample, and compare the hybridization profile of the test sample with a hybridization profile generated from a control sample or reference standard, in which an alteration of the signal of at least one miRNA is indicative that the subject has, or is at risk of developing, a solid cancer.

In one embodiment, the microarray comprises miRNA-specific probe oligonucleotides for a substantial portion of all known human miRNAs. In a particular embodiment, the microarray comprises miRNA-specific probe oligonucleotides for one or more miRNAs selected from the group consisting of miR20a, miR-21, miR-106a, miR-181b, miR-203 and combinations thereof.

The microarray can be prepared from gene-specific oligonucleotide probes generated from known miRNA sequences. The array may contain two different oligonucleotide probes for each miRNA, one containing the active mature sequence and the other which is specific for the miRNA precursor. The array can also contain controls, such as one or more mouse sequences that differ from human orthologs by only a few bases, that can act as controls for stringency conditions of hybridization. Also tRNAs or other RNAs (eg, rRNA, mRNA) from both species can be printed on the microplate, providing a relatively stable internal positive control for specific hybridization. One or more appropriate controls for specific hybridization may also be included on the microplate. For this purpose, the sequences are selected based on the absence of any homology to any known miRNA.

Microarrays can be manufactured using techniques known in the art. For example, probe oligonucleotides of the appropriate length, eg 40 nucleotides, are modified with 5 'amine at position C6 and printed using commercially available microarray systems, eg the GenuMachine OmniGrid^TM 100 Microarrayer and Slides Activated by Amersham CodeLink^TM. The labeled cDNA oligomer corresponding to the target RNAs is prepared by reverse transcription of the target RNA with a labeled primer. After first strand synthesis, the RNA / DNA hybrids are denatured to degrade the RNA templates. The labeled target cDNAs prepared in this way are then hybridized on the microarray plate.

ES 2 425 387 T3 under hybridization conditions, for example, 6X SSPE / 30% formamide at 25 ° C for 18 hours, followed by washing in 0.75X TNT (Tris HCl / NaCl / Tween 20) at 37 ° C for 40 minutes. At positions on the array where the immobilized probe DNA recognizes a complementary target cDNA in the sample, hybridization occurs. The labeled target cDNA marks the exact position on the matrix where binding occurs, allowing automatic detection and quantification. The result consists of a list of hybridization events, indicating the relative abundance of specific cDNA sequences, and thus the relative abundance of the corresponding complementary miRs in the patient sample.

According to one embodiment, the labeled cDNA oligomer is a biotin-labeled cDNA, prepared from a biotin-labeled primer. The microarray is then processed by direct detection of biotin-containing transcripts using, for example, Streptavidin-Alexa647 conjugate, and screened using standard screening procedures. The image intensities of each point in the matrix are proportional to the abundance of the corresponding miR in the patient sample.

The use of the array has several advantages for detection of miRNA expression. First, the global expression of several hundred genes can be identified in the same sample at one point in time. Second, through careful design of the oligonucleotide probes, the expression of both mature and precursor molecules can be identified. Third, compared to Northern blot analysis, the microplate requires a small amount of RNA, and provides reproducible results using 2.5 pg of total RNA. The relatively limited number of miRNAs (several hundred per species) allows the construction of a common microarray for several species, with different oligonucleotide probes for each. This tool enables the analysis of trans-species expression for each known miR under various conditions.

In addition to use for specific miR quantitative expression level assays, a microplate containing miRNA-specific probe oligonucleotides corresponding to a substantial part of the miRNoma, preferably the entire miRNoma, can be used to perform miR gene expression profiling. , for analysis of miR expression patterns. Different miR identifications can be associated with established disease markers, or directly with a pathology.

In accordance with the expression profiling procedures described herein, total RNA from a sample from a subject suspected of having colon cancer-related disease was quantitatively reverse transcribed to provide a set of labeled target oligodeoxynucleotides. complementary to the RNA in the sample. The target oligodeoxynucleotides are then hybridized to a microarray comprising miRNA-specific probe oligonucleotides to provide a hybridization profile for the sample. The result is a hybridization profile for the sample showing the miRNA expression pattern in the sample. The hybridization profile comprises the signal of the binding of the target oligodeoxynucleotides in the sample with the miRNA-specific probe oligonucleotides on the microarray. The profile can be recorded as the presence or absence of binding (signal vs. zero signal).

Most preferably, the profile recorded includes the intensity of the signal from each hybridization. The profile is compared to the hybridization profile generated from a normal, ie, non-cancerous, control sample. An alteration of the signal is indicative of the presence of, or propensity to develop, cancer in the subject.

Other techniques for measuring miR gene expression are also within the skill of the art, and include various techniques for measuring RNA transcription and degradation rates.

Also provided herein are methods of determining the prognosis of a colon cancer subject, comprising measuring the level of at least one miR gene product, which is associated with a particular prognosis in a colon cancer-related disease ( eg, a good or positive prognosis, a negative or adverse prognosis), in a test sample of the subject.

According to these procedures, an alteration in the level of a miR gene product that is associated with a particular prognosis in the test sample, compared to the level of a corresponding miR gene product in a control sample, is indicative. that the subject has a solid cancer with a particular prognosis. In one embodiment, the miR gene product is associated with an adverse (ie, negative) prognosis. Examples of an adverse prognosis include, but are not limited to, low survival rate and rapid disease progression. In certain embodiments, the level of the at least one miR gene product is measured by reverse transcription of RNA from a test sample obtained from the subject to provide a set of target oligodeoxynucleotides, hybridizing the target oligodeoxynucleotides to a microarray comprising probe-specific oligonucleotides of miRNA to provide a hybridization profile for the test sample, and comparing the hybridization profile of the test sample with a hybridization profile generated from a control sample.

Without wishing to be bound by theory, it is believed that alterations in the level of one or more miR gene products in cells can result in the dysregulation of one or more intended targets for these miRs, which can lead to the formation of solid cancers. Therefore, altering the level of the miR gene product (for example, reducing the level of a miR gene product that is upregulated in cells of

ES 2 425 387 T3 solid cancer, by increasing the level of a miR gene product that is negatively regulated in solid cancer cells) can successfully treat solid cancer.

Accordingly, methods for inhibiting tumorigenesis in a subject who has, or is suspected of having, a solid cancer in which at least one miR gene product is deregulated (eg, down-regulated, down-regulated) are further provided herein. positively) in the subject's cancer cells. When the at least one isolated miR gene product is down-regulated in cancer cells (eg, miR-21), the method comprises administering an effective amount of the at least one isolated miR gene product, or an isolated variant, or a variant. isolated or biologically active fragment thereof, so as to inhibit the proliferation of cancer cells in the subject.

For example, when a miR gene product is downregulated in a cancer cell in a subject, administering an effective amount of an isolated miR gene product to the subject can inhibit proliferation of the cancer cell. The isolated miR gene product administered to the subject may be identical to the endogenous natural miR gene product (eg, a miR gene product) that is down-regulated in the cancer cell or it may be a biologically active variant or fragment thereof. .

As defined herein, a "variant" of a miR gene product refers to a miRNA that has less than 100% identity to a corresponding natural miR gene product and possesses one or more biological activities of the miR gene product. miR natural. Examples of such biological activities include, but are not limited to, inhibition of the expression of a target RNA molecule (e.g., inhibition of translation of a target RNA molecule, modulation of the stability of a target RNA molecule, inhibition of processing of a target RNA molecule) and inhibition of a solid cancer-associated cellular process (eg, cell differentiation, cell growth, cell death). These variants include species variants and variants that result from one or more mutations (eg, a substitution, a deletion, an insertion) in a miR gene. In certain embodiments, the variant is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to a corresponding natural miR gene product.

As defined herein, a "biologically active fragment" of a miR gene product refers to an RNA fragment of a miR gene product that possesses one or more biological activities of a corresponding natural miR gene product. As described above, examples of such biological activities include, but are not limited to, inhibition of the expression of a target RNA molecule and inhibition of a cellular process associated with colon cancer. In certain embodiments, the biologically active fragment is at least about 5, 7, 10, 12, 15, or 17 nucleotides in length. In a particular embodiment, an isolated miR gene product can be administered to a subject in combination with one or more additional antineoplastic treatments. Suitable antineoplastic treatments include, but are not limited to, chemotherapy, radiation therapy, and combinations thereof (eg, chemoradiation).

When the at least one isolated miR gene product is up-regulated in cancer cells, the method comprises administering to the subject an effective amount of at least one compound to inhibit the expression of the at least one miR gene product, referred to herein as miR gene expression inhibition compounds, so as to inhibit the proliferation of solid cancer cells. In a particular embodiment, the at least one miR expression inhibition compound is specific for a miR gene product selected from the group consisting of miR20a, miR-21, miR-106a, miR-181b, miR-203 and combinations thereof.

A miR gene expression inhibitor compound can be administered to a subject in combination with one or more additional antineoplastic treatments. Suitable antineoplastic treatments include, but are not limited to, chemotherapy, radiation therapy, and combinations thereof (eg, chemoradiation).

The terms "treat", "treating" and "treatment" as used herein refer to alleviating symptoms associated with a disease or condition, eg, solid cancer, including preventing or delaying the onset of symptoms. disease, and / or reduce the severity or frequency of symptoms of the disease or condition. The terms "subject", "patient" and "individual" are defined herein to include animals, such as mammals, including, but not limited to, primates, cows, sheep, goats, horses, dogs, cats, rabbits, guinea pigs. , rats, mice or other bovine, ovine, equine, canine, feline, rodent or murine species. In a preferred embodiment, the animal is human.

As used herein, an "effective amount" of an isolated miR gene product is an amount sufficient to inhibit the proliferation of a cancer cell in a subject suffering from a solid cancer. One skilled in the art can easily determine an effective amount of a miR gene product to administer to a given subject, taking into account factors such as the subject's height and weight; the extent of disease penetration; the age, health and sex of the subject; the route of administration; and if the administration is regional or systemic.

For example, an effective amount of an isolated miR gene product can be based on the approximate weight of a tumor mass to be treated. The approximate weight of a tumor mass can be determined by calculating the volume

ES 2 425 387 T3 approximate of the mass, being a cubic centimeter of volume approximately equivalent to one gram. An effective amount of the isolated miR gene product based on the weight of a tumor mass may be in the range of about 10-500 micrograms / gram of tumor mass. In certain embodiments, the tumor mass can be at least about 10 micrograms / gram of tumor mass, at least about 60 micrograms / gram of tumor mass, or at least about 100 micrograms / gram of tumor mass.

An effective amount of an isolated miR gene product can also be based on the approximate or estimated body weight of a subject to be treated. Preferably, such effective amounts are administered parenterally or enterally, as described herein. For example, an effective amount of the isolated miR gene product is administered to a subject that can range from about 5 to about 3,000 micrograms / kg of body weight, from about 700-1,000 micrograms / kg of body weight, or greater than about 1,000 micrograms / kg of body weight.

One of skill in the art can also readily determine an appropriate dosage regimen for the administration of an isolated miR gene product to a given subject. For example, a miR gene product can be administered to the subject once (eg, as a single injection or bowel movement). Alternatively, a miR gene product can be administered once or twice daily to a subject for a period of about 3 to about 28 days, more particularly about seven to about ten days. In a particular dosage regimen, a miR gene product is administered once daily for seven days. When a dosage regimen comprises multiple administrations, it is understood that the effective amount of miR gene product administered to the subject may comprise the total amount of gene product administered during the entire dosage regimen.

As used herein, an "isolated" miR gene product is one that is synthesized, or altered, or removed from the natural state by human intervention. For example, a synthetic miR gene product, or a miR gene product partially or completely separated from coexisting materials from its natural state, is considered to be "isolated". An isolated miR gene product may exist in substantially purified form, or it may exist in a cell into which the miR gene product has been delivered. Therefore, a miR gene product that is deliberately delivered to, or expressed in, a cell is considered an "isolated" miR gene product. A miR gene product produced within a cell from a miR precursor molecule is also considered to be an "isolated" molecule. According to a particular embodiment, the isolated miR gene products described herein can be used for the manufacture of a drug for treating solid cancer in a subject (eg, a human).

Isolated miR gene products can be obtained using various standard techniques. For example, miR gene products can be chemically synthesized or produced recombinantly using procedures known in the art. In one embodiment, miR gene products are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA / RNA synthesizer. Commercial suppliers of synthetic RNA molecules or synthesis reagents include, for example, Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL, USA) , Glen Research (Sterling, VA, United States), ChemGenes (Ashland, MA, United States) and Cruachem (Glasgow, United Kingdom).

Alternatively, miR gene products can be expressed from recombinant linear or circular DNA plasmids using any suitable promoter. Promoters suitable for expressing RNA from a plasmid include, for example, RNA pol III U6 or H1 promoter sequences, or cytomegalovirus promoters. Selection of suitable promoters is within the skill of the art. The recombinant plasmids of the invention may also comprise inducible or regulatable promoters for expression of miR gene products in cancer cells.

The miR gene products that are expressed from recombinant plasmids can be isolated from cultured cell expression systems by standard techniques. The miR gene products that are expressed from recombinant plasmids can also be delivered to, and directly expressed in, cancer cells. The use of recombinant plasmids to deliver miR gene products to cancer cells is discussed in more detail below.

The miR gene products can be expressed from a separate recombinant plasmid or they can be expressed from the same recombinant plasmid. In one embodiment, the miR gene products are expressed as RNA precursor molecules from a single plasmid, and the precursor molecules are processed into the functional miR gene product by a suitable processing system, including, but not limited to, processing systems. existing within a cancer cell. Other suitable processing systems include, for example, the in vitro Drosophila cell lysate system (for example, as described in US Published Patent Application No. 2002/0086356 to Tuschl et al. And the RNAse system III from E. coli (eg, as described in US Published Patent Application No. 2004/0014113 to Yang et al).

ES 2 425 387 T3

Selection of suitable plasmids to express the miR gene products, methods of inserting the nucleic acid sequences into the plasmid to express the gene products, and methods of delivering the recombinant plasmid to cells of interest are within the skill of the art. See, for example, Zeng et al. (2002), Molecular Cell 9: 1327-1333; Tuschl (2002), Nat. Biotechnol, 20: 446-448; Brummelkamp et al. (2002), Science 296: 550-553; Miyagishi et al. (2002), Nat. Biotechnol. 20: 497-500; Paddison et al. (2002), Genes Dev. 16: 948-958; Lee et al. (2002), Nat. Biotechnol. 20: 500-505; and Paul et al. (2002), Nat. Biotechnol. 20: 505-508.

In one embodiment, a plasmid expressing miR gene products comprises a sequence encoding a miR precursor RNA under the control of the CMV early-intermediate promoter. As used herein, "under the control" of a promoter means that the nucleic acid sequences encoding the miR gene product are located 3 'to the promoter, so that the promoter can initiate transcription of the sequences. encoding the miR gene product.

The miR gene products can also be expressed from recombinant viral vectors. It is contemplated that miR gene products can be expressed from two separate recombinant viral vectors or from the same viral vector. RNA expressed from recombinant viral vectors can be isolated from cultured cell expression systems by standard techniques or can be directly expressed in cancer cells. The use of recombinant viral vectors to deliver miR gene products to cancer cells is discussed in more detail below.

The recombinant viral vectors of the invention comprise sequences encoding the miR gene products and any suitable promoter to express the RNA sequences. Suitable promoters include, but are not limited to, RNA pol III U6 or H1 promoter sequences, or cytomegalovirus promoters. The selection of other suitable promoters is within the skill of the art. The recombinant viral vectors of the invention may also comprise inducible or regulatable promoters for expression of miR gene products in a cancer cell.

Any viral vector capable of accepting the coding sequences for miR gene products can be used; for example, vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (eg lentivirus (LV), Rhabdovirus, murine leukemia virus); herpes viruses and the like. The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins as appropriate.

For example, the lentiviral vectors of the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. The AAV vectors of the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector that expresses a serotype 2 capsid in a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors expressing different capsid protein serotypes are within the skill of the art; see, for example, Rabinowitz, JE, et al. (2002), J. Virol. 76: 791-801.

Selection of suitable recombinant viral vectors for use in the invention, methods of inserting nucleic acid sequences to express RNA into the vector, methods of delivering the viral vector to cells of interest, and methods are within the skill of the art. recovery of expressed RNA products. See, for example, Dornburg (1995), Gene Therapy 2: 301-310; Eglitis (1988). Biotechniques 6: 608614; Miller (1990), Hum. Gene Therapy 1: 5-14; and Anderson (1998), Nature 392: 25-30.

Particularly suitable viral vectors are those derived from AV and AAV. A suitable AV vector for expressing miR gene products, a method for constructing the recombinant AV vector, and a method for delivering the vector to target cells are described in Xia et al. (2002), Nat. Biotech. 20: 1006-1010. Suitable AAV vectors for expressing miR gene products, methods for constructing the recombinant AAV vector, and methods for delivering the vectors to target cells are described in Samulski et al. (1987), J. Virol. 61: 3096-3101; Fisher et al. (1996), J. Virol., 70: 520-532; Samulski et al. (1989), J. Virol. 63: 3822-3826; US Patent No. 5,252,479; US Patent No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641. In one embodiment, the miR gene products are expressed from a single recombinant AAV vector comprising the CMV intermediate early promoter.

In a certain embodiment, a recombinant AAV viral vector of the invention comprises a nucleic acid sequence encoding a miR precursor RNA in operable connection with a polyT termination sequence under the control of a human U6 RNA promoter. As used herein, "in operable connection with a polyT termination sequence" means that the nucleic acid sequences encoding the sense and antisense strands are immediately adjacent to the polyT termination signal in the 5 'direction. During transcription of the vector's miR sequences, polyT termination signals act to terminate transcription.

In another embodiment of the treatment methods of the invention, an amount

ES 2 425 387 T3 efficacy of at least one compound that inhibits miR expression. As used herein, "inhibit miR expression" means that the production of the precursor and / or active mature form of the miR gene product after treatment is less than the amount produced before treatment. One skilled in the art can easily determine whether miR expression has been inhibited in a cancer cell, using, for example, the techniques for determining the level of miR transcript discussed above for the diagnostic procedure. Inhibition can occur at the level of gene expression (i.e., inhibiting the transcription of a miR gene encoding the miR gene product) or at the level of processing (for example, inhibiting the processing of a miR precursor into an active mature miR ).

As used herein, an "effective amount" of a compound that inhibits miR expression is an amount sufficient to inhibit the proliferation of a cancer cell in a subject suffering from cancer (eg, colon cancer). One skilled in the art can easily determine an effective amount of a miR expression inhibiting compound to administer to a given subject, taking into account factors such as the subject's height and weight; the degree of penetration of disease; the age, health and sex of the subject; the route of administration; and if the administration is regional or systemic.

For example, an effective amount of the expression inhibiting compound can be based on the approximate weight of a tumor mass to treat, as described herein. An effective amount of a compound that inhibits miR expression can also be based on the approximate or estimated body weight of a subject to be treated, as described herein.

One skilled in the art can also readily determine an appropriate dosage regimen for administering a compound that inhibits miR expression to a given subject.

Suitable compounds for inhibiting miR gene expression include double-stranded RNA (such as small or short interference RNA or "siRNA"), antisense nucleic acids, and enzymatic RNA molecules, such as ribozymes. Each of these compounds can target a given miR gene product and interfere with the expression (eg, inhibit translation of, induce cleavage or destruction of) the target miR gene product.

For example the expression of a given miR gene can be inhibited by inducing RNA interference of the miR gene with an isolated double-stranded RNA ("dsRNA") molecule that has at least 90%, for example at least 95%, to the least 98%, at least 99% or 100% sequence homology with at least a part of the miR gene product. In a particular embodiment, the dsRNA molecule is a "short or small interference RNA" or "siRNA".

SiRNAs useful in the present methods comprise short double stranded RNAs from about 17 nucleotides to about 29 nucleotides in length, preferably from about 19 nucleotides to about 25 nucleotides in length. The siRNA comprises a sense RNA strand and a complementary antisense RNA strand hybridized to each other by interactions of standard Watson-Crick base pair arrays (hereinafter "with base pairs"). The sense strand comprises a nucleic acid sequence that is substantially identical to a nucleic acid sequence contained within the target miR gene product.

As used herein, a nucleic acid sequence in a siRNA that is "substantially identical" to a target sequence contained within the target mRNA is a nucleic acid sequence that is identical to, or differs from, the target sequence. target sequence at one or two nucleotides. The sense and antisense strands of siRNA may comprise two complementary single-stranded RNA molecules, or they may comprise a single molecule in which two complementary parts are base-paired and are covalently linked by a single-stranded "hairpin" area.

SiRNA can also be altered RNA that differs from naturally occurring RNA by adding, deleting, substituting, and / or altering one or more nucleotides. Such alterations may include the addition of non-nucleotide material, such as the end or ends of the siRNA or to one or more internal nucleotides of the siRNA or modifications that render the siRNA resistant to nuclease digestion, or the substitution of one or more nucleotides in the siRNA. SiRNA with deoxyribonucleotides.

One or both strands of the siRNA may also comprise a 3 'overhang. As used herein a "3 'overhang" refers to at least one mismatched nucleotide extending from the 3' end of a double stranded RNA strand. Thus, in certain embodiments, the siRNA comprises at least one 3 'overhang of one to about 6 nucleotides (including ribonucleotides or deoxyribonucleotides) in length, 1 to about 5 nucleotides in length, 1 to about 4 nucleotides in length, or about 2 to about 4 nucleotides in length. In a particular embodiment, the 3 'overhang is present on both strands of the siRNA and is 2 nucleotides long. For example, each strand of siRNA may comprise 3 'overhangs of dithymidylic acid ("TT") or diuridylic acid ("uu").

SiRNA can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as described above for isolated miR gene products. They describe

ES 2 425 387 T3 Exemplary procedures for producing and testing dsRNA or siRNA molecules in the Patent Application

Gewirtz US Published No. 2002/0173478 and US Published Patent Application

No. 2004/0018176 to Reich et al.

The expression of a given miR gene can also be inhibited by an antisense nucleic acid. As used herein, an "antisense nucleic acid" refers to a nucleic acid molecule that binds to target RNA via RNA-RNA, RNA-DNA, or DNA-peptide nucleic acid interactions, which alters activity of the target RNA. Suitable antisense nucleic acids for use in the present methods are single-stranded nucleic acids (eg, RNA, DNA, RNA-DNA chimeras, peptide nucleic acid (PNA)) that generally comprise a nucleic acid sequence complementary to an acid sequence. contiguous nucleic acid in a miR gene product. The antisense nucleic acid can comprise a nucleic acid sequence that is 50-100% complementary, 75-100% complementary, or 95-100% complementary to a contiguous nucleic acid sequence in a miR gene product.

Without wishing to be bound by theory, it is believed that antisense nucleic acids activate RNase H or other cellular nuclease that digests the miR gene / antisense nucleic acid double strand.

Antisense nucleic acids may also contain modifications of the nucleic acid backbone or sugar and base moieties (or their equivalents) to enhance target specificity, nuclease resistance, delivery, or other properties related to the efficacy of the molecule. Such modifications include cholesterol moieties, double-stranded intercalators, such as acridine, or one or more nuclease resistant groups.

Antisense nucleic acids can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as described above for isolated miR gene products. Procedures for producing and testing are within the skill of the art; see, for example, Stein and Cheng (1993), Science 261: 1004 and US Patent No. 5,849,902 to Woolf et al., the full disclosures of which are incorporated herein by reference.

The expression of a given miR gene can also be inhibited by an enzymatic nucleic acid. As used herein, an "enzymatic nucleic acid" refers to a nucleic acid that comprises a substrate-binding region that has complementarity to a contiguous nucleic acid sequence of a miR gene product, and that is capable of specifically cleave the miR gene product. The enzyme nucleic acid substrate binding region can be, for example, 50-100% complementary, 75-100% complementary, or 95-100% complementary to a contiguous nucleic acid sequence in a miR gene product. Enzymatic nucleic acids can also comprise modifications to the base, sugar, and / or phosphate groups. An exemplary enzymatic nucleic acid for use in the present methods is a ribozyme.

Enzymatic nucleic acids can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as described above for isolated miR gene products. Exemplary procedures for producing and testing dsRNA or siRNA molecules are described in Werner and Uhlenbeck (1995), Nucl. Acids Res. 23: 2092-96; Hammann et al. (1999), Antisense and Nucleic Acid Drug Dev. 9: 25-31; and US Patent No. 4,987,071 to Cech et al.

Administration of at least one miR gene product, or at least one compound to inhibit miR expression, will inhibit the proliferation of cancer cells in a subject having a solid cancer. As used herein, "inhibiting the proliferation of a cancer cell" means destroying the cell, or permanently or temporarily halting or slowing the growth of the cell. Inhibition of cancer cell proliferation can be inferred if the number of such cells in the subject remains constant or is reduced after administration of miR gene products, or miR gene expression inhibition compounds. An inhibition of cancer cell proliferation can also be inferred if the absolute number of cancer cells increases, but the tumor growth rate is reduced.

The number of cancer cells in a subject's body can be determined by direct measurement, or by estimating the size of primary or metastatic tumor masses. For example, the number of cancer cells in a subject can be measured by immunohistological procedures, flow cytometry, or other techniques designed to detect characteristic surface markers of cancer cells.

The size of a tumor mass can be determined by direct visual information, or by diagnostic imaging procedures such as X-rays, magnetic resonance imaging, ultrasound, and scintigraphy. Diagnostic imaging procedures can be used to determine the size of the tumor mass with or without contrast agents, as is known in the art. The size of a tumor mass can also be determined by physical means, such as palpation of the tissue mass or measurement of the tissue mass with a measuring instrument, such as a caliper.

The miR gene products or miR gene expression inhibition compounds can be administered to a subject by any suitable means to deliver these compounds to cancer cells of the subject. For example, miR gene products or miR expression inhibition compounds can be administered by suitable procedures to transfect cells of the subject with these compounds, or with nucleic acids that

ES 2 425 387 T3 comprise sequences encoding these compounds.

In one embodiment, cells are transfected with a plasmid or viral vector comprising sequences encoding at least one miR gene product or miR gene expression inhibition compound.

Transfection procedures for eukaryotic cells are well known in the art, and include, for example, direct injection of nucleic acid into the nucleic or pronucleus of a cell; electroporation; liposome transfer or transfer mediated by lipophilic materials; receptor-mediated nucleic acid delivery, biobalistics, or particle acceleration; calcium phosphate precipitation and viral vector-mediated transfection.

For example, cells can be transfected with a liposome transfer compound, for example, DOTAP (N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium methylsulfate, Boehringer-Mannheim) or an equivalent, such as LIPOFECTIN. The amount of nucleic acid used is not critical to the practice of the invention; Acceptable results can be achieved with 0.1-100 micrograms nucleic acid / 10⁵ cells. For example, a ratio of approximately 0.5 micrograms of plasmid vector in 3 micrograms of DOTAP per 10⁵ cells.

A miR gene product or miR gene expression inhibition compound can also be administered to a subject by any suitable enteral or parenteral route of administration. Enteric routes of administration suitable for the present procedures include, for example, oral, rectal, or intranasal delivery. Suitable parenteral routes of administration include, for example, intravascular administration (eg, intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion, and catheter instillation into the vasculature); peri- and intratissue injection (eg, peritumoral and intratumoral injection, intraretinal injection or subretinal injection); subcutaneous injection or deposition, including subcutaneous infusion (such as by osmotic pumps); direct application to the tissue of interest, for example by a catheter or other delivery device (for example, a retinal pellet or suppository or an implant comprising a porous, non-porous or gelatinous material); and inhalation. Particularly suitable routes of administration are injection, infusion and direct injection into the tumor.

In the present methods, a miR gene product or miR gene product expression inhibition compound can be administered to the subject as naked RNA, in combination with a delivery reagent, or as a nucleic acid (eg, a recombinant plasmid or viral vector) comprising sequences expressing the miR gene product or miR gene product expression inhibiting compound. Suitable delivery reagents include, for example, lipophilic Mirus Transit TKO reagent; lipofectin; lipofectamine; celfectin; polycations (eg, polylysine) and liposomes.

Recombinant plasmids and viral vectors comprising sequences expressing miR gene products or miR gene expression inhibiting compounds, and techniques for delivering such plasmids and vectors to miR gene expression inhibitors, are discussed herein and / or are well known in the art. cancer cells.

In a particular embodiment, liposomes are used to deliver a miR gene product or miR gene expression inhibition compound (or nucleic acids comprising sequences that encode them) to a subject. Liposomes can also increase the blood half-life of gene products or nucleic acids. Suitable liposomes for use in the invention can be formed from conventional vesicle-forming lipids, which generally include negatively charged or neutral phospholipids and a sterol, such as cholesterol. Lipid selection is generally guided by consideration of factors, such as the desired liposome size and the half-life of the liposomes in the bloodstream. A variety of procedures are known to prepare liposomes, for example, as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng. 9: 467; and United States Patent Nos. 4,235,871,4,501,728, 4,837,028 and 5,019,369.

Liposomes for use in the present methods may comprise a ligand molecule that targets liposome to cancer cells. Ligands that bind to receptors prevalent on cancer cells, such as monoclonal antibodies that bind to tumor cell antigens, are preferred.

Liposomes for use in the present methods can also be modified to avoid removal by the mononuclear macrophage system ("MMS") and reticulum endothelial system ("RES"). Such modified liposomes have opsonization inhibiting moieties on the surface or incorporated into the structure of the liposome. In a particularly preferred embodiment, a liposome of the invention may comprise both an opsonization inhibiting moiety and a ligand.

The opsonization inhibiting moieties for use in preparing the liposomes of the invention are typically large hydrophilic polymers that bind to the liposome membrane. As used herein, an opsonization inhibiting moiety is "bound" to a liposome membrane when it is chemically or physically bound to the membrane, for example, by intercalation of a lipid soluble anchor on the membrane at itself, or by directly binding to active membrane lipid groups. These opsonization inhibitor hydrophilic polymers form a protective surface layer that significantly reduces the uptake of liposomes by MMS and RES; for example, as described in US Patent No. 4,920,016.

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Suitable opsonization inhibiting moieties for modifying liposomes are preferably water soluble polymers with a number average molecular weight of from about 500 to about 40,000 daltons and more preferably from about 2,000 to about 20,000 daltons. Such polymers include derivatives of polyethylene glycol (PEG) or polypropylene glycol (PPG); for example, methoxy PEG or PPG and PEG or PPG stearate; synthetic polymers, such as polyacrylamide or poly N-vinyl pyrrolidone; linear, branched or dendrimeric polyamidoamines; polyacrylic acids; polyalcohols, for example polyvinyl alcohol and polyxyylitol with which carboxylic or amino groups are chemically linked, as well as gangliosides, such as ganglioside GM1. Also suitable are copolymers of PEG, methoxy PEG or methoxy PPG or derivatives thereof. Furthermore, the opsonization inhibiting polymer can be a block copolymer of PEG and a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide. The opsonization inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, for example galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; amino polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, for example, that react with derivatives of carbonic acids with resulting linkage of carboxylic groups. Preferably, the opsonization inhibitory moiety is a PEG, PPG, or a derivative thereof. PEG-modified or PEG-derived liposomes are sometimes referred to as "pegylated liposomes."

The opsonization inhibiting moiety can be attached to the liposome membrane by any of a number of well-known techniques. For example, the N-hydroxysuccinimide ester of pEg can be attached to a lipid soluble phosphatidyl ethanolamine anchor and then attached to a membrane. Similarly, a dextran polymer can be derivatized with a lipid soluble stearyl amine anchor by reductive amination using Na (CN) BH3 and a mixed solvent, such as tetrahydrofuran and water in a 30:12 ratio at 60 ° C.

Liposomes modified with opsonization inhibition moieties remain in circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes referred to as "stealth" liposomes. Stealth liposomes are known to accumulate in tissues fed by porous or "filtering" microvasculature. Therefore, tissue characterized by said microvasculature defects, eg solid tumors, will efficiently accumulate these liposomes; see Gabizon, et al. (1988), Proc. Natl. Acad. Sci., USA, 18: 6949-53. Furthermore, the reduced uptake by RES reduces the toxicity of stealth liposomes by preventing significant accumulation of liposomes in the liver and spleen. Therefore, liposomes that are modified with opsonization inhibitory moieties are particularly suitable for delivering miR gene products or miR gene expression inhibition compounds (or nucleic acids comprising sequences encoding them) to tumor cells.

The miR gene products or miR gene expression inhibition compounds can be formulated as pharmaceutical compositions, sometimes referred to as "drugs", prior to administration to a subject, according to techniques known in the art. Accordingly, the invention encompasses pharmaceutical compositions for treating solid cancer. In one embodiment, the pharmaceutical composition comprises at least one isolated miR gene product, or an isolated variant or biologically active fragment thereof, and a pharmaceutically acceptable carrier. In a particular embodiment, the at least one miR gene product corresponds to a miR gene product that has a reduced level of expression in solid cancer cells relative to suitable control cells. In certain embodiments, the isolated miR gene product is selected from the group consisting of miR20a, miR-21, miR-106a, miR-181b, miR-203, and combinations thereof.

In other embodiments, the pharmaceutical compositions of the invention comprise at least one miR expression inhibiting compound. In a particular embodiment, the at least one miR gene expression inhibition compound is specific for a miR gene whose expression is greater in colon cancer cells than in control cells. In certain embodiments, the miR gene expression inhibitor compound is specific for one or more miR gene products selected from the group consisting of miR20a, miR-21, miR-106a, miR181b, miR-203, and combinations thereof.

The pharmaceutical compositions of the present invention are characterized by being at least sterile and without pyrogens. As used herein, "pharmaceutical compositions" include formulations for human and veterinary use. Procedures for preparing pharmaceutical compositions of the invention are within the skill of the art, for example as described in Remington's Pharmaceutical Science, 17^to ed., Mack Publishing Company, Easton, Pa. (1985).

The present pharmaceutical compositions comprise at least one miR gene product or miR gene expression inhibition compound (or at least one nucleic acid comprising sequences encoding them) (eg 0.1 to 90% by weight) , or a physiologically acceptable salt thereof, in admixture with a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical compositions of the invention additionally comprise one or more antineoplastic agents (eg, chemotherapeutic agents). The pharmaceutical formulations of the invention may also comprise at least one miR gene product or miR gene expression inhibition compound (or at least one nucleic acid comprising sequences encoding them), which are encapsulated by liposomes and a pharmaceutically carrier. acceptable. In one embodiment, the pharmaceutical composition comprises a miR gene or gene product that is miR-21.

ES 2 425 387 T3

Especially suitable pharmaceutically acceptable carriers are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid, and the like.

In a particular embodiment, the pharmaceutical compositions of the invention comprise at least one miR gene product or miR gene expression inhibition compound (or at least one nucleic acid comprising sequences that encode them) that is resistant to degradation by nucleases. . One skilled in the art can easily synthesize nucleic acids that are nuclease resistant, for example by incorporating one or more ribonucleotides that are modified at the 2 'position in the miR gene product. Suitable 2 'modified ribonucleotides include those modified at the 2' position with fluoro, amino, alkyl, alkoxy and O-allyl.

The pharmaceutical compositions of the invention may also comprise conventional pharmaceutical excipients and / or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolarity adjusting agents, buffers, and pH adjusting agents. Suitable additives include, for example, physiologically biocompatible buffers (for example, tromethamine hydrochloride), additions of chelators (such as, for example, DTPA or DTPA-bisamide) or complexes of calcium chelates (such as, for example, DTPA calcium, CaNaDTPA-bisamide) or, optionally, additions of calcium or sodium salts (for example calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). The pharmaceutical compositions of the invention can be packaged for use in liquid form, or they can be lyophilized.

For solid pharmaceutical compositions of the invention, conventional non-toxic solid pharmaceutically acceptable carriers can be used; for example, pharmaceutical uses of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like.

For example, a solid pharmaceutical composition for oral administration may comprise any of the carriers and excipients listed above and 10-95%, preferably 25-75% of the at least one miR gene product or gene expression inhibition compound of miR (or at least one nucleic acid comprising sequences encoding them). A pharmaceutical composition for administration by aerosol (by inhalation) may comprise 0.01-20% by weight, preferably 1% -10% by weight, of the at least one miR gene product or gene expression inhibition compound of miR (or at least one nucleic acid comprising sequences encoding them) encapsulated in a liposome as described above, and a propellant. A vehicle can also be included as desired; for example, lecithin for intranasal delivery.

The pharmaceutical compositions of the invention may further comprise one or more antineoplastic agents. In a particular embodiment, the compositions comprise at least one miR gene product or miR gene expression inhibition compound (or at least one nucleic acid comprising sequences that encode them) and at least one chemotherapeutic agent. Chemotherapeutic agents that are suitable for the methods of the invention include, but are not limited to, DNA alkylating agents, antitumor antibiotic agents, anti-metabolic agents, tubulin stabilizing agents, tubulin destabilizing agents, hormone antagonizing agents, topoisomerase inhibitors, protein kinase inhibitors, HMG-CoA inhibitors, CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinase inhibitors, antisense nucleic acids, triple helix DNA, nucleic acid aptamers, and molecularly modified viral, bacterial, and exotoxic agents. Examples of suitable agents for the compositions of the present invention include, but are not limited to, cytidine arabinoside, methotrexate, vincristine, etoposide (VP-16), doxorubicin (adriamycin), cisplatin (CDDP), dexamethasone, arglabine, cyclophosphamide, sarcolysin, methylnitrosourea, fluorouracil, 5-fluorouracil (5FU), vinblastine, camptothecin, actinomycin-D, mitomycin C, hydrogen peroxide, oxaliplatin, irinotecan, topotecan, leucovorin, carmustine, streptozocin, CPT-11, taxol, tamoxifen, dacarbazine, rituximab, daunorubicin, 1-β-D-arabinofuranosylcytosine, imatinib, fludarabine, docetaxel, FOLFOX4.

Also provided herein are methods of identifying an inhibitor of tumorigenesis, comprising providing a test agent to a cell and measuring the level of at least one miR gene product in the cell. In one embodiment, the method comprises providing a test agent to a cell and measuring the level of at least one miR gene product associated with reduced expression levels in cancer cells. An increase in the level of the miR gene product in the cell after the agent is provided, relative to a suitable control cell (eg, no agent is provided), is indicative that the test agent is an inhibitor of tumorigenesis. In a particular embodiment, at least one miR gene product associated with reduced expression levels in cancer cells is selected from the group consisting of miR20a, miR-21, miR-106a, miR-181b, miR-203 and combinations thereof .

In other embodiments, the method comprises providing a test agent to a cell and measuring the level of at least one miR gene product associated with increased expression levels in cancer cells. A reduction in the level of the miR gene product in the cell after the agent is provided, relative to a suitable control cell (eg, no agent is provided), is indicative that the test agent is an inhibitor. of tumorigenesis. In a particular embodiment, at least one miR gene product associated with increased expression levels in cancer cells is selected from the group consisting of miR20a, miR-21, miR-106a, miR-181b, miR-203 and combinations thereof .

Suitable agents include, but are not limited to, drugs (eg, small molecules, peptides), and

ES 2 425 387 T3 biological macromolecules (eg proteins, nucleic acids). The agent can be produced recombinantly, synthetically, or can be isolated (ie, purified) from a natural source. Various procedures for delivering such agents to a cell (eg, transfection) are well known in the art, and several such procedures have been described hereinbefore. Methods for detecting the expression of at least one miR gene product (eg Northern blot, in situ hybridization, RT-PCR, expression profiling) are also well known in the art. Several of these procedures have also been described previously herein.

The invention will now be illustrated by the following non-limiting examples.

Example 1

MicroRNA expression patterns are altered in control tumors

The inventors compared microRNA profiles of 84 pairs of colon tumor and adjacent non-tumor tissues using microRNA microarrays.³⁰. These 84 subjects were patients recruited from the greater Baltimore, Maryland area with incident colon adenocarcinoma and is termed the Maryland trial cohort (Table 1).

Table 1- Population and tumor characteristics

<td></td><td>Maryland Trial Cohort</td><td>Hong Kong Validation Cohort</td>

<td></td><td>N = 84</td><td>N = 113</td>

<td>Recruitment area</td><td>Baltimore, Maryland, United States</td><td>Hong Kong, China</td>

<td>Age at admission years Mean ± SD</td><td> 64,6 ± 10,7</td><td> 55,8 ± 15</td>

<td>Interval</td><td> 32-87</td><td> 32-84</td>

<td>Sex - N ° (%)</td><td></td><td></td>

<td>Men</td><td> 66 (79)</td><td> 56 (50)</td>

<td>Woman</td><td> 18 (21)</td><td> 57 (50)</td>

<td>Race - N ° (%)</td><td></td><td></td>

<td>White</td><td> 52 (62)</td><td> 0 (0)</td>

<td>Black</td><td> 32 (38)</td><td> 0 (0)</td>

<td>Asian</td><td> 0 (0)</td><td> 113 (100)</td>

<td>Tumor location - No. (%)</td><td></td><td></td>

<td>Distal</td><td> 48 (59)</td><td> 90 (80)</td>

<td>Proximal</td><td> 34 (41)</td><td> 23 (20)</td>

<td>Histology of adenocarcinoma - No. (%) Adenocarcinoma</td><td> 75 (89)</td><td> 105 (93)</td>

<td>Mucinous adenocarcinoma</td><td> 8 (10)</td><td> 7 (6)</td>

<td>Adenosquamous carcinoma</td><td> 1 (1)</td><td> 0 (0)</td>

<td>Signet ring and mucinous cell</td><td> 0 (0)</td><td> 1 (1)</td>

<td>Adjuvant chemotherapy² - N ° (%) Received</td><td> 22 (37)</td><td> 40 (35)</td>

<td>Did not receive</td><td> 37 (63)</td><td> 73 (65)</td>

<td>TNM stage - N^s (%)</td><td></td><td></td>

<td>II</td><td> 29 (34)</td><td> 37 (33)</td>

<td>III</td><td> 36 (43)</td><td> 48 (42)</td>

<td>IV</td><td> 10 (12)</td><td> 19 (17)</td>

<td colspan="3">'Distal includes tumors located in or distant from the descending colon. Proximal tumors include tumors in or near the splenic flexure. Tumor location was available for 82 subjects in the original cohort and all subjects in the validation cohort.²Detailed information concerning the receipt of chemotherapy was available for 59 subjects in the trial cohort and all subjects in the validation cohort.</td>

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Chemotherapy was primarily fluorouracil-based (in forms of 5-fluorouracil intravenous or oral drugs including tegafur with uracil [UFT]) with or without Levamisole or Leucovorin.

The tumor microRNA profiles were clearly different from non-tumor profiles. 37 independent microRNAs were found to be differentially expressed in tumors (p &lt;0.001 with false discovery rate &lt;0.5%); Table 2.

Table 2 - MicroRNAs that are differentially expressed in tumors

<td>Probe</td><td>look mature</td><td>p-value¹</td><td>Fdr²</td><td>Change factor</td><td>Chromosomal location</td>

<td>hsa-mir-21 No1</td><td>miR-21</td><td>&lt;1e-07</td><td>&lt;1e-07</td><td> 1,7</td><td>17q23.2</td>

<td>hsa-mir-021-prec-17No1</td><td>miR-21</td><td>&lt;1e-07</td><td>&lt;1e-07</td><td> 1,8</td><td>17q23.2</td>

<td>hsa-mir-092-prec-13 = 092-1No2</td><td>miR-92</td><td>&lt;1e-07</td><td>&lt;1e-07</td><td> 1,4</td><td>13g31.3</td>

<td>hsa-mir-222-precNo2</td><td>miR-222</td><td>1.40E-06</td><td>8.05E-05</td><td> 1,2</td><td>Xp11.3</td>

<td>hsa-mir-181b-2No1</td><td>miR-181b</td><td>1.90E-06</td><td>8.74E-05</td><td> 1,2</td><td>9q33.3</td>

<td>hsa-mir-210-prec</td><td>mIR-210</td><td>1.12E-05</td><td> 0,00032</td><td> 1,2</td><td>11p15.5</td>

<td>hsa-mir-020-prec</td><td>miR-20a</td><td>2.53E-05</td><td> 0,00057</td><td> 1,5</td><td>13q31.3</td>

<td>hsa-mir-106-prec-X</td><td>miR-106a</td><td>3.30E-05</td><td> 0,00058</td><td> 1,4</td><td>X26.2</td>

<td>hsa-mir-106aNo1</td><td>miR-106a</td><td>3.51E-05</td><td> 0,00058</td><td> 1,4</td><td>X26.2</td>

<td>hsa-mir-093-prec-7.1 = 093-1</td><td>miR-93</td><td>3.52E-05</td><td> 0,00058</td><td> 1,2</td><td>7q22.1</td>

<td>hsa-mir-335No2</td><td>miR-335</td><td>3.55E-05</td><td> 0,00058</td><td> 1,2</td><td>7q32.2</td>

<td>hsa-mir-222-precNol</td><td>miR-222</td><td>4.27E-05</td><td> 0,00065</td><td> 1,2</td><td>Xp11.3</td>

<td>hsa-mir-338No1</td><td>miR-338</td><td>5.78E-05</td><td> 0,00074</td><td> 1,1</td><td>17q25.3</td>

<td>hsa-mir-133bNo2</td><td>miR-133b</td><td>6.50E-05</td><td> 0,00079</td><td> 1,1</td><td>6p12.2</td>

<td>hsa-mir-092-prec-X = 092-2</td><td>miR-92</td><td>7.95E-05</td><td> 0,00083</td><td> 1,4</td><td>Xq26.2</td>

<td>hsa-mir-346No1</td><td>miR-346</td><td>8.42E-05</td><td> 0,00084</td><td> 1,2</td><td>10q23.2</td>

<td>hsa-mir-106bNo1</td><td>miR-106b</td><td> 0,0002091</td><td> 0,00178</td><td> 1,2</td><td>7q22.1</td>

<td>hsa-mir-135-2-prec</td><td>miR-153a</td><td> 0,0002363</td><td> 0,00194</td><td> 1,1</td><td>12q23.1</td>

<td>hsa-mir-219-1No2</td><td>miR-219</td><td> 0,0002515</td><td> 0,00199</td><td> 1,3</td><td>9q34.11</td>

<td>hsa-mir-34aNo1</td><td>miR-34a</td><td> 0,000265</td><td> 0,00203</td><td> 1,1</td><td>1p36.22</td>

<td>hsa-mir-099b-prec-19No1</td><td>miR-99b</td><td> 0,0003758</td><td> 0,00259</td><td> 1,1</td><td>19q13.41</td>

<td>hsa-mir-185-precNo2</td><td>miR-185</td><td> 0,0003827</td><td> 0,00259</td><td> 1,2</td><td>22q11.21</td>

<td>hsa-mir-223-prec</td><td>miR-223</td><td> 0,0004038</td><td> 0,00265</td><td> 1,4</td><td>Xq12</td>

<td>hsa-mir-211 -precNo2</td><td>miR-211</td><td> 0,0004338</td><td> 0,00277</td><td> 1,1</td><td>15q13.3</td>

<td>hsa-mir-135-1-prec</td><td>miR-135a</td><td> 0,0004648</td><td> 0,00287</td><td> 1,1</td><td>3p21.1</td>

<td>hsa-mir-127-prec</td><td>miR-127</td><td> 0,0004748</td><td> 0,00287</td><td> 1,1</td><td>14q32.31</td>

<td>hsa-mir-203-precNo1</td><td>miR-203</td><td> 0,0009933</td><td> 0,00294</td><td> 1,4</td><td>14q32.33</td>

<td>hsa-mir-212-precNo1</td><td>miR-212</td><td> 0,0006339</td><td> 0,00364</td><td> 1,1</td><td>17p13.3</td>

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<td>hsa-mir-095-prec-4</td><td>miR-95</td><td> 0,0006996</td><td> 0,00392</td><td> 1,2</td><td>4p16.1</td>

<td>hsa-mir-017-precNo2</td><td>miR-17-5p</td><td> 0,0007252</td><td> 0,00392</td><td> 1,3</td><td>13q31.3</td>

<td colspan="6">microRNA with reduced expression in tumors</td>

<td>Probe</td><td>look mature</td><td>p-value¹</td><td>Fdr²</td><td>Change factor</td><td>Chromosomal location</td>

<td>hsa-mir-342No2</td><td>miR-342</td><td>4.00E-06</td><td> 0,0005</td><td> 0,9</td><td>14q32.2</td>

<td>hsa-mir-192-2 / 3Nol</td><td>miR-192</td><td>8.70E-06</td><td> 0,00029</td><td> 0,7</td><td>11q13.1</td>

<td>hsa-mir-1-2No2</td><td>miR-1</td><td>2.22E-05</td><td> 0,00057</td><td> 0,9</td><td>18g11.2</td>

<td>hsa-mir-34bNo2</td><td>miR-34b</td><td>4.78E-05</td><td> 0,00069</td><td> 0,8</td><td>11q23.1</td>

<td>hsa-mir-215-precNo1</td><td>miR-215</td><td>5.26E-05</td><td> 0,00071</td><td> 0,7</td><td>1q41</td>

<td>hsa-mir-192No1</td><td>miR-192</td><td>7.36E-05</td><td> 0,00081</td><td> 0,7</td><td>11q13.1</td>

<td>hsa-mir-301 No2</td><td>miR-301</td><td>7.44E-05</td><td> 0,00081</td><td> 0,7</td><td>17q23.2</td>

<td>hsa-miR-324-5pNo2</td><td>miR-324-5p</td><td>1.00E-04</td><td> 0,00096</td><td> 0,9</td><td>17p13.1</td>

<td>hsa-mir-030a-precNo2</td><td>miR-30a-3p</td><td> 0,0001933</td><td> 0,00171</td><td> 0,9</td><td>6q13</td>

<td>hsa-mir-1-1 No2</td><td>miR-1</td><td> 0,0002906</td><td> 0,00216</td><td> 0,9</td><td>20q13.33</td>

<td>hsa-mir-34cNo2</td><td>miR-34c</td><td> 0,0007334</td><td> 0,00392</td><td> 0,9</td><td>11q23.1</td>

<td>hsa-mir-331 No2</td><td>miR-331</td><td> 0,0008555</td><td> 0,00446</td><td> 0,9</td><td>12q22</td>

<td>hsa-mir-148bNo2</td><td>miR-148b</td><td> 0,0008726</td><td> 0,00446</td><td> 0,9</td><td>12q13.13</td>

<td colspan="6">The p-values ​​presented are the result of paired class comparison analysis of microRNA expression patterns from 84 pairs of colon adenocarcinomas and non-tumor tissue. ²FDR = false discovery rate</td>

26 microRNAs were expressed at higher levels in tumors with miR21 enriched more than 1.8 times. Global microRNA profiles distinguish between tumor and matched non-tumor tissue with 89% accuracy using closest centroid class prediction algorithms or 3 nearest neighbors (10 5 times cross validation repeated 100 times), suggesting a systematic change in microRNA expression patterns during tumor formation.

The inventors selected miR-20a, miR-21, miR-106a, miR-181b, and miR-203 for validation based on their expression differences between matched tumor and non-tumor tissue combined with their association with poor survival. For validation, the inventors measured the expression levels of these microRNAs with qRT-PCR in paired tumor and non-tumor tissue from an independent cohort. The validation cohort consists of 113 patients recruited from Hong Kong, China with incident colon cancer (Table 1).

MiR-20a (2.3-times), miR-21 (2.8-times), miR106a (2.4-times), miR-18 Ib (1.4-times) and miR-203 (1.8 -times) were all expressed at higher levels in tumors (p &lt;0.001, Wilcoxon paired pair test) (Table 3a).

Table 3 - Expression of microRNA in tumors versus paired non-tumor tissue

Table 3a - The Hong Kong Validation Cohort

<td></td><td></td><td></td><td>Change factor</td><td></td>

<td>MicroRNA</td><td>ΔΔ Ct¹</td><td>DT (ΔΔ Ct)</td><td>tumors²</td><td>p - value³</td>

<td>miR-20a</td><td> 1,18</td><td> 0,97</td><td>2.3 times</td><td>p &lt;0.001</td>

<td>miR-21</td><td> 1,47</td><td> 1,20</td><td>2.8 times</td><td>p &lt;0.001</td>

<td>miR-106a</td><td> 1,25</td><td> 0,94</td><td>2.4 times</td><td>p &lt;0.001</td>

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<td>miR-181 b</td><td> 0,47</td><td> 1,03</td><td>1.4 times</td><td>p &lt;0.001</td>

<td>miR-203</td><td> 0,83</td><td> 1,40</td><td>1.8 times</td><td>p &lt;0.001</td>

Table 3b Expression of microRNA in adenoma vs. matched non-adenoma tissue

<td></td><td>Half</td><td></td><td>Change factor</td><td></td>

<td>MicroRNA</td><td>ΔΔ Ct¹</td><td>SD (ΔΔ Ct)</td><td>adenomas²</td><td>p - value³</td>

<td>miR-20a</td><td> -0,11</td><td> 0,97</td><td>0.9 times</td><td>p = 0.82</td>

<td>miR-21</td><td> 0,64</td><td> 0,90</td><td>1.6 times</td><td>p = 0.006</td>

<td>miR-106a</td><td> 0,28</td><td> 1,22</td><td>1.2 times</td><td>p = 0.19</td>

<td>miR-181 b</td><td> 0,30</td><td> 1,24</td><td>1.2 times</td><td>p = 0.27</td>

<td>miR-203</td><td> 0,77</td><td> 1,98</td><td>1.7 times</td><td>p = 0.14</td>

<td colspan="5">¹Mean (tumor ACt - non-tumor matched ACt) or mean (adenoma ACt - non-adenoma matched ACt) of qRT-PCR. ²Calculated by Wilcoxon 2 Paired Pair Test^ΔΔ. DT = standard deviation. Numbers in bold are statistically significant. For tumor / non-tumor comparisons, 113 tissue pairs were used for miR-20a and miR-203 while 111 tissue pairs were used for miR-21, miR-106a, and miR-181b. For all adenoma / non-adenoma comparisons, 18 tissue pairs were used.</td>

Most tumors (89% for miR-20a, 87% for miR-21, 90% for miR-106a, 71% for miR-181b and 74% for miR-203) had higher expression of these microRNAs than tissue paired non-tumor. The expression patterns for these five microRNAs distinguish tumor versus non-tumor matched state with 96% or 98% accuracy based on 3-nearest neighbors or closest centroid algorithms, respectively (10-fold crossover variation, 100-fold repeated).

The inventors used in situ hybridization to visualize miR-21 expression in tumor and adjacent non-tumor tissue (see Figure 1a-f).

MiR-21 is expressed at high levels both in the nuclei and in the cytoplasm of colonic epithelial cells in human tumor tissue compared to adjacent non-tumor tissue. These results are consistent with qRT-PCR and microarray data and support a role for microRNAs in carcinogenesis.

MiR-21 is overexpressed at higher levels in colon adenomas.

Adenomas represent a precursor stage for colon adenocarcinomas³. The inventors tested the expression levels of miR-20a, miR21, miR-106a, miR-181b and miR-203 by qRT-PCR in 18 pairs of adenoma and adjacent non-adenoma tissue. Although four of the five microRNAs showed increased levels in adenoma tissue, only miR-21 was significantly enriched at 1.6 times more (p = 0.006, Wilcoxon paired pair test) (see Table 3b).

The adenoma tissue expressed higher levels of miR-21 in 15/18 matched pairs. More advanced stages of tumors express higher levels of miR-21. Subjects were stratified based on adenoma diagnosis and TNM stages when adenoma was considered the least advanced and TNM stage IV was the most advanced. Adenomas expressed lower levels of miR-21 expression than tumors in the validation cohort (p &lt;0.001, Mann-Whitney test). Most advanced tumors expressed higher levels of miR-21 expression (trend test, p &lt;0.001) (see Figure 1g).

This trend was also observed using microRNA microarray data from the Maryland test cohort (p = 0.04) (see Figure 2).

High miR-21 expression predicts a negative prognosis in two independent cohorts.

The inventors analyzed tumor / non-tumor (T / N) expression ratios of individual microRNAs to determine if any were associated with negative prognosis. MicroRNA T / N expression ratios were rated high based on the highest tertile. The inventors searched for any microRNAs in which high TIN ratios were associated with cancer survival (p &lt;0.05). From these, the inventors selected microRNAs that were differentially expressed in tumors (p &lt;0.001). Five microRNAs met these criteria. Kaplan-Meier analysis indicated that high T / N ratios for miR-20a (p = 0.02), miR-21

ES 2 425 387 T3 (p = 0.004), miR-106a (p = 0.01), miR-181b (p = 0.04), and miR-203 (p = 0.004) were each associated with poor survival. These five microRNAs were selected for further analysis.

Colon adenocarcinomas of 89-93% of the subjects in this study were of typical histology. A minority of tumors were from mucinous adenocarcinoma, adenosquamous carcinoma, or signet ring 5-cell carcinoma histologies (see Table 1). Different subtypes of adenocarcinomas can be associated with different chemical outcomes, including survival prognosis.³¹. To eliminate the potential confusion associated with histology, the inventors excluded all subjects with mucinous adenocarcinomas, adenosquamous carcinomas, and signet ring cell carcinomas from the initial analysis.

Associations of T / N ratios with poor survival could be due to levels of expression of microRNA 10 in tumor tissue, surrounding non-tumor tissue, or a combination of both. To distinguish these possibilities the inventors analyzed the association of microRNA expression in paired tumors and non-tumors separately. High levels of tumor expression (based on the largest tertile) for miR-20a, miR-21, miR106a, miR-181b, and miR-203 were each associated with poor survival in the Maryland trial cohort (see Figure 3a, also from data not shown). No significant association with expression of 15 microRNAs in non-tumor tissue was observed for any of the five microRNAs.

Univariate and multivariate Cox proportional hazard analysis was used to assess the association of tumor expression levels with prognosis in individuals with typical adenocarcinoma (Table 4a).

Table 4 - Univariate and multivariate Cox regression analysis of miR-21 expression levels and overall cancer survival in subjects with colon adenocarcinoma¹

<td>Table 4a</td><td colspan="4">Maryland Trial Cohort</td>

<td></td><td colspan="2">Univariate analysis</td><td colspan="2">Multivariate analysis²</td>

<td>Characteristic</td><td>HR (95% CI)</td><td>p-value</td><td>HR (95% CI)</td><td>p-value</td>

<td>Expression of miR- twenty-one³ N = 71</td><td></td><td></td><td></td><td></td>

<td>Under</td><td> 1,0</td><td></td><td> 1,0</td><td></td>

<td>High</td><td> 2,5 (1,2-5,2)</td><td> 0,01</td><td> 2,9 (1,4-6,1) 0,004</td><td></td>

<td>Stage I-II TNM</td><td> 1,0</td><td></td><td> 1,0</td><td></td>

<td>III-IV</td><td> 3,5 (1,6-7,9)</td><td> 0,002</td><td> 3,4 (1,5-7,8)</td><td> 0,004</td>

<td>Age at admission &lt;50</td><td> 1,0</td><td></td><td></td><td></td>

<td> &gt;50</td><td> 0,7 (0,2-2,3)</td><td> 0,52</td><td></td><td></td>

<td>Female gender</td><td> 1,0</td><td></td><td></td><td></td>

<td>Male</td><td> 1,4 (0,5-3,9)</td><td> 0,57</td><td></td><td></td>

<td>White race</td><td> 1,0</td><td></td><td></td><td></td>

<td>Black</td><td> 1,0 (0,5-2,1)</td><td> 0,97</td><td></td><td></td>

<td>Distal tumor location</td><td> 1,0</td><td></td><td></td><td></td>

<td>Proximal</td><td> 0,6 (0,3-1,4)</td><td> 0,26</td><td></td><td></td>

<td>Table 4b</td><td colspan="4">Hong Kong Validation Cohort</td>

<td></td><td colspan="2">Univariate analysis</td><td colspan="2">Multivariate analysis²</td>

<td>Characteristic</td><td>HR (95% CI)</td><td>p-value</td><td>HR (95% CI)</td><td>p-value</td>

<td>MiR21 expression³ n = 103</td><td></td><td></td><td></td><td></td>

<td>Short</td><td> 1,0</td><td></td><td> 1,0</td><td></td>

<td>high</td><td> 2,4 (1,4-3,9)</td><td> 0,002</td><td> 2,4 (1,4-4,1)</td><td> 0,002</td>

<td>Stage I-II TNM</td><td> 1,0</td><td></td><td> 1,0</td><td></td>

<td>III-IV</td><td> 4,7 (2,4-9,5)</td><td> &lt;0,001</td><td> 4,7 (2,4-9,5)</td><td> &lt;0,001</td>

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<td>Age at admission &lt;50</td><td> 1,0</td><td></td><td></td><td></td>

<td> &gt;50</td><td> 1,5(0,9-2,6)</td><td> 0,14</td><td></td><td></td>

<td>Female gender</td><td> 1,0</td><td></td><td></td><td></td>

<td>Male</td><td> 1,4 (0,8-2,3)</td><td> 0,29</td><td></td><td></td>

<td>Distal tumor location</td><td> 1,0</td><td></td><td></td><td></td>

<td>Proximal</td><td> 0,7 (0,3-1,4)</td><td> 0,27</td><td></td><td></td>

<td colspan="5">MicroRNA expression was measured with miRNA microarrays for the Maryland cohort and with qRT-PCR for the Hong Kong cohort. ¹Cases with mucinous adenocarcinoma, adenosquamous carcinoma, or signet ring cell carcinoma were excluded from this analysis. ⁷Multivariate analysis used stepwise addition and the withdrawal of clinical co-variants was found to be associated with survival in univariate models (p &lt;0.10) and final models include only co-variants that were significantly associated with survival ( Wald statistic p &lt;0.05). ³High expression in tumors for all miRNAs was defined based on the highest tertile.</td>

Individuals with tumors expressing high levels of mir-21 had a significantly higher risk of dying from colon cancer in both univariate (HR = 2.5 [1.2-5.2], p = 0.01) and multivariate ( HR = 2.9 [1.46.1], p = 0.004).

To validate these findings, the inventors used qRT-PCR to measure tumor and non-tumor expression levels for these five microRNAs in the Hong Kong validation cohort and analyzed the associations with prognosis. High tumor expression of miR-21 predicts a negative prognosis in the Hong Kong validation cohort (p = 0.001, Kaplan-Meier log-rank test) while expression in non-tumor tissue does not (see Figure 3b).

The inventors found no statistically significant associations with prognosis and expression of miR20a, miR-106a, 181b, or miR-203 in this cohort.

High miR-21 expression in tumors was not significantly associated with age, gender, tumor histology, or tumor location (Fisher's exact test) in the Hong Kong validation cohort. All co-variants were examined by Cox proportional hazard analysis (Table 4b).

The high expression of miR-21 in tumors (HR = 2.4 [1.4-3.9], p = 0.002) and TNM stages (HR = 4.7 [2.4-9.5], p &lt;0.001) was significantly associated with survival in univariate models. Multivariate Cox regression analysis demonstrated that high expression of miR-21 in tumors predicts poor survival prognosis (HR = 2.4 [1.4-4.1], p = 0.002), independently of other co-variants This is consistent with the inventors' findings in the Maryland trial cohort.

The inventors repeated the analysis including all subjects regardless of tumor histology. In both cohorts, the association with high expression of miR-21 and prognosis was maintained (see Figure 4, see Table 5).

Table 5 - Univariate and multivariate Cox regression analysis of miR-21 expression levels and overall cancer survival in subjects with all subjects

<td>Table 5a</td><td colspan="4">Maryland Trial Cohort</td>

<td></td><td colspan="2">Univariate analysis</td><td colspan="2">Multivariate analysis²</td>

<td>Characteristic</td><td>HR (95% CI)</td><td>p-value</td><td>HR (95% CI)</td><td>p-value</td>

<td>Expression of miR- twenty-one³ N = 79</td><td></td><td></td><td></td><td></td>

<td>Under</td><td> 1,0</td><td></td><td> 1,0</td><td></td>

<td>High</td><td> 2,0 (1,1-4,0)</td><td> 0,04</td><td> 2,1 (1,1-4,0)</td><td> 0,03</td>

<td>Stage I-II TNM</td><td> 1,0</td><td></td><td> 1,0</td><td></td>

<td>III-IV</td><td> 3,2 (1,5-6,9)</td><td> 0,002</td><td> 3,2 (1,5-6,8)</td><td> 0,003</td>

<td>Age at the time</td><td> 1,0</td><td></td><td></td><td></td>

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<td>admission &lt;50</td><td></td><td></td><td></td><td></td>

<td> &gt;50</td><td> 0,7 (0,2-2,4)</td><td> 0,59</td><td></td><td></td>

<td>Female gender</td><td> 1,0</td><td></td><td></td><td></td>

<td>Male</td><td> 1,6 (0,7-4,2)</td><td> 0,33</td><td></td><td></td>

<td>White race</td><td> 1,0</td><td></td><td></td><td></td>

<td>Black</td><td> 1,0 (0,5-2,0)</td><td> 0,99</td><td></td><td></td>

<td>Distal tumor location</td><td> 1,0</td><td></td><td></td><td></td>

<td>Proximal</td><td> 0,8 (0,3-2,1)</td><td> 0,65</td><td></td><td></td>

<td>Histology of Adenocarcinoma</td><td> 1,0</td><td></td><td></td><td></td>

<td>Mucinous or Adenosquamous</td><td> 0,7 (0,3-2,1)</td><td> 0,57</td><td></td><td></td>

<td>Table 5b</td><td colspan="4">Hong Kong Validation Cohort</td>

<td></td><td colspan="2">Univariate analysis</td><td colspan="2">Multivariate analysis²</td>

<td>Characteristic</td><td>HR (95% CI)</td><td>p-value</td><td>HR (95% CI)</td><td>p-value</td>

<td>MiR21 expression³ n = 111</td><td></td><td></td><td></td><td></td>

<td>Short</td><td> 1,0</td><td></td><td> 1,0</td><td></td>

<td>high</td><td> 2,3 (1,4-3,9) 23 (1,4- 3,9)</td><td> 0,002</td><td> 23 (1,4-3,9)</td><td> 0,002</td>

<td>Stage I-II TNM</td><td> 1,0</td><td></td><td> 1,0</td><td></td>

<td>III-IV</td><td> 4,9 (2,5-97)</td><td> &lt;0,001</td><td> 4,9 (2,5-98)</td><td> &lt;0,001</td>

<td>Age at admission &lt;50</td><td> 1,0</td><td></td><td></td><td></td>

<td> &gt;50</td><td> 1,4 (0,8-2,4)</td><td> 0,20</td><td></td><td></td>

<td>Female gender</td><td> 1,0</td><td></td><td></td><td></td>

<td>Male</td><td> 1,3 (0,8-2,3)</td><td> 0,27</td><td></td><td></td>

<td>Distal tumor location</td><td> 1,0</td><td></td><td></td><td></td>

<td>Proximal</td><td> 0,7 (0,3-1,4)</td><td> 0,27</td><td></td><td></td>

<td>Histology of adenocarcinoma</td><td> 1,0</td><td></td><td></td><td></td>

<td>Mucinous or adenosquamous</td><td> 1,2 (0,4-3,3)</td><td> 0,74</td><td></td><td></td>

<td colspan="5">MicroRNA expression was measured with miRNA microarrays for the Maryland cohort and with qRT-PCR for the Hong Kong cohort. ¹All individuals were included in this analysis regardless of tumor histology. The multivariate analysis used stepwise addition and it was found that the withdrawal of clinical covariates was associated with survival in univariate models (p &lt;0.10) and the final models included only the covariates that were significantly associated with survival (Wald statistic p &lt;0.05). The tall expression³ in tumors for all miRNAs it was defined based on the highest tertile.</td>

MiR-21 expression levels and response to therapy.

Identifying biomarkers associated with a response to adjuvant chemotherapy will allow clinicians to better predict the benefits of therapy. To this end, the inventors analyzed associations with expression of 5 miR-21 and the response to adjuvant chemotherapy in patients with stage II and III cancer. Information on the administration of adjuvant chemotherapy was available for 47 of 65 stage II or III subjects in the cohort.

ES 2 425 387 T3 from Maryland trial and all subjects in the Hong Kong validation cohort.

In both cohorts, chemotherapy regimens were primarily fluorouracil-based (in forms of intravenous 5-fluorouracil or oral drugs including uracil tegafur [UFT]) with or without Levamisole or Leocovorin. Only subjects with typical adenocarcinoma histology were used for this analysis, leaving 20 of the 42 stage II / III individuals who received chemotherapy in the Maryland cohort. For those who received chemotherapy, high miR-21 expression in tumors predicted worse overall survival (p = 0.01, Kaplan-Meier log-rank test) providing preliminary support that high miR-21 is associated with poor response to adjuvant chemotherapy.

For the Hong Kong validation cohort, 77 individuals with stage II / III cancer with typical adenocarcinoma histology were used for this analysis. Stage II / III subjects who received adjuvant chemotherapy had a better prognosis for survival than those who did not (p = 0.02, Kaplan-Meier log-rank test). Among subjects receiving adjuvant chemotherapy (n = 36), high expression of miR-21 in tumors was associated with poor response to treatment (p = 0.03, Kaplan-Meier log-rank test), consistent with observations in the Maryland cohort (see Figure 5a).

In this cohort, all stage II subjects who received adjuvant chemotherapy (n = 11) survived (see Figure 5b), but for stage III subjects who received adjuvant chemotherapy (n = 25) the high expression of miR-21 was associated with poor survival (p = 0.02, Kaplan-Meier log-rank test) (see Figure 5c).

Multivariate Cox regression analysis was used to analyze these observations to show that high miR-21 expression predicted a negative prognosis (HR = 3.1 [1.5-6.1]; P = 0.001) and receiving chemotherapy predicted improved survival outcome (HR = 0.3 [0.1-0.5]; p &lt;0.001) regardless of other clinical co-variants (Table 6a).

Table 6 - Univariate and Multivariate Cox Regression Analysis of miR-21 Expression, Adjuvant Chemotherapy Receipt, and Cancer Survival in Stage I / III Subjects¹ with adenocarcinoma

<td>Table 6a</td><td colspan="4">Maryland Trial Cohort</td>

<td></td><td colspan="2">Univariate analysis</td><td colspan="2">Multivariate analysis²</td>

<td>Characteristic</td><td>HR (95% CI)</td><td>p-value</td><td>HR (95% CI)</td><td>p-value</td>

<td>MiR21 expression³ N = 77</td><td></td><td></td><td></td><td></td>

<td>Under</td><td> 1,0</td><td></td><td> 1,0</td><td></td>

<td>High</td><td> 2,6 (1,3-5,1)</td><td> 0,005</td><td> 3,1 (1,5-6,1)</td><td> 0,001</td>

<td>Did not receive adjuvant chemotherapy</td><td> 1,0</td><td></td><td> 1,0</td><td></td>

<td>He received</td><td> 04, (0,2-0,8)</td><td> 0,01</td><td> 0,3 (0,1-0,5)</td><td> &lt;0,001</td>

<td>Stage II TNM</td><td> 1,0</td><td></td><td> 1,0</td><td></td>

<td>III</td><td> 2,8 (1,3-6,0)</td><td> 0,008</td><td> 5,4 (2,4-12)</td><td> &lt;0,001</td>

<td>Distal tumor location</td><td> 1,0</td><td></td><td> 1,0</td><td></td>

<td>Proximal</td><td> 0,3 (0,1-10)</td><td> 0,04</td><td> 0,2 (0,1-0,8)</td><td> 0,02</td>

<td>Age at admission &lt;50</td><td> 1,0</td><td></td><td></td><td></td>

<td> &gt;50</td><td> 1,6 (0,8,3,1)</td><td> 0,20</td><td></td><td></td>

<td>Female gender</td><td> 1,0</td><td></td><td></td><td></td>

<td>Male</td><td> 1,2 (0,6-2,3)</td><td> 0,61</td><td></td><td></td>

<td>Table 6b</td><td colspan="4">Hong Kong Validation Cohort</td>

<td></td><td colspan="2">Univariate analysis</td><td colspan="2">Multivariate analysis²</td>

<td>Characteristic</td><td>HR (95% CI)</td><td>p-value</td><td>HR (95% CI)</td><td>p-value</td>

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<td>MiR21 expression³ N = 119</td><td></td><td></td><td></td><td></td>

<td>Short</td><td> 1,0</td><td></td><td> 1,0</td><td></td>

<td>high</td><td> 2,6 (1,5-4,5)</td><td> 0,001</td><td> 3,0 (1,7-5,4)</td><td> &lt;0,001</td>

<td>Did not receive adjuvant chemotherapy</td><td> 1,0</td><td></td><td> 1,0</td><td></td>

<td>He received</td><td> 07, (0,4-1,2)</td><td> 0,21</td><td> 0,4 (0,2-0,8)</td><td> 0,004</td>

<td>Stage II TNM</td><td> 1,0</td><td></td><td> 1,0</td><td></td>

<td>III</td><td> 3,2 (1,7-6,1)</td><td> 0,001</td><td> 5,2 (2,6-11)</td><td> &lt;0,001</td>

<td>Distal tumor location</td><td> 1,0</td><td></td><td> 1,0</td><td></td>

<td>Proximal</td><td> 0,4 (0,2-0,8)</td><td> 0,02</td><td> 0,3 (0,1-0,7)</td><td> 0,007</td>

<td>Age at admission &lt;50</td><td> 1,0</td><td></td><td></td><td></td>

<td> &gt;50</td><td> 1,4 (0,7-2,5)</td><td> 0,32</td><td></td><td></td>

<td>Female gender</td><td> 1,0</td><td></td><td></td><td></td>

<td>Male</td><td> 1,3 (0,7-2,2)</td><td> 0,44</td><td></td><td></td>

<td colspan="5">The expression of miRNA was measured with a qRT-PCR. '' TNM stage II / III subjects with typical adenocarcinoma histology were included in this analysis.²The multivariate analysis used stepwise addition and removal of clinical covariates that were found to be associated with survival in univariate models (p &lt;0.10) and the final models included only the covariates that were significantly associated with survival (Wald statistic p &lt;0.05). ³High expression in tumors for all miRNAs was defined based on the highest profile. Race was not associated with a negative prognosis.</td>

Analyzes using cancer relapse as an endpoint rather than cancer death resulted in similar associations with high miR-21 expression in tumors predicting faster disease recurrence (data not shown).

An analysis combining both cohorts resulted in similar associations. Kaplan-Meier analysis demonstrated that high miR-21 expression predicted a negative prognosis in stage II (p = 0.02) or stage III (p = 0.004) subjects (see Figure 6).

High miR-21 expression predicted poor response to chemotherapy in stage II / III subjects (p = 0.003) or in stage III subjects only (p = 0.007). Multivariate Cox regression demonstrated that high miR21 expression predicted negative prognosis (HR = 3.0 [1.7-5.4]; P &lt;0.001) and adjuvant chemotherapy treatment predicted improved survival (HR = 0.4 [0.2-0.8]; p = 0.004) regardless of other clinical qualities (Table 6b).

Analysis

The inventors analyzed microRNA profiles in colon cancer tissues using two independent cohorts. Thirty-seven microRNAs were differentially expressed in tumor tissues by microRNA microarray analysis. The expression patterns of the five microRNAs tested in the Hong Kong cohort were validated. The differentiating power of five microRNAs to differentiate between tumor and non-tumor tissue indicates that predictable and systematic changes in microRNA expression patterns occur during tumorigenesis and are probably representative of most sporadic colon adenocarcinomas.

All miR-20a and miR-21, miR-106a, miR-181b and miR-203 were found to be expressed at higher levels in colon tumors. These changes in microRNA expression patterns may simply be associated with colon cancer or cause histological progression to cancer. There is strong evidence to suggest that changes in microRNA expression patterns promote tumor formation, especially for miR-20a and miR-21. MiR-20a is part of the miR-17-92 polycistronic microRNA cluster³².

Overexpression of this group enhances cell proliferation in vitro³³ and accelerates tumor formation in animal models¹⁶. Obligate expression of the miR-17-92 group causes increased tumor size and tumor vascularization in mice through down-regulation of the antiangiogenic TspI protein²⁴. Experimental evidence also suggests that increased expression of miR-21 promotes tumor development. MiR-21

ES 2 425 387 T3 is expressed at high levels in most solid tumors^19,34. MiR-21 overexpression acts as an anti-apoptotic factor in human glioblastoma cells¹³. Inhibition of miR-21 inhibits cell growth in vitro and inhibits tumor growth in xenograft mouse models through indirect down-regulation of anti-apoptotic factor Bc1-2³⁵. Studies in human cell lines have shown that miR-21 can also target tumor suppressor genes PTEN³⁶ and TPM1³⁷. All of these data taken together support a causative role for altered microRNA expression during tumorigenesis.

Adenomas represent a precursor stage of adenocarcinoma. Adenomas express high levels of miR-

twenty-one. If increased expression of miR-21 promotes colon tumor progression, increased expression in adenomas may be an early cellular event in progression to cancer. Inhibition of miR-21 activity may help prevent tumor promotion in populations at high risk for colon cancer, such as individuals with familial adenomatous polyposis^38.

Therefore, evidence demonstrating an association with microRNA expression patterns with colon cancer prognosis and response to adjuvant chemotherapy is presented herein. More advanced tumors express higher levels of miR-21. A robust association of high miR-21 expression in tumors and poor survival was observed in the Maryland trial cohort and the Hong Kong validation cohort, separately.

In each cohort, these associations were independent of all other clinical covariates, indicating that miR-21 expression may be a useful prognostic indicator, in addition to TNM staging and other clinical parameters, to help identify patients. with an increased risk of terminal cancer. These observations were made in two independent cohorts with very different racial and geographic compositions. Therefore, the inventors' observations are likely to be generally applicable to other populations.

High miR-21 expression in tumors was associated with poor response to adjuvant chemotherapy in both cohorts. These results may help predict the benefits of therapy in individuals whose miR-21 expression status is known. Furthermore, if the high expression of miR-21 is the cause of the poor survival of patients with colon cancer, the antagomir^29,39 or other antisense therapeutics that target miR-21 may have therapeutic benefits in subjects with tumors that express high miR-21. These can also be used for current therapies to improve survival outcomes.

The inventors herein have discovered systematic differences in microRNA expression patterns between colon tumors and paired non-tumor tissue. The high expression of miR-21 in tumors predicts the outcome of poor survival and poor response to adjuvant chemotherapy in two independent cohorts, regardless of stage and other clinical covariates, suggesting that it may be a useful diagnostic biomarker for colon adenocarcinomas and prognosis of survival including response to therapy.

Procedures

Tissue collection and RNA isolation:

Pairs of primary colon tumor and adjacent non-tumor tissues came from 84 patients recruited from the University of Maryland Medical Center between 1993 and 2002, and from 113 patients recruited from Queen Mary Hospital, Hong Kong between 1991 and 2000. It has been collected Detailed information for each tissue donor, including age, sex, clinical stage, tumor location, survival times from diagnosis, and receipt of adjuvant chemotherapy. Tumor histopathology was classified according to the World Health Organization tumor classification system¹. The adenoma tissue was obtained from the Cooperative Human Tissue Network. This study was approved by the Institutional Evaluation Board of the National Institute of Health, the Institutional Evaluation Board of the University of Hong Kong / Hong Kong Hospital Authority Western group, and the Institutional Evaluation Board for human subjects research in the University of Maryland.

RNA isolation and microRNA profiling:

RNA was extracted from tissue using standard TRIZOL procedures (Invitrogen, Carlsbad). MicroRNA microarray profiles were performed as previously described³⁰. Briefly, 5 lag of total RNA was labeled and hybridized with each microRNA microarray containing quadruplicates of approximately 400 human microRNA probes. The slides were scanned using a PerkinElmer ScanArray LX5K scanner. MicroRNA qRT-PCR was performed using Taqman microRNA assays (Applied Biosystems, Foster City) according to the manufacturer's instructions with the 7500 real-time RT-PCR system (Applied Biosystems, Foster City). U6B was the normalization control for all qRT-PCR experiments. All tests were performed in duplicate (miR-20a and miR-203) or in triplicate (miR-21, miR-106a, miR-181b), qRT-PCR for miR-21, miR-106a and miR-181b was performed by AJS, blinded to survival outcomes and clinical data for members of the validation cohort at that time.

Microarray Analysis:

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The data analyzed in this publication have been deposited with the NCBI Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through the GEO serial reference number GSE7828 .

LOESS normalized microarray data was imported into BRB 3.5.0 matrix tools (http://linus.nci.nih.gov/.BRB-ArrayTools.html) and all subsequent microarray analyzes were performed with this software.

Microarray analyzes were performed. Probes with missing values ​​were removed from&gt; 20% of the assay arrays leaving 230 probes. Paired class comparison analysis identified microRNAs that were differentially expressed in tumors (p &lt;0.001).

To initially search for microRNAs associated with poor survival, tumor / non-tumor (T / N) microRNA expression ratios were analyzed in the Maryland cohort using microarray data. TN to microRNA expression ratios were created by subtracting non-tumor log2 expression values ​​from tumor log2. MicroRNAs lacking&gt; 25% T / N ratios were removed by filtering leaving 208. The T / N expression ratios were dichotomized with the major tertile classified as high and the 2 minor tertiles classified as low (see Complementary Procedures). This high / low cut-off point was used universally throughout the present study. Tumor and non-tumor microRNA expression levels were batch normalized based on the date of the microarray experiments for all survival association analyzes.

In situ hybridization:

In situ hybridization (ISH) was performed with probes for human miR-21, mixed and U6 (Exiqon, Woburn) with a modified version of the manufacturer's protocol for formalin-fixed paraffin embedded tissue (FFPE) written by W. Kloosterman (http : //www.exiqon.com/uploads/.LNA 52-FFPE miRNA in situj.rotocol.pdf) in human colon tissue. Modifications included the use of rabbit polyclonal anti-DIG HRP-conjugated antibody and DakoCytomation GenPoint tyramide signal amplification system (DakoCytomation, Carpintería), and VECTOR® NovaRed ™ substrate (Vector Laboratories, Burlingame). Images were taken with an Olympus BX40 microscope using the Olympus DP70 digital camera and DP driver software (Olympus, Champaign).

Statistic analysis:

Statistical analyzes were carried out. Wilcoxon matched pair assays were used to analyze differences in microRNA expression between tumors and matched non-tumor tissue as well as differences between adenoma and matched non-adenoma tissue for all qRT-PCR data. All trend tests presented are non-parametric tests with respect to trend between ordered groups. All Kaplan-Meier analysis was performed with WINSTAT 2001 (R. Fitch Software). Multivariate Cox regression analysis was performed using Intercooled Stata 9.2 (StataCorp LP, College Station). The final multivariate models were based on the stepwise addition and removal of clinical covariates that had been found to be associated with poor survival in univariate models (p &lt;0.10). A Wald statistic of p &lt;0.05 was used as a criterion for inclusion in final multivariate models. All p values ​​presented are 2-sided. Hazard relationships are presented with 95% confidence intervals in parentheses. Expression graphs were made using GraphPad Prism 4.0 (GraphPad Software Inc., San Diego).

Additional microarray analysis

The microarrays used for this analysis were spot application microRNA microarrays (from The Ohio State University Comprehensive Cancer Center, version 2.0). The intensities of each point were the median of the intensities of the foreground. Each of the 170 microarrays used for this study contained 11,520 dots. All points where foreground intensity was less than background intensity were reassigned as NA (NA marks missing data points). All points marked as deficient by the scout were also reassigned as NA. All white dots (no oligo) with high foreground intensity were reassigned as NA. Each microRNA oligo is represented by quadruplicate dots on these arrays as two distant pairs of two adjacent dots. If there was 0 or 1 NA for a quadruple oligo, and the mean of the distant oligo pairs differed by&gt; 1 on the log2 scale, all quadrupled points were reassigned as NA. If there were 2 NAs for a quadruple oligo and the two non-NA point intensities differed by&gt; 1 on the log2 scale, all quadrupled points were reassigned NA. If there were 3 NA points for a quad, the end point was reassigned as NA. In total, 1,082,689 out of 1,958,400 points were reassigned as NA using these procedures. Normalization was performed by LOESS (locally weighted scatter plot smoothing) using the R software package. All data was then imported into BRB Matrix Tools version 3.5.0 for analysis and all repeated points were averaged. Eighty-five pairs (tumor and matched non-tumor tissue) of arrays were originally used. It was later discovered that a case that was originally identified as an incident colon carcinoma patient had been diagnosed as carcinoma in situ and was therefore withdrawn from the analysis, leaving the study population at 84 subjects. The microRNA lists were filtered to include only the 389 human hsa-miR probe sets. They were further filtered to remove any missing probe sets from more than 25% of the arrays, leaving 230 human microRNA probe sets. Paired class comparison analysis was used to identify microRNAs that were differentially expressed between tumor and

ES 2 425 387 T3 paired non-tumor tissue. For two microRNAs (miR-181b and miR-338), two independent probes measuring each gave conflicting results showing a probe higher expression in tumors and a probe showing lower expression in tumors for each microRNA. For each, the inventors discarded the least significant result designating both miR-181b and miR-388 as enriched in tumors.

Additionally, qRT-PCR confirmed that miR-181b was enriched in tumors.

The inventors initially used tumor / non-tumor (T / N) expression profiles for each microRNA to search for microRNAs that were associated with poor survival. For this analysis, the inventors decided to dichotomize all expression data with a universal high and low cut-off point to look for associations with poor survival. To determine which universal high / low cutoff to use, the inventors dichotomized the T / N expression data in three separate ways and determined which procedure provided the most significant number of results in the test cohort. High expression was ranked based on greater than median, major tertile, or highest quartile and the inventors tested associations with these cutoff points with poor survival using univariate Cox regression analysis. Of the 37 microRNAs that were differentially expressed in tumors, four high expression was associated with poor survival based on greater than median, five based on the largest tertile, and two based on the largest quartile (p &lt;0.05, data not shown). Dichotomization based on the largest tertile provided the highest amount of microRNAs associated with poor survival based on these criteria in the Maryland trial cohort; therefore, the classification based on the major tertile was used uniformly throughout the present study to analyze the associations between microRNA expression levels and a negative prognosis in both the Maryland trial cohort and the validation cohort of Hong Kong.

The inventors used microRNA microarrays to compare miR 21 expression levels in prognostic tumors. The microarray probe used for this analysis was hsa-miR-21-precl7Nol. This analysis required batch normalization of the data based on the date of the microarray experiment. To normalize by date, the arrays expressing the largest 1/3 of a given microRNA were ranked high for each day, separately. Up to twelve tissue pairs were profiled on any given day. For any day in which fewer than 10 pairs of microarrays were made, the matrices made on those days were discarded, resulting in the loss of 5 pairs of matrices. These data are then combined with each other for analysis of associations with survival outcomes. The inventors checked and found no significant differences in the frequency distribution of age, sex, race, tumor location, TNM stage, or cancer survival between categorized groups based on the date of the microarray experiment (Fisher's exact test).

Statistical analysis

Cox proportional hazard regression was used to analyze the effect of miR-21 expression levels and another clinical variable on patient survival. The clinical variables included were: age, sex, race, tumor location, tumor histology, receipt of adjuvant therapy, and TNM stage. For these models, the inventors chose to dichotomize age as age&gt; 50 versus age &lt;50 since the recommended screening age for colon cancer is 50 years; tumor location was defined as proximal if the tumor was located within or close to the splenic flexure and distant if the tumor was located within or distant from the descending colon; TNM stage was dichotomized based on metastatic versus non-metastatic disease resulting in stage I-II versus III-IV. One patient in the Maryland cohort died on the day of surgery resulting in a survival time of 0 months. This case was included in the Kaplan-Meier analysis and was withdrawn for the Cox regression analysis causing the difference in cases between the expression of miR-21 in tumors for Figure 2 (n = 72) and the number of cases in Cox regression analysis in Table 4 (n = 71). Univariate Cox regression was performed on each clinical covariate to examine the influence of each on patient survival. The final multivariate models were based on the stepwise addition and removal of clinical covariates that had been found to be associated with poor survival in univariate models (p &lt;0.10). A Wald statistic of p &lt;0.05 was used as a criterion for inclusion in final multivariate models. The most parsimonious Cox regression model was used for the final multivariate model.

Example 2 - Initial results

MiRNAs are differentially expressed in colon tumors

The inventors analyzed miRNA profiles of 85 pairs of adjacent cancerous and non-cancerous colon tissues using miRNA microarrays. The inventors found that the miRNA expression profiles of tumors were quite different from normal tissues suggesting that miRNAs may play significant roles in colon carcinogenesis. Paired class comparison analysis identified 27 independent miRNAs that were differentially expressed in these tumors (Table 7).

Table 7 - 27 miRNAs are differentially expressed in colon tumors compared to matched normal tissue. 27 miRNAs were found to be differentially expressed in tumors using paired class comparison analysis on BRB 3.4 matrix tools. A significance value of p &lt;0.001 was used as the criterion for differential expression resulting in a false discovery rate.

ES 2 425 387 T3 estimated at 0.08%. Positive refers to miRNAs that were expressed at higher levels in tumors while negative indicates that miRNA levels were lower in tumors.

<td>Table 7</td><td>MicroRNA</td><td>Positively / negatively regulated</td><td>P-value</td>

<td> 1</td><td>miR-331</td><td>Negative</td><td>1.00 E-07</td>

<td> 2</td><td>miR-21</td><td>Positive</td><td>1.00 E-07</td>

<td> 3</td><td>miR-34b</td><td>Negative</td><td>2.00e-07</td>

<td> 4</td><td>miR-342</td><td>Negative</td><td>2.00e-07</td>

<td> 5</td><td>miR-215</td><td>Negative</td><td>2.20E-05</td>

<td> 6</td><td>miR-371</td><td>Negative</td><td>7.00E-07</td>

<td> 7</td><td>miR-373</td><td>Negative</td><td>6.30E-06</td>

<td> 8</td><td>miR-192</td><td>Negative</td><td>7.70E-06</td>

<td> 9</td><td>miR-148b</td><td>Negative</td><td>1.03E-05</td>

<td> 10</td><td>miR-138</td><td>Negative</td><td>1.49 E-05</td>

<td> 11</td><td>miR-301</td><td>Negative</td><td>1.85E-05</td>

<td> 12</td><td>miR-338</td><td>Negative</td><td>2.63E-05</td>

<td> 13</td><td>miR-153</td><td>Negative</td><td>2.67E-05</td>

<td> 14</td><td>miR-129</td><td>Negative</td><td>3.20E-05</td>

<td> 15</td><td>miR-222</td><td>Positive</td><td>9.08E-05</td>

<td> 16</td><td>miR-346</td><td>Positive</td><td> 0,000126</td>

<td> 17</td><td>miR-204</td><td>Positive</td><td> 0,000244</td>

<td> 18</td><td>miR. 181 b</td><td>Positive</td><td> 0,000263</td>

<td> 19</td><td>let-7a-2</td><td>Negative</td><td> 0,000272</td>

<td> 20</td><td>miR-106a</td><td>Positive</td><td> 0,000305</td>

<td> 21</td><td>miR-093</td><td>Positive</td><td> 0,000334</td>

<td> 22</td><td>miR-34c</td><td>Negative</td><td> 0,000341</td>

<td> 23</td><td>miR-219</td><td>Positive</td><td> 0,000352</td>

<td> 24</td><td>miR-019b</td><td>Positive</td><td> 0,000364</td>

<td> 25</td><td>miR-210</td><td>Positive</td><td> 0,000389</td>

<td> 26</td><td>miR-185</td><td>Positive</td><td> 0,000516</td>

<td> 27</td><td>miR-1</td><td>Negative</td><td> 0,00064</td>

The false discovery rate, to cover the multiple comparison trials, was approximately 0.8% indicating that most if not all of these miRNAs are differentially expressed and are not the result of multiple comparison trials. Eleven miRNAs were found to have elevated expression levels in tumors while 16 miRNAs were found to be depleted in tumors. Additionally, miRNA profiles could be used to predict whether or not the tissue was tumorous or non-tumorous with 92% accuracy. Based on

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2000 random permutations, the probability that these predictions occurred by chance was extremely low (p &lt;0.0005). These results show that there are systematic differences in miRNA expression profiles between tumors and normal tissue, indicating that miRNA expression profiles have been altered during colon carcinogenesis.

Global miRNA Expression Profiles Predict Colon Cancer Survival Prognosis

The inventors determined whether miRNA expression profiles predict patient survival. For this analysis the inventors calculated tumor versus normal miRNA expression ratios (TIN ratio) for each miRNA for each individual. Hierarchical unsupervised clustering of all miRNA TIN relationships groups individuals into two arbitrarily labeled groups group A and group B (Figure 7).

These two groups differ significantly both in clinical stage (p = 0.009; Figure 1b) and in survival prognosis (p = 0.026; Figure 7c).

This indicated that miRNA profiles were predictive of clinical stage and, more importantly, prognosis of survival.

Univariate and multivariate Cox regression analysis was used to investigate this relationship in more detail (Table 8).

Table 8 Cox regression analysis of global miRNA profiles

Univariate (above) and multivariate (later) Cox regression analyzes were performed to show that individuals classified in miRNA group B had an increased risk of dying from colon cancer. Neither age, gender nor race contributed significantly to the risk of survival. For the purposes of these analyzes, age was dichotomized into plus or minus 50 and race was dichotomized into African American (AA) and Caucasian.

<td colspan="3">Univariate analysis</td>

<td>Variable</td><td>HR (95% CI)</td><td>p value</td>

<td>group B / A</td><td> 2,6 (1,0-6,3)</td><td> 0,042</td>

<td>age 2 50 / age &lt;50</td><td> 0,62 (0,14-2,7)</td><td> 0,53</td>

<td>male / female</td><td> 1,4 (0,48-4,0)</td><td> 0,54</td>

<td>AA / Caucasian</td><td> 1,1 (0,83-2,3)</td><td> 0,83</td>

<td colspan="3">Multivariate, with adjustment for age, sex, and race</td>

<td>group B / A</td><td>2.7 (01.01 to 06.08)</td><td> 0,034</td>

<td>age 2 50 / age &lt;50</td><td> 0,49 (0,11-2,2)</td><td> 0,35</td>

<td>male / female</td><td> 1,5 (0,52-4,4)</td><td> 0,45</td>

<td>AA / Caucasian</td><td> 1,0 (0,45-2,2)</td><td> 0,99</td>

Group B individuals were to have a significantly higher risk of dying from colon cancer (hazard ratio [HR] = 2.6 (p = 0.04)). This risk remained significantly high after adjusting for age, ethnicity, and sex (HR = 2.7, p = 0.03). These results demonstrate the potential to use miRNA profiles of colon tumors to predict prognosis. These results suggest that miRNAs may also play a role in colon carcinogenesis.

The profiles of miR-21, miR-106a, miR-181b, miR-16h, miR-203, let-7tg, miR-29a, miR-103-2 and miR-10a predict prognosis of colon cancer

The inventors identified individual miRNAs whose expression levels were predictive of colon cancer prognosis. The inventors used Kaplan Meier survival plot and multivariate Cox regression analysis on TIN relationships to identify miRNA expression patterns that were associated with poor survival prognosis. BRB matrix tools were used to identify TIN relationships correlated with poor survival (data not shown). The inventors chose to analyze these miRNAs in more detail. The inventors also analyzed any miRNA that was differentially expressed in tumors (p &lt;0.01). The TIN relationships for each individual were dichotomized based on the median or highest quartile of

ES 2 425 387 T3 TIN relationships. The inventors also removed any miRNAs from the analysis in which TIN relationships were absent in more than 18 individuals. The inventors identified at least 9 miRNAs, including miR-21, miR-106a, miR-181b, miR-16h, miR-203, let-7g, miR-29a, miR-103-2, and miR-10a whose TIN ratios were predictors of colon cancer prognosis (Figure 8, Table 9).

Cox regression analysis of TIN ratios for individual miRNAs.

Univariate and multivariate Cox regression analyzes were performed to show that individual miRNA TIN ratios could be used to rank individuals at increased risk of dying from colon cancer. The TIN relationships for these 9 miRNAs were significant predictors of survival prognosis regardless of TNM stage, age, sex, and race. Note that the high / low distinctions for miR-16b, 10 miR-21, miR-29a, miR-103-2, miR-106a and miR-203 were classified based on the median of the TIN ratio values ​​while Iet- 7g, miR-10a and miR -1815 were classified based on the TIN ratios of the highest quartile.

Table 9: Cox regression analysis of TIN ratios for individual miRNAs

<td colspan="2">Variable HR (95% CI)</td><td>p =</td><td>n</td>

<td colspan="4">Univariate analysis</td>

<td>miR-21 high / low</td><td> 3,0 (11,3 7,0)</td><td> 0,01</td><td> 80</td>

<td colspan="4">Multivariate analysis</td>

<td>miR-21 high / low</td><td> 2,8 (01,02 - 06,08)</td><td> 0,02</td><td></td>

<td>age 2 50 / age &lt;50</td><td> 0,46 (0,10-2,1)</td><td> 0,32</td><td></td>

<td>male / female</td><td> 3,1 (0,9-11,0)</td><td> 0,07</td><td></td>

<td>AA / Caucasian</td><td> 1,2 (0,5-2,7)</td><td> 0,66</td><td></td>

<td>Stage III-IV / stage I-II</td><td> 4,4 (1,6 - 11,9)</td><td> 0,004</td><td></td>

<td colspan="4">Univariate analysis</td>

<td>miR-181b high / low</td><td> 3,4 (1,6 - 7,5)</td><td> 0,002</td><td> 78</td>

<td></td><td>Multivariate analysis</td><td></td><td></td>

<td>miR-181b high / low</td><td> 3,3 (1,3 - 8,2)</td><td> 0,01</td><td></td>

<td>age 2 50 / age &lt;50</td><td> 0,39 (0,08-1 0,8)</td><td> 0,23</td><td></td>

<td>Male / female</td><td> 2,2 (0,7-7,2)</td><td> 0,17</td><td></td>

<td>AA / Caucasian</td><td> 1,1 (0,5 - 2,5)</td><td> 0,82</td><td></td>

<td>Stage III-IV / Stage I-II</td><td> 3,1 (1,2 - 8,1)</td><td> 0,02</td><td></td>

<td colspan="4">Univariate analysis</td>

<td>let-7g high / low</td><td> 2,7 (1,3 - 5,9)</td><td> 0,01</td><td> 84</td>

<td colspan="4">Multivariate analysis</td>

<td>let-7q high / low</td><td> 2,5 (1,1 - 5,5)</td><td> 0,03</td><td></td>

<td>age 2 50 / age &lt;50</td><td> 0,5 (0,1-2,4)</td><td> 0,39</td><td></td>

<td>Male / female</td><td> 1,5 (0,5-4,4)</td><td> 0,50</td><td></td>

<td>AA / Caucasian</td><td> 1,3 (0,6-2,9)</td><td> 0,50</td><td></td>

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<td>Stage III-VI / Stage I-II</td><td> 3,6 (1,4 - 9,2)</td><td> 0,006,</td><td></td>

<td colspan="4"></td>

<td colspan="2">Variable HR (95% CI)</td><td>p =</td><td>n</td>

<td colspan="4">Univariate analysis</td>

<td>miR-103-2 high / low</td><td> 2,5 (1,1 - 5,6)</td><td> 0,03</td><td> 81</td>

<td colspan="4">Multivariate analysis</td>

<td>miR-103-2 high / low</td><td> 3,1 (1,3 - 7,5)</td><td> 0,01</td><td></td>

<td>age 2 50 / age &lt;50</td><td> 0,5 (0,1-2,2)</td><td> 0,36</td><td></td>

<td>male / female</td><td> 1,6 (0,6-4,9)</td><td> 0,38</td><td></td>

<td>AA / Caucasian</td><td> 0,8 (0,4-1,9)</td><td> 0,69</td><td></td>

<td>Stage III-IV / Stage I - II</td><td> 4,4 (1,7 -11,1)</td><td> 0,002</td><td></td>

<td colspan="4"></td>

<td colspan="2">Variable HR (95% CI)</td><td>p =</td><td>n</td>

<td colspan="4">Univariate analysis</td>

<td>miR-16b high / low</td><td> 4,6 (01,07 - 12,05)</td><td> 0,003</td><td> 69</td>

<td colspan="4">Multivariate analysis</td>

<td>miR-16b high / low</td><td> 5,1 (1,8-15,9)</td><td> 0,003</td><td></td>

<td>age 2 50 / age &lt;50</td><td> 0,4 (0,08-1,7)</td><td> 0,20</td><td></td>

<td>male / female</td><td> 3,2 (0,8-1,7)</td><td> 0,12</td><td></td>

<td>AA / Caucasian</td><td> 0,9 (1,9-22,4)</td><td> 0,003</td><td></td>

<td>Stage III-IV / Stage I - II</td><td> 6,5 (1,9-22,4</td><td> 0,003</td><td></td>

<td colspan="4"></td>

<td colspan="2">Variable HR (95% CI)</td><td>p =</td><td>n</td>

<td colspan="4">Univariate analysis</td>

<td>miR-106a high / low</td><td> 2,6 (1,1 - 6,1)</td><td> 0,01</td><td> 82</td>

<td colspan="4">Multivariate analysis</td>

<td>miR-106a high / low</td><td> 2,4 (1,0-5,7)</td><td> 0,05</td><td></td>

<td>age 2 50 / age &lt;50</td><td> 0,54 (0,11-2,5)</td><td> 0,44</td><td></td>

<td>male / female</td><td> 1,8 (0,5-6,5)</td><td> 0,34</td><td></td>

<td>AA / Caucasian</td><td> 1,1 (0,5 - 2,5)</td><td> 0,84</td><td></td>

<td>Stage III-IV / Stage I - II</td><td> 5,4 (1,8-16,0)</td><td> 0,002</td><td></td>

<td colspan="4"></td>

<td colspan="2">Variable HR (95% CI)</td><td>p =</td><td>n</td>

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<td colspan="4">Univariate analysis</td>

<td>miR-203 high / low</td><td> 3,8 (01,04 - 10,05)</td><td> 0,01</td><td> 57</td>

<td colspan="4">Multivariate analysis</td>

<td>miR-203 high / low</td><td> 3,2 (01,01 - 09,04)</td><td> 0,03</td><td></td>

<td>age 2 50 / age &lt;50</td><td> 1,0 (0,1-8,1)</td><td> 0,97</td><td></td>

<td>male / female</td><td> 1,4 (0,4 -5,1)</td><td> 0,61</td><td></td>

<td>AA / Caucasian</td><td> 0,9 (0,4-2,3)</td><td> 0,83</td><td></td>

<td>Stage III-IV / Stage I - II</td><td> 3,9 (01,03 - 11,08)</td><td> 0,02</td><td></td>

<td colspan="4"></td>

<td colspan="2">Variable HR (95% CI)</td><td>p =</td><td>n</td>

<td colspan="4">Univariate analysis</td>

<td>miR-29a high / low</td><td> 3,1 (1,3 - 7,3)</td><td> 0,01</td><td> 77</td>

<td colspan="4">Multivariate analysis</td>

<td>miR-29a high / low</td><td> 3,2 (1,3 - 7,9)</td><td> 0,01</td><td></td>

<td>age 2 50 / age &lt;50</td><td> 0,5 (0,1-2,2)</td><td> 0,35</td><td></td>

<td>male / female</td><td> 2,2 (0,6-7,4)</td><td> 0,22</td><td></td>

<td>AA / Caucasian</td><td> 0,9 (0,4 2,1)</td><td> 0,76</td><td></td>

<td>Stage III-IV / Stage I - II</td><td> 4,5 (1,7 - 12,2)</td><td> 0,003</td><td></td>

<td colspan="4"></td>

<td colspan="2">Variable HR (95% CI)</td><td>p =</td><td>n</td>

<td colspan="4">Univariate analysis</td>

<td>miR-10a high / low</td><td> 2,7 (1,3 - 5,7)</td><td> 0,01</td><td> 84</td>

<td colspan="4">Multivariate analysis</td>

<td>miR-10a high / low</td><td> 3,5 (1,5 - 7,8)</td><td> 0,003</td><td></td>

<td>age 2 50 / age &lt;50</td><td> 0,4 (0,1-1,9)</td><td> 0,26</td><td></td>

<td>male / female</td><td> 1,7 (0,6-5,0)</td><td> 0,34</td><td></td>

<td>AA / Caucasian</td><td> 1,0 (0,45-2,3)</td><td> 0,98</td><td></td>

<td>Stage III-IV / Stage I - II</td><td> 4,9 (01,09 - 12,02)</td><td> 0,001</td><td></td>

The expression of miR-21 is elevated in tumors (Table 7). MiR-21 TIN relationships are also associated with clinical stages and survival prognosis for colon cancer patients, (Table 9, Figure 8a).

There was a tendency for individuals with a more advanced TNM stage to have higher TIN ratios (p = 0.034). The TIN relationships were dichotomized based on the median of the values ​​for each of the 80 individuals with the data. Individuals with high miR-21 TIN expression ratios had a poorer survival prognosis based on Kaplan-Meier analysis (p = 0.004) suggesting that tumors expressing high levels of miR-21 are predictive of negative prognosis. . These results were further analyzed with

ES 2 425 387 T3 Cox regression analysis.

Individuals with high miR-21 TIN ratios were at higher risk with both univariate (HR = 3.0; p = 0.01) and multivariate (HR = 2.8; p = 0.02) analyzes adjusted for age , sex, race and TNM stage (Table 9).

This result suggested that miR-21 expression levels may be useful as prognostic prediction procedures and may provide more predictive value for survival prognosis than TNM staging alone. MiR-21 has been found to be differentially expressed in many tumor types^12-18.

Studies have also shown that high levels of miR-21 can lead to an inhibition of apoptosis in glioblastoma cells.⁵ while inhibition of miR-21 can lead to increased cell proliferation in HeLa cells¹⁹.

The inventors found herein that miR-21 is now believed to contribute to colon carcinogenesis in a similar manner.

The inventors have discovered that elevated miR-106a in tumors (Table 7) and TIN ratios of miR-106a are associated with survival prognosis (Table 9, Figure 8b).

MiR-106a is a member of a class of miRNA paralogs that include miR-17, miR-20, miR-106a, and miR-106h²⁰. These miRNAs are very similar to each other because they differ only by 1-2 nucleotides. Due to their similarity, they are all likely to have similar targets. Interestingly, all four of these miRNAs show similar expression patterns and prognostic associations (data not shown). The inventors here present associations for miR-106a, but do not formally rule out the possibility that any or all of the other paralogs miRNAs contribute to this association. The miR-106a TIN ratios were dichotomized based on the median of the values ​​for each of the 82 individuals with data. Individuals with high miR-106a TIN expression ratios had a worse survival prognosis based on the Kaplan-Meier analysis (p = 0.013; Figure 8b).

This suggests that tumors expressing high levels of miR-106a are predictive of poor survival prognosis. Individuals with high miR-106a TIN ratios had a higher risk with both univariate (FIR = 2.6, p = 0.01) and multivariate (HR = 2.4; p = 0.05) analyzes adjusted for age , sex, race and TNM stage (Table 7). Therefore, miR-106a may be a useful prognostic predictor for colon cancer prognosis independent of TNM stage. Interestingly, the retinoblastoma tumor suppressor gene has been shown to be a functional target of miR-106a.¹², which supports a mechanism for how miR-106a may mechanically contribute to colon carcinogenesis.

Overexpression of the miR-17-92 cluster, containing miR-106a paralogs, resulted in accelerated tumor development in mice.¹⁰. This shows experimentally that miRNAs of the miR-106a family are capable of affecting carcinogenesis further strengthening the hypothesis that miR-106a may contribute to carcinogenesis and tumor progression.

Expression patterns of seven additional miRNAs were associated with the determination of clinical staging and poor survival prognosis (Table 9, Figures 8c-8i).

There is a tendency for individuals who have been diagnosed with a more advanced TNM stage to have higher TIN ratios for let-7a (p = 0.010), miR-10a (p = 0.008), miR-16h (p = 0.048), miR-29a (p = 0.005), miR103-2 (p = 0.033), miR-181H (p = 0.016), and miR-203 (p = 0.016) (Figure 8).

TIN ratios were dichotomized based on the median (miR-16h, miR-29a, miR-103-2, miR-203) or major quartile (let-7 g, miR-10a, miR-181H) and Kaplan analysis Meier revealed that high TIN ratios for each were found to be predictors of poor survival prognosis (Figures 8c-8i).

Univariate and multivariate Cox regression analysis confirmed that high TIN ratios of any one of these miRNAs were predictive of negative colon cancer prognosis regardless of TNM status (Table 9). Multivariate Cox regression models that were adjusted for age, sex, race, and TNM stage showed that high TIN ratios for miR-16b (HR = 5.1, p = 0.003), Jet-7g (HR = 2.5; p = 0.03), miR-10a (HR = 3.4, p = 0.003), miR-29a (HR = 3.2; p = 0.01), miR-103-2 (HR = 3.1; p = 0.01), miR-181H (HR = 3.2; p = 0.01) and miR-203 (HR = 3.2; p = 0.03) were each predictive of poor survival prognosis. These results suggest that patients with tumors expressing high levels of any of these miRNAs are at increased risk of dying from colon cancer. Therefore, the expression levels of any of these miRNAs can be useful biomarkers that can help predict survival risks for colon cancer patients regardless of stage.

Identification of miRNA expression of 9 miRNA predicts survival prognosis:

The inventors used the TIN ratios of the previously mentioned 9 miRNAs to develop a

ES 2 425 387 T3 identification of miRNA that could be used to predict prognosis of colon cancer. Individuals lacking more than 2 out of 9 of these values ​​were excluded from this analysis. Hierarchical grouping of the TIN ratios of the 9 miRNAs resulted in grouping the remaining 78 patients into two groups (Figure 9a).

These groups had significantly different survival prognoses (Figure 9b; p = 0.004). Univariate (HR = 3.2, p = 0.008) and multivariate (HR = 2.8; p = 0.04) Cox regression analysis showed that miRNA identification was associated with poor survival prognosis regardless of the stage of TNM (Table 10).

Table 10 - MicroRNA identification Cox regression analysis

<td colspan="3">Univariate analysis</td>

<td>Variable</td><td>HR (95% CI)</td><td>p value</td>

<td>9 miR B / A group</td><td> 3,2 (1,4-7,8)</td><td> 0,008</td>

<td colspan="3">Multivariate, adjusted for age, sex, and race</td>

<td>Variable</td><td>HR (95% CI)</td><td>p value</td>

<td>9 miR B / A group</td><td> 2,8 (1,0-7,4)</td><td> 0,043</td>

<td>age 2 50 / age &lt;50</td><td> 0,4, (0,08-1,8)</td><td> 0,23</td>

<td>male / female</td><td> 1,9 (0,6-6,6)</td><td> 0,29</td>

<td>AA / Caucasian</td><td> 0,9 (01,04 - 10,07)</td><td> 0,82</td>

<td>Stage III-IV / Stage I - II</td><td> 3,9 (01,04 - 10,07)</td><td> 0,007</td>

Univariate (previously) and multivariate (adjusted for age, sex, race, and stage; later) Cox regression analyzes were performed to show that individuals classified in group B using the identification of 9 miRNAs had a higher risk of dying from colon cancer. Neither age, sex nor race contributed significantly to the risk of survival. This risk associated with group assignment is independent of stage.

These results demonstrate that miRNA identifications can be used as a biomarker to predict the survival prognosis of colon cancer patients.

Analysis

Individual miRNAs are differentially expressed in colon tumors^12,13 suggesting that the altered expression of these miRNAs may be part of the cellular changes responsible for colon carcinogenesis. In addition to these findings, the inventors show herein that miRNA expression profiles are associated with identification of colon cancer stage and prognosis. Therefore miRNAs, analyzed individually or as part of a miRNA identification, can be used as biomarkers that will allow clinicians to predict patient survival risk with greater precision.

The strong associations of miRNA TIN relationships with survival prognosis suggest that altered miRNA expression may be part of the causal pathway in colon carcinogenesis and progression. If altered expression of any of these miRNAs is causative of carcinogenesis, it may be possible to design antagomir-type pharmaceuticals that can be used to treat cancer. Using miRNA profiles and miRNA-based therapeutics, it is possible to design personalized drug treatment strategies based on which these nine miRNAs are altered. Additionally, these strategies may be helpful in preventing colon cancer in people who are at high risk due to genetically inherited risks or a history of prior cancer.

Example 3

Procedures, reagents, and kits for diagnosis, staging, prognosis, monitoring, and treatment of colon cancer-related diseases.

In one embodiment, a diagnostic method is provided for evaluating whether a patient has a colon cancer-related disease or is at higher than normal risk for developing colon cancer-related disease, comprising the steps of comparing the expression level of a marker in a patient sample and the normal level of marker expression in a control, for example, a sample from a

ES 2 425 387 T3 patient without colon cancer related disease. A significantly higher level of marker expression in the patient sample compared to the normal level is an indication that the patient is afflicted with a colon cancer-related disease or has a higher-than-normal risk of developing a cancer-related disease. colon.

Markers are selected so that the positive predictive value of the procedures is at least about 10%, and in certain non-limiting embodiments, about 25%, about 50%, or about 90%. They are also preferred for use in marker methods that are differentially expressed, compared to normal cells, at least twice by at least about 20%, and in certain non-limiting embodiments, by about 50% or about 75%.

In a diagnostic procedure for evaluating whether a patient is afflicted with a colon cancer-related disease (eg, re-detection ("scan"), recurrence detection, reflex test), the method comprises comparing: a) in the level of expression of a marker in a patient sample and b) the normal level of expression of the marker in a control non-colon cancer-related disease sample. A significantly higher level of marker expression in the patient sample compared to the normal level is an indication that the patient is afflicted with a disease related to colon cancer.

Diagnostic methods are also provided for evaluating the efficacy of a therapy to inhibit a colon cancer-related disease in a patient. Said methods comprise comparing: a) the expression of a marker in a first sample obtained from the patient before providing at least a part of the patient's therapy and b) expression of the marker in a second sample obtained from the patient after provision of the part of the therapy. A significantly lower level of marker expression in the second sample relative to that of the first sample is an indication that the therapy is effective in inhibiting a colon cancer-related disease in the patient.

It will be appreciated that in these methods the "therapy" can be any therapy for treating colon cancer related disease including, but not limited to, pharmaceutical compositions, gene therapy, and biological therapy such as administration of antibodies and chemokines. Therefore, the procedures described herein can be used to assess a patient before, during, and after therapy, for example, to assess reduction in pathology.

In certain aspects, diagnostic procedures refer to therapy using a chemical or biological agent. These methods comprise comparing: a) the expression of a marker in a first sample obtained from the patient and maintained in the presence of the chemical or biological agent, and b) expression of the marker in a second sample obtained from the patient and maintained in the absence of the agent. A significantly lower expression level of the marker in the second sample relative to that in the first sample is an indication that the agent is effective in inhibiting a colon cancer-related disease in the patient. In one embodiment, the first and second samples may be parts of a single sample obtained from the patient or parts of pooled samples obtained from the patient.

Also provided is a monitoring method for evaluating the progression of a colon cancer-related disease in a patient, the method comprising: a) detecting in a patient sample at a first time point, the expression of a marker; b) repeating step a at a later time point in time; and c) comparing the level of expression detected in steps a) and b), and from them monitoring the progression of a disease related to colon cancer in the patient. A significantly higher level of marker expression in the sample at a later time point than the sample at the first time point is an indication that colon cancer-related disease has progressed, while a significantly lower level of expression is a an indication that colon cancer-related disease has regressed.

A diagnostic method is further provided to determine whether a colon cancer-related disease has worsened or is likely to worsen in the future, the method comprising comparing: a) the level of expression of a marker in a patient sample and b) the normal level of marker expression in a control sample. A significantly higher level of expression in the patient sample compared to the normal level is an indication that colon cancer-related disease has worsened or is likely to worsen in the future.

Also provided is a test method for selecting a composition for inhibiting a colon cancer-related disease in a patient. This method comprises the steps of: a) obtaining a sample comprising cells from the patient, b) maintaining separate aliquots of the sample in the presence of a plurality of test compositions; c) comparing the expression of a marker in each of the aliquots; and d) selecting one of the test compositions that significantly reduces the level of marker expression in the aliquot containing this test composition, relative to the marker expression levels in the presence of the other test compositions.

ES 2 425 387 T3

A test procedure is further provided to evaluate the deleterious potential of a compound to cause colon cancer related disease. This procedure comprises the steps of: a) maintaining separate aliquots of cells in the presence and absence of the compound; and b) comparing the expression of a marker in each of the aliquots. A significantly higher level of marker expression in the aliquot maintained in the presence of the compound, relative to that of the aliquot maintained in the absence of the compound, is an indication that the compound possesses such harmful potential.

In addition, a method of inhibiting a colon cancer-related disease in a patient is further provided. This procedure comprises the steps of: a) obtaining a sample comprising cells from the patient; b) maintaining separate aliquots of the sample in the presence of a plurality of compositions; c) comparing the expression of a marker in each of the aliquots; and d) administering to the patient at least one of the compositions that significantly reduces the level of marker expression in the aliquot containing this composition, relative to the marker expression levels in the presence of the other compositions.

The level of expression of a marker in a sample can be assessed, for example, by detecting the presence in the sample of: the corresponding marker protein or a fragment of the protein (for example, using a reagent, such as an antibody, an antibody derivative, an antibody fragment, or a single chain antibody, that specifically binds to the protein or protein fragment), the acid corresponding marker nucleic acid (for example, a nucleotide transcript, or a complement thereof), or a fragment of the nucleic acid (for example, by contacting transcribed polynucleotides obtained from the sample with a substrate that has one or more nucleic acids having the complete nucleic acid sequence or a segment of it or a complement thereof attached to it), a metabolite that is produced directly ( that is, it is catalyzed) or indirectly by the corresponding marker protein.

Any of the above procedures can be performed using at least one or a plurality (eg, 2, 3, 5, or 10 or more) of colon cancer-related disease markers, including colon cancer-related disease markers.

In such procedures, the level of expression in the sample of each of a plurality of markers, at least one of which is a marker, is compared with the normal level of expression of each of the plurality of markers in samples thereof. type obtained from control humans not afflicted with colon cancer-related disease. A significantly altered level of expression (i.e., increased or decreased as specified in the procedures described above using a single marker) in the sample of one or more markers, or some combination thereof, relative to the normal or normal level. Corresponding control of that marker is an indication that the patient is suffering from a disease related to colon cancer. For all of the above-mentioned procedures, the marker (s) are selected so that the positive predictive value of the procedure is at least about 10%.

In another aspect, various diagnostic and test kits are provided. In one embodiment, a kit is useful for evaluating whether a patient is afflicted with a colon cancer-related disease. The kit comprises a reagent for evaluating the expression of a marker. In another embodiment, a kit is useful for evaluating the suitability of a chemical or biological agent to inhibit a colon cancer-related disease in a patient. Said kit comprises a reagent for evaluating the expression of a marker, and may also comprise one or more of said agents.

In a further embodiment, the kits are useful for evaluating the presence of colon cancer-related disease cells or treating colon cancer-related diseases. Such kits comprise an antibody, an antibody derivative or an antibody fragment, which specifically binds to a marker protein or a fragment of the protein. Such kits may also comprise a plurality of antibodies, antibody derivatives or antibody fragments with the plurality of said antibody agents specifically binding to a marker protein or a fragment of the protein.

In a further embodiment, the kits are useful for evaluating the presence of colon cancer-related disease cells, the kit comprising a nucleic acid probe that specifically binds to a marker nucleic acid or nucleic acid fragment. The kit may also comprise a plurality of probes, each of the probes being specifically linked with a marker nucleic acid, or a fragment of the nucleic acid.

In a further aspect, methods are provided for treating a patient afflicted with a colon cancer-related disease or at risk of developing a colon cancer-related disease. Such methods may comprise reducing the expression and / or interfering with the biological function of a marker. In one embodiment, the method comprises providing the patient with an antisense oligonucleotide or polynucleotide complementary to a marker nucleic acid, or a segment thereof. For example, an antisense polynucleotide can be provided to the patient by providing a vector that expresses an antisense polynucleotide of a marker nucleic acid or a fragment thereof. In another embodiment, the method comprises providing the patient with an antibody, an antibody derivative, or an antibody fragment, which binds

ES 2 425 387 T3 specifically with a marker protein or a protein fragment.

In a general aspect, a method is provided for producing a non-human animal model for evaluation of at least one colon cancer-related disease. The method includes exposing the animal to repeated doses of at least one chemical compound believed to cause colon cancer. In certain aspects, the method further includes collecting one or more selected samples from the animal; and comparing the collected sample to one or more indications of potential colon cancer initiation or development.

In general aspects, a method of producing the animal model is provided which includes: keeping the animal in an environment without specific chemical compounds and sensitizing the animal with at least one chemical compound believed to cause colon cancer. In certain embodiments, at least a portion of the animal's colon is sensitized by multiple sequential exposures.

In a general aspect, a method is provided for screening an agent for efficacy against at least one colon cancer-related disease. The method generally includes: administering at least one agent to the animal, determining whether the agent reduces or aggravates one or more symptoms of colon cancer-related disease; correlating a reduction in one or more symptoms with efficacy of the agent against colon cancer-related disease; or correlate a lack of reduction in one or more symptoms with agent ineffectiveness.

The animal model is useful for evaluating one or more metabolic pathways that contribute to at least one of onset, progression, severity, pathology, aggressiveness, grade, activity, disability, mortality, morbidity, disease subclassification, or other underlying pathogenic or pathological characteristic of at least one disease related to colon cancer. The analysis can be by one or more of: hierarchical grouping, construction of identification networks, proteomic analysis by mass spectroscopy, surface plasmon resonance, performance of linear statistical models, differential analysis of partial least squares, and multiple linear regression analysis. .

In a particular aspect, the animal model is evaluated with respect to at least one colon cancer-related disease, examining a level of expression of one or more markers, or a functional equivalent thereof.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (eg, in cell culture, molecular genetics, nucleic acid chemistry , hybridization techniques and biochemistry). Conventional techniques are used for molecular, genetic, and biochemical procedures that are within the skill of the art. These techniques are fully explained in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2^to Ed., Ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (Glover ed., 1985); Oligonucleotide Synthesis (Gait ed., 1984); Mullis et al. US Patent No. 4,683,195; Nucleic Acid Hybridization (Hames &amp; Higgins eds., 1984); Transcription And Translation (Hames &amp; Higgins eds., 1984); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., NY); Gene Transfer Vectors For Mammalian Cells (Miller and Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (Weir and Blackwell, eds., 1986); The Laboratory Rat, Editor-in-Chief: Mark A. Suckow; authors: Sharp and LaRegina. CRC Press, Boston, 1988 and chemical procedures.

Newly discovered markers associated with a colon cancer-induced state of various cells are described herein. The higher than normal expression level of any of these markers or combination of these markers has been found to correlate with the presence of a colon cancer-related disease in a patient. Methods are provided for detecting the presence of a colon cancer-related disease in a sample; the absence in a sample; the stage of a colon cancer-related disease; and other characteristics of a colon cancer-related disease that are relevant to the evaluation, prevention, diagnosis, characterization, and therapy of a colon cancer-related disease in a patient. Methods for treating a disease related to colon cancer are also provided.

Definitions As used herein, each of the following terms has the meaning associated with it in this section.

The article "a" is used herein to refer to one or more than one (ie, at least one) of the article's grammatical object. As an example, "one item" means one item or more than one item.

A "marker" is a gene or protein whose altered level of expression in a tissue or cell from its level of expression in normal or healthy tissue or cell is associated with a pathology.

The "normal" level of expression of a marker is the level of expression of the marker in cells of the colon system

ES 2 425 387 T3 from a human subject or patient not afflicted with a disease related to colon cancer.

An "overexpression" or "significantly higher expression level" of a marker refers to a level of expression in a test sample that is greater than the standard error of the assay used to assess expression, and in certain embodiments, at least two times, and in other embodiments, three, four, five, or ten times the expression level of the marker in a control sample (e.g., sample from a healthy subject that does not have the disease associated with the marker) and in certain embodiments, the mean expression level of the marker in various control samples.

A "significantly lower expression level" of a marker refers to a level of expression in a test sample that is at least two times, and in certain embodiments, three, four, five or ten times lower than the expression level of the marker in a control sample (eg, sample from a healthy subject that does not have the disease associated with the marker) and in certain embodiments, the mean expression level of the marker in various control samples.

A kit is any manufactured product (for example a container or container) that comprises at least one reagent, for example a probe, to specifically detect the expression of a marker. The kit can be promoted, distributed, or sold as a unit to perform the methods of the present invention.

"Proteins" encompasses marker proteins and their fragments; variant marker proteins and their fragments; peptides and polypeptides comprising a segment of at least 15 amino acids of a variant marker or marker protein; and fusion proteins comprising a variant marker or marker protein, or a segment of at least 15 amino acids from a variant marker or marker protein.

The compositions, kits, and methods described herein have the following uses, among others: 1) evaluating whether a patient is afflicted with a colon cancer-related disease; 2) assessing the stage of a colon cancer-related disease in a human patient; 3) assessing the degree of a colon cancer-related disease in a patient; 4) assessing the nature of a colon cancer-related disease in a patient; 5) assessing the potential for developing a colon cancer-related disease in a patient; 6) evaluate the histological type of cells associated with a colon cancer-related disease in a patient, 7) make antibodies, antibody fragments or antibody derivatives that are useful to treat a colon cancer-related disease and / or evaluate whether a patient is afflicted with a disease related to colon cancer; 8) evaluating the presence of colon cancer-related disease cells; 9) evaluating the efficacy of one or more test compounds in inhibiting a colon cancer-related disease in a patient; 10) evaluating the efficacy of a therapy to inhibit a colon cancer-related disease in a patient; 11) monitoring the progression of a colon cancer-related disease in a patient; 12) selecting a composition or therapy to inhibit a colon cancer-related disease in a patient; 13) treating a patient afflicted with a colon cancer-related disease; 14) inhibiting a colon cancer-related disease in a patient; 15) evaluating the harmful potential of a test compound; and 16) preventing the occurrence of a colon cancer-related disease in a patient at risk of developing a colon cancer-related disease.

Scan procedures

Animal models created by the procedures described herein will allow the exploration of therapeutic agents useful for treating or preventing a disease related to colon cancer. Consequently, the methods are useful for identifying therapeutic agents to treat or prevent a disease related to colon cancer. The methods comprise administering a candidate agent to an animal model performed by the procedures described herein, evaluating at least one colon cancer-related disease response in the animal model compared to a control animal model that has not been tested. administered the candidate agent. If symptoms are reduced or the onset of at least one colon cancer-related disease response is delayed, the candidate agent is an agent for treating or preventing colon cancer-related disease.

Candidate agents can be pharmacological agents already known in the art or they can be agents that have been previously unknown to have no pharmacological activity. The agents can be of natural origin or designed in the laboratory. They can be isolated from microorganisms, animals or plants, or they can be produced recombinantly, or synthesized by any suitable chemical method. They can be small molecules, nucleic acids, proteins, peptides, or peptidomimetics. In certain embodiments, the candidate agents are small organic compounds that have a molecular weight of greater than 50 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins. Candidate agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.

Candidate agents are obtained from a wide variety of sources including libraries of compounds.

ES 2 425 387 T3 synthetic or natural. There are, for example, numerous means available for random and targeted synthesis of a wide variety of organic compounds and biomolecules, including expression of random oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds are readily available or produced in the form of bacterial, fungal, plant and animal extracts. Additionally, naturally or synthetically produced libraries and compounds are readily modified by conventional chemical, physical, and biochemical means, and can be used to produce combinatorial libraries. In certain embodiments, candidate agents can be obtained using any of the numerous approaches in the art of combinatorial library procedures, including by way of example but not limitation: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library procedures requiring deconvolution; the "one bead one compound" library method; and synthetic library procedures using affinity chromatography selection.

In certain additional embodiments, certain pharmacological agents may be subjected to random or targeted chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

The same procedures for identifying therapeutic agents to treat colon cancer-related disease can also be used to validate candidate compounds / agents generated from in vitro studies.

The candidate agent may be an agent that positively or negatively regulates one or more colon cancer-related disease response pathways. In certain embodiments, the candidate agent can be an antagonist that affects said pathway.

Procedures to treat a disease related to colon cancer

Provided herein are methods of treating, inhibiting, alleviating, or reversing a response to colon cancer-related disease. In the methods described herein, an agent that interferes with a signaling cascade is administered to an individual in need thereof, such as, but not limited to, patients with colon cancer-related disease in which such complications are not yet e sighted and those who already have at least one colon cancer-related disease response.

In the above case, said treatment is useful to prevent the appearance of said disease response related to colon cancer and / or reduce the degree to which it occurs. In the latter case, such treatment is useful to reduce the degree to which said colon cancer-related disease response occurs, prevent its further development, or reverse the colon cancer-related disease response.

In certain embodiments, the agent that interferes with the colon cancer-related disease response cascade may be an antibody specific for said response.

Expression of a marker

Expression of a marker can be inhibited in a number of ways, including, by way of non-limiting example, being able to provide an antisense oligonucleotide to colon cancer-related disease cells to inhibit transcription, translation, or both, of the marker (s). Alternatively, a polynucleotide can be provided that encodes an antibody, an antibody derivative, or an antibody fragment that specifically binds to a marker protein, and operably linked with an appropriate promoter / regulatory region to the cell to generate intracellular antibodies that will inhibit the function or activity of the protein. The expression and / or function of a marker can also be inhibited by treating the colon cancer-related disease cell with an antibody, antibody derivative, or antibody fragment that specifically binds to a marker protein. Using the methods described herein, a variety of molecules, particularly including molecules small enough that can cross the cell membrane, can be screened to identify molecules that inhibit the expression of a marker or inhibit the function of a marker protein. The compound thus identified can be provided to the patient to inhibit colon cancer-related disease cells of the patient.

Any marker or combination of markers, as well as any of certain markers in combination with the markers, can be used in the compositions, kits, and procedures described herein. In general, it is desirable to use markers for which the difference between the level of expression of the marker in colon cancer-related disease cells and the level of expression of the same marker in cells of the normal colon system is as high as possible. Although this difference can be as small as the detection limit of the procedure for evaluating marker expression, it is desirable that the difference be at least greater than the standard error of the evaluation procedure and, in certain embodiments, a difference of at least 2 , 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 100, 500, 1000 times or more than the level of expression of the same marker in normal tissue.

It is recognized that certain marker proteins are secreted into the extracellular space surrounding cells. These markers are used in certain embodiments of compositions, kits, and methods, due to the fact that such marker proteins can be detected in a body fluid sample associated with cancer of the

ES 2 425 387 T3 colon, which can be more easily collected from a human patient than a tissue biopsy sample. In addition, in vivo techniques for detection of a marker protein include introducing into a subject a labeled antibody directed against the protein. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by conventional imaging techniques.

To determine whether any particular marker protein is a secreted protein, the marker protein is expressed, for example, in a mammalian cell, such as a human colon line, extracellular fluid is collected, and the presence or absence of the protein is evaluated in the extracellular fluid (eg using a labeled antibody that specifically binds to the protein).

It will be appreciated that patient samples containing colon cells can be used in the procedures described herein. In these embodiments, the level of marker expression can be assessed by evaluating the amount (eg, absolute amount or concentration) of the marker in a sample. The cell sample can, of course, undergo a variety of preparatory and storage techniques after collection (e.g., nucleic acid and / or protein extraction, fixation, storage, freezing, ultrafiltration, concentration, evaporation, centrifugation, etc. .) before evaluating the amount of the marker in the sample.

It will also be appreciated that the markers can be shed from cells in the digestive system, bloodstream, and / or interstitial spaces. Detached markers can be tested, for example, by examining serum or plasma.

The compositions, kits, and methods can be used to detect expression of marker proteins that have at least a portion that is displayed on the surface of cells that express them. For example, immunological procedures can be used to detect such proteins in whole cells, or computer-based sequence analysis procedures can be used to predict the presence of at least one extracellular domain (i.e., including both secreted proteins and proteins having at least a cell surface domain). Expression of a marker protein that has at least a portion that is displayed on the surface of a cell that expresses it can be detected without necessarily lysing the cell (for example, using a labeled antibody that specifically binds to a cell surface domain of the protein).

The expression of a marker can be evaluated by any of a wide variety of methods to detect the expression of a transcribed nucleic acid or protein. Non-limiting examples of such procedures include immunological procedures for detection of secreted, cell surface, cytoplasmic, or nuclear proteins, protein purification procedures, protein activity or function assays, nucleic acid hybridization procedures, acid reverse transcription procedures. nucleic and nucleic acid amplification procedures.

In a particular embodiment, the expression of a marker is evaluated using an antibody (for example, a radiolabeled, chromophore-labeled, fluorophore-labeled or enzyme-labeled antibody), an antibody derivative (for example, an antibody conjugated to a substrate or with the protein or ligand of a protein-ligand pair) or an antibody fragment (for example, a single chain antibody, an isolated antibody hypervariable domain, etc.) that specifically binds to a marker protein or fragment thereof, including a marker protein that has undergone all or part of its normal post-translational modification.

In another particular embodiment, the expression of a marker is evaluated by preparing mRNA / cDNA (that is, a transcribed polynucleotide) from cells in a patient sample, and hybridizing the mRNA / cDNA with a reference polynucleotide that is a complement of a marker nucleic acid, or a fragment thereof. The cDNA can, optionally, be amplified using any of a variety of polymerase chain reaction procedures prior to hybridization to the reference polynucleotide; preferably, it is not amplified. The expression of one or more markers can be similarly detected using quantitative PCR to assess the level of expression of the marker (s). Alternatively, any of the many methods for detecting mutations or variants (eg, single nucleotide polymorphisms, deletions, etc.) of a marker can be used to detect the appearance of a marker in a patient.

In a related embodiment, a mixture of transcribed polynucleotides obtained from the sample is contacted with a substrate that has a complementary or homologous polynucleotide of at least one part attached thereto (for example, at least 7, 10, 15, 20 , 25, 30, 40, 50, 100, 500 or more nucleotide residues) of a marker nucleic acid. If the complementary or homologous polynucleotides are differentially detectable on the substrate (e.g., detectable using different chromophores or fluorophores, or fixed at different selected positions), then the expression levels of a plurality of markers can be simultaneously assessed using a single substrate (eg, a "gene chip" microarray of polynucleotides attached at selected positions). When using a method to assess marker expression that involves hybridization of one nucleic acid to another, it is desired that the hybridization be performed under stringent hybridization conditions.

ES 2 425 387 T3

In certain embodiments, biomarker assays can be performed using mass spectrometry or surface plasmon resonance. In various embodiments, the method of identifying an active agent against a colon cancer-related disease may include a) providing a sample of cells that contains one or more markers or derivatives thereof; b) preparing an extract of said cells; c) mixing said extract with a labeled nucleic acid probe containing a marker binding site; and d) determining the formation of a complex between the marker and the nucleic acid probe in the presence or absence of the test agent. The determination step may include subjecting said nucleic acid probe / extract mixture to an electrophoretic mobility shift assay.

In certain embodiments, the determination step comprises an assay selected from an enzyme-linked immunosorbent assay (ELISA), fluorescence-based assays, and ultra-high throughput assays, for example surface plasmon resonance (SPR) assays or correlation spectroscopy. fluorescence (FCS). In such embodiments, the SPR sensor is useful for direct real-time observation of biomolecular interactions since SPR is sensitive to minimal refractory index changes on a metal dielectric surface. SPR is a surface technique that is sensitive to changes of 10⁵ to 10^-6 Refractory Index (IR) units at a distance of approximately 200 nm from the SPR sensor / sample interface. Therefore, SPR spectroscopy is useful to monitor the growth of thin organic films deposited on the sensor layer.

Because the compositions, kits and procedures are based on the detection of a difference in the expression levels of one or more markers, it is desired that the marker expression level be significantly greater than the minimum detection limit of the procedure. used to assess expression in at least one of normal cells and cells affected by colon cancer.

It is understood that by routine screening of additional patient samples using one or more of the markers, some of the markers will be expected to be overexpressed in cells of various types, including specific colon cancer-related diseases.

In addition, since a greater number of patient samples are evaluated for marker expression and the results of the individual patients from whom samples were obtained are correlated, it will also be confirmed that the altered expression of some of the markers is strongly correlated with a disease related to colon cancer and that the altered expression of other markers is strongly correlated with other diseases. Compositions, kits, and methods are therefore useful for characterizing one or more of the stage, grade, histological type, and nature of colon cancer-related disease in patients.

When the compositions, kits, and methods are used to characterize one or more of the stage, grade, histological type, and nature of a colon cancer-related disease in a patient, it is desired that the marker or panel of markers be selected so that a positive result is obtained by at least about 20%, and in certain embodiments, at least about 40%, 60%, or 80%, and in substantially all patients afflicted with colon cancer-related disease of the corresponding stage, grade, histological type, or nature. The marker or panel of markers of the invention can be selected such that a positive predictive value of greater than about 10% is obtained for the general population (in a non-limiting example, coupled with an assay specificity greater than 80%).

When a plurality of markers are used in the compositions, kits, and methods, the level of expression of each marker in a patient sample can be compared to the normal level of expression of each of the plurality of markers in non-cancer samples. colon of the same type, in a single reaction mix (i.e. using reagents, such as different fluorescent probes, for each label) or in individual reaction mixtures that correspond to one or more of the labels. In one embodiment, a significantly increased expression level of more than one of the plurality of markers in the sample, relative to corresponding normal levels, is an indication that the patient is afflicted with a colon cancer-related disease. When using a plurality of markers, 2, 3, 4, 5, 8, 10, 12, 15, 20, 30 or 50 or more individual markers can be used; In certain embodiments, the use of fewer markers may be desired.

To maximize the sensitivity of compositions, kits, and methods (i.e., by interference attributable to cells of a non-colon system origin in a patient sample), it is desirable that the marker used therein is a marker that has a restricted tissue distribution, for example, not normally expressed in non-colon system tissue.

It is recognized that the compositions, kits, and methods will be particularly useful for patients who are at increased risk of developing colon cancer-related disease and their medical advisers. Patients who are recognized to be at increased risk of developing colon cancer-related disease include, for example, patients who have a family history of colon cancer-related disease.

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The level of expression of a marker in normal human colon system tissue can be assessed in a variety of ways. In one embodiment, the normal level of expression is assessed by evaluating the level of expression of the marker in a part of cells of the colon system that appear to be normal and comparing this normal level of expression with the level of expression in a part of the cells of the colon. colon system suspected to be abnormal. Alternatively, and particularly as additional information becomes available as a result of routine performance of the procedures described herein, population mean values ​​for normal expression of the markers can be used. In other embodiments, the "normal" level of expression of a marker can be determined by evaluating the expression of the marker in a patient sample obtained from a patient afflicted with non-colon cancer, from a patient sample obtained from a patient prior to onset. suspected colon cancer-related disease in the patient, from archived patient specimens, and the like.

Also provided herein are compositions, kits, and methods for evaluating the presence of colon cancer-related disease cells in a sample (eg, an archived tissue sample or a sample obtained from a patient). These compositions, kits, and procedures are substantially the same as those described above, except that, where necessary, the compositions, kits, and procedures are adapted for use with samples other than patient samples. For example, when the sample to be used is an archived, paraffinized human tissue sample, it may be necessary to adjust the ratio of compounds in the compositions, in the kits, or in the procedures used to assess levels of marker expression in the sample.

Kits and reagents

The kits are useful for evaluating the presence of colon cancer-related disease cells (eg, in a sample, such as a patient sample). The kit comprises a plurality of reagents, each of which is capable of specifically binding a marker nucleic acid or protein. Suitable reagents for binding to a marker protein include antibodies, antibody derivatives, antibody fragments, and the like. Suitable reagents for binding to a marker nucleic acid (eg, genomic DNA, mRNA, spliced ​​mRNA, cDNA, or the like) include complementary nucleic acids. For example, nucleic acid reagents can include oligonucleotides (labeled or unlabeled) bound to a substrate, labeled oligonucleotides not bound to a substrate, pairs of PCR primers, molecular beacon probes, and the like.

Kits may optionally comprise additional components useful to perform the procedures described herein. As an example, the kit may comprise fluids (e.g. SSC buffer) suitable for hybridizing complementary nucleic acids or for binding an antibody to a protein to which it specifically binds, one or more sample compartments, an instructional material describing the performance of the procedure, a sample of cells from the normal colon system, a sample of cells from colon cancer-related disease, and the like.

Procedure to produce antibodies

Also provided herein is a method of making an isolated hybridoma that produces an antibody useful for evaluating whether a patient is afflicted with a colon cancer-related disease. In this method, a protein or peptide comprising all or a segment of a marker protein is synthesized or isolated (for example by purification of a cell in which it is expressed or by transfection and translation of a nucleic acid encoding the protein or peptide in vivo or in vitro). A vertebrate, eg, a mammal such as a mouse, rat, rabbit, or sheep is immunized using the protein or peptide. The vertebrate may optionally (and preferably) be immunized an additional time with the protein or peptide, such that the vertebrate displays a robust immune response to the protein or peptide. Splenocytes are isolated from the immunized vertebrate and fused with an immortalized cell line to form hybridomas, using any of a variety of procedures. Hybridomas formed in this manner are then screened using standard procedures to identify one or more hybridomas that produce an antibody that specifically binds to the marker protein or a fragment thereof. Also provided herein are hybridomas made by this method and antibodies made using such hybridomas.

Procedure for evaluating effectiveness

Also provided herein is a method of evaluating the efficacy of a test compound in inhibiting colon cancer-related disease cells. As described above, differences in the level of expression of the markers correlate with the abnormal state of cells of the colon system. Although it is recognized that changes in the levels of expression of some of the markers probably result from the abnormal state of the cells of the colon system, it is probably recognized that changes in the levels of expression of another of the markers induce, maintain and promote the abnormal state of those cells. Therefore, compounds that inhibit a colon cancer-related disease in a patient will cause the level of expression of one or more of the markers to change to a level closer to the normal level of expression for that marker (i.e. the level of expression for the marker in cells

ES 2 425 387 T3 of the normal colon system).

This method therefore comprises comparing the expression of a marker in a first colon cell sample and maintained in the presence of the test compound and expression of the marker in a second colon cell sample and maintained in the absence of the test compound. A significantly reduced expression of a marker in the presence of the test compound is an indication that the test compound inhibits a colon cancer-related disease. Colon cell samples can, for example, be aliquots of a single sample of normal colon cells obtained from a patient, pooled samples of normal colon cells obtained from a patient, cells from a normal colon cell line, aliquots of a single sample of colon cancer-related disease cells obtained from a patient, pooled samples of colon cancer-related disease cells obtained from a patient, cells of a colon cancer-related disease cell line or the like.

In one embodiment, the samples are colon cancer related disease cells obtained from a patient and a plurality of compounds believed to be effective in inhibiting various colon cancer related diseases are tested to identify the compound that is likely to best inhibit the colon cancer-related disease in the patient.

This procedure can similarly be used to evaluate the efficacy of a therapy to inhibit a colon cancer-related disease in a patient. In this procedure, the expression level of one or more markers is evaluated in a pair of samples (one subjected to therapy, the other not subjected to therapy). As with the method of evaluating the efficacy of test compounds, if the therapy induces a significantly lower level of expression of a marker then the therapy is effective in inhibiting a colon cancer-related disease. As before, if samples from a selected patient are used in this procedure, then alternative in vitro therapies can be evaluated to select a therapy that is most likely effective in inhibiting a colon cancer-related disease in the patient.

As described herein, the abnormal condition of human colon cells correlates with changes in the expression levels of the markers. A method for evaluating the harmful potential of a test compound is also provided. This procedure comprises maintaining separate aliquots of human colon cells in the presence and absence of the test compound. The expression of a marker in each of the aliquots is compared. A significantly higher level of expression of a marker in the aliquot maintained in the presence of the test compound (relative to the aliquot maintained in the absence of the test compound) is an indication that the test compound possesses deleterious potential. The relative harmful potential of various test compounds can be assessed by comparing the degree of enhancement or inhibition of the expression level of the relevant markers, comparing the number of markers with which the expression level is either enhanced or inhibited, or by comparing both.

Various aspects are described in more detail in the following subsections.

Isolated proteins and antibodies

One aspect concerns isolated marker proteins and biologically active parts thereof, as well as polypeptide fragments suitable for use as immunogens to induce antibodies directed against a marker protein or a fragment thereof. In one embodiment, the native marker protein can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, a protein or peptide comprising the entire marker protein or a segment thereof is produced by recombinant DNA techniques. As an alternative to recombinant expression, said protein or peptide can be chemically synthesized using standard peptide synthesis techniques.

An "isolated" or "purified" protein or biologically active part thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemical compounds when synthesized chemically. The term "substantially free of cellular material" includes protein preparations in which the protein is separated from cellular components of the cells from which it is isolated or is produced recombinantly. Thus, protein that is substantially free of cellular material includes protein preparations that have less than about 30%, 20%, 10%, or 5% (by dry weight) heterologous protein (also referred to herein as "protein pollutant").

When the protein or biologically active part thereof is produced recombinantly, it is also preferably substantially free of culture medium, that is, the culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When the protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemical compounds, that is, it is separated from chemical precursors or other chemical compounds that are involved in the synthesis of the protein. Consequently such protein preparations have less than about 30%, 20%, 10%, 5% (by dry weight) of protein precursors or compounds other than the polypeptide of interest.

Biologically active parts of a marker protein include polypeptides comprising sequences of

ES 2 425 387 T3 amino acids sufficiently identical to or derived from the amino acid sequence of the marker protein, that they include fewer amino acids than the full-length protein, and show at least one activity of the corresponding full-length protein. Typically, the biologically active parts comprise a domain or motif with at least one activity of the corresponding full-length protein. A biologically active part of a marker protein can be a polypeptide that is, for example, 10, 25, 50, 100 or more amino acids in length. In addition, other biologically active parts, in which other regions of the marker protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of the native form of the marker protein. In certain embodiments, useful proteins are substantially identical (eg, at least about 40%, and in certain embodiments, 50%, 60%, 70%, 80%, 90%, 95%, or 99%) at a of these sequences and retain the functional activity of the corresponding naturally occurring marker protein but differ in amino acid sequence due to natural allelic variation or mutagenesis.

In addition, libraries of marker protein segments can be used to generate a motley population of polypeptides for screening and subsequent selection of variant marker proteins or segments thereof.

Predictive medicine

Also provided herein are uses of animal models and markers in the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and clinical trial monitoring are used for prognostic (predictive) purposes to try to this way to an individual prophylactically. Accordingly, diagnostic assays are also provided herein to determine the level of expression of one or more marker proteins or nucleic acids, to determine whether an individual is at risk of developing a colon cancer-related disease. Such assays can be used for prognostic or predictive purposes to thus prophylactically treat an individual prior to the onset of colon cancer-related disease.

In another aspect, the methods are useful for at least periodic screening of the same individual to see if that individual has been exposed to chemicals or toxins that change their expression patterns.

Yet another aspect concerns monitoring the influence of agents (for example, drugs or other compounds administered to inhibit a colon cancer-related disease or to treat or prevent any other disorder (for example, to understand any system effects that may have such treatment) in the expression or activity of a marker in clinical trials.

Pharmacogenomics

The markers are also useful as pharmacogenomic markers. As used herein, a "pharmacogenomic marker" is a target biochemical marker whose level of expression correlates with a specific clinical drug response or susceptibility in a patient. The presence or amount of the expression of the pharmacogenomic marker is related to the predicted response of the patient and more particularly the patient's tumor to therapy with a specific drug or class of drugs. By evaluating the presence or amount of expression of one or more pharmacogenomic markers in a patient, a drug therapy can be selected that is more appropriate for the patient, or that is predicted to have a greater degree of success.

Supervision of clinical trials

Monitoring the influence of agents (eg, drug compounds) on the level of expression of a marker can be applied not only in basic drug screening, but also in clinical trials. For example, the efficacy of an agent to affect marker expression can be monitored in clinical trials of subjects receiving treatment for a disease related to colon cancer.

In a non-limiting embodiment, the present invention provides a method of monitoring the efficacy of treating a subject with an agent (eg, an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other pharmacological candidate) that comprises the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detect the level of expression of one or more selected markers in the pre-administration sample; (iii) obtain one or more post-administration samples from the subject; (iv) detect the level of expression of the marker (s) in the post-administration samples; (v) comparing the expression level of the marker (s) in the pre-administration sample with the expression level of the markers in the post-administration sample (s); and (vi) altering the administration of the agent to the subject accordingly.

For example, increased expression of the marker gene (s) during the course of treatment may indicate ineffective dosing and the desirability of increasing dosing. Conversely, reduced expression of the marker gene (s) may indicate effective treatment and no need to change the dosage.

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Readable media in electronic devices, systems, matrices and procedures for their use

As used herein, "electronically readable media" refers to any medium suitable for storing, maintaining, or containing data or information that can be read and directly accessed by an electronic device. Such media may include, but are not limited to: magnetic storage media, such as floppy disks, hard disk storage media, and magnetic tape; optical storage media such as compact disc; electronic storage media such as RAM, ROM, EPROM, EEPROM, and the like; and general and hybrid hard drives of these categories such as magnetic / optical storage media. The medium is adapted or configured for a marker to be recorded thereon as described herein.

As used herein, the term "electronic apparatus" is intended to include any suitable computing or processing apparatus or other device configured or adapted to store data or information. Examples of electronic apparatus suitable for use with the present invention include self-contained computing apparatus; networks, including a local area network (LAN), a wide area network (WAN), the Internet, Intranet, and Extranet; electronic devices such as personal digital assistants (PDAs), mobile phones, pagers and the like; and local and distributed processing systems.

As used herein "proprietary" refers to a method for storing or encoding information on the electronically readable medium. Those skilled in the art can readily adopt any method of recording information in the media for generating materials comprising the markers described herein.

A variety of computer programs and formats can be used to store the marker information of the present invention on the electronically readable medium. Any variety of data processor structuring formats (eg, text file or database) can be used to obtain or create a medium that has markers registered on it. By providing the markers in readable form, marker sequence information can be routinely accessed for a variety of purposes. For example, one of skill in the art can use nucleotide or amino acid sequences in readable form to compare a target sequence or target structural motif with sequence information stored within the data storage medium. Search means are used to identify fragments or regions of the sequences that match a particular target sequence or target motif.

Therefore, also provided herein is a means of maintaining instructions for performing a procedure to determine whether a subject has a colon cancer-related disease or a predisposition to a colon cancer-related disease, wherein the procedure comprises the steps of determining the presence or absence of a marker and based on the presence or absence of the marker, determining whether the subject has a colon cancer-related disease or a predisposition to a colon cancer-related disease and / or recommending a particular treatment for a colon cancer-related disease or colon cancer-related pre-disease condition.

Also provided herein is an electronic and / or networked system, a method for determining whether a subject has a colon cancer-related disease or a predisposition to a colon cancer-related disease associated with a marker in which The method comprises the steps of determining the presence or absence of the marker, and based on the presence or absence of the marker, determining whether the subject has a colon cancer-related disease or a predisposition to a colon cancer-related disease and / or recommending a particular treatment for the colon cancer-related disease or colon cancer-related pre-disease condition. The method may further comprise the step of receiving phenotypic information associated with the subject and / or acquiring phenotypic information associated with the subject from a network.

Also provided herein is a network, a method of determining whether a subject has a colon cancer related disease or a predisposition to a colon cancer related disease associated with a marker, the method comprising the steps of receiving associated information with the marker, receive phenotypic information associated with the subject, acquire information from the network corresponding to the colon cancer-related marker and / or disease and based on one or more of the phenotypic information, the marker, and the acquired information, determine whether the subject has a colon cancer-related disease or predisposition to a disease related to colon cancer. The method may further comprise the step of recommending a particular treatment for colon cancer-related disease or colon cancer-related pre-disease condition.

Also provided herein is a business method for determining whether a subject has a colon cancer-related disease or a predisposition to a colon cancer-related disease, the method comprising the steps of receiving information associated with the marker, receiving phenotypic information associated with the subject, acquire information from the network corresponding to the marker and / or a disease related to colon cancer, and based on one or more of the phenotypic information, the marker, and the acquired information, determining whether the subject has a colon cancer-related disease or a

ES 2 425 387 T3 predisposition to colon cancer related disease. The method may further comprise the step of recommending a particular treatment for colon cancer related disease or colon cancer related pre-disease condition.

Also provided herein is an array that can be used to test the expression of one or more genes on the array. In one embodiment, the array can be used to test for gene expression in tissue to determine tissue specificity of genes on the array. In this manner, up to about 7000 or more genes can be tested simultaneously for expression. This allows a profile to be developed that shows a battery of genes specifically expressed in one or more tissues.

In addition to such a qualitative determination, quantification of gene expression is provided herein. Therefore, not only tissue specificity, but also the level of expression of a battery of genes in the tissue can be determined. Therefore, genes can be grouped based on their expression in the tissue itself and the level of expression in that tissue. This is useful, for example, when determining the relationship of gene expression between or within tissues. Therefore, one tissue can be altered and the effect on gene expression in a second tissue can be determined. In this context, the effect of one cell type on another cell type in response to a biological stimulus can be determined.

Such a determination is useful, for example, to know the effect of cell-cell interaction at the level of gene expression. If an agent is administered therapeutically to treat one cell type but has an undesirable effect on another cell type, the method provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer one compensatory agent or treat another. mode the unwanted effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level. Therefore, the effects of an agent on the expression of a gene other than the target can be determined and counteracted.

In another embodiment, the array can be used to monitor the time course of expression of one or more genes on the array. This can be done in various biological contexts, as disclosed herein, for example development of a colon cancer-related disease, progression of a colon cancer-related disease, and processes, such as cell transformation associated with a disease related to colon cancer.

The matrix is ​​also useful for determining the effect of the expression of one gene or the expression of other genes in the same cell or in different cells. This enables, for example, a selection of alternative molecular targets for therapeutic intervention if the last or downstream target cannot be regulated.

The matrix is ​​also useful for determining differential expression patterns of one or more genes in normal and abnormal cells. This provides a battery of genes that could act as a molecular target for diagnostic or therapeutic intervention.

Substitute markers

The markers can act as surrogate markers for one or more disorders or pathologies or for conditions leading to colon cancer-related pathology. As used herein, a "surrogate marker" is a target biochemical marker that correlates with the absence or presence of a disease or disorder, or with the progression of a disease or disorder. The presence or quantity of these markers is independent of the disease. Therefore, these markers can act to indicate whether a particular course of treatment is effective in reducing a pathology or disorder. Surrogate markers are particularly useful when the presence or degree of a pathology or disorder is difficult to assess by conventional methodologies, or when an assessment of disease progression is desired before a potentially dangerous clinical end point is reached.

Markers are also useful as pharmacodynamic markers. As used herein, a "pharmacodynamic marker" is a target biochemical marker that specifically correlates with pharmacological effects. The presence or amount of a pharmacodynamic marker is not related to the pathology or disorder for which the drug is administered; therefore, the presence or amount of the marker is indicative of the presence or activity of the drug in a subject. For example, a pharmacodynamic marker can be indicative of the concentration of the drug in a biological tissue, because the marker is expressed or transcribed or not expressed or transcribed in that tissue relative to the level of the drug. In this way, the distribution or adaptation of the drug can be monitored by the pharmacodynamic marker. Similarly, the presence or amount of the pharmacodynamic marker can be related to the presence or amount of the metabolic product of a drug, such that the presence or amount of the marker is indicative of the relative degradation rate of the drug in vivo.

Pharmacodynamic markers are particularly useful to increase the sensitivity of detection of pharmacological effects, particularly when the drug is administered in low doses. Since even a small amount of a drug may be sufficient to activate multiple cycles of marker transcription or expression, the amplified marker may be in an amount that is more easily detectable than the drug itself.

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Furthermore, the marker can be more easily detected due to the nature of the marker itself; for example, using the methods described herein, antibodies can be employed in an immune system-based detection system for a protein marker or marker-specific radiolabeled probes can be used to detect an mRNA marker. In addition, the use of a pharmacodynamic marker may offer mechanism-based risk prediction due to drug treatment beyond the range of possible direct observations.

Protocols to test

The test procedure for colon cancer-related diseases comprises, for example, measuring the level of expression of each marker gene in a biological sample from a subject over time and comparing the level with that of the marker gene in a biological sample. of control.

When the marker gene is one of the genes described herein and the expression level is differentially expressed (for example, it is higher or lower than in the control), the subject is considered to be affected by a cancer-related disease of colon. When the expression level of the marker gene falls within the permissible range, it is unlikely that the subject is affected by a disease related to colon cancer.

The standard value for the control can be predetermined by measuring the expression level of the marker gene in the control, to compare the expression levels. For example, the standard value can be determined based on the expression level of the above-mentioned marker gene in the control. For example, in certain embodiments, the allowable range is taken as ± 2 SD based on the conventional value. Once the standard value has been determined, the assay procedure can be performed by only measuring the level of expression in a biological sample from a subject and comparing the value with the standard value determined for the control.

The levels of marker gene expression include transcription of marker genes to mRNA and translation into proteins. Therefore, a method for assays to detect colon cancer-related disease is performed based on a comparison of the intensity of mRNA expression corresponding to the marker genes, or the level of expression of proteins encoded by the marker genes.

Measurement of marker gene expression levels in the assay for colon cancer-related disease can be carried out according to various gene analysis procedures. Specifically, one can use, for example, a hybridization technique that uses nucleic acids that hybridize to these genes as probes, or a gene amplification technique that uses DNA that hybridizes with the marker genes as primers.

The probes or primers used for the assays can be designed based on the nucleotide sequences of the marker genes. Identification numbers for the nucleotide sequences of the respective marker genes are described herein.

Furthermore, it should be understood that higher animal genes generally accompany the polymorphism at a high frequency. There are also many molecules that produce isoforms that comprise mutually different amino acid sequences during the splicing process. Any gene associated with a colon cancer-related disease that has activity similar to that of a marker gene is included in marker genes, even if it has nucleotide sequence differences due to polymorphism or that it is an isoform.

It should also be understood that marker genes can include homologues from species other than humans. Therefore, unless otherwise specified, the term "marker gene" refers to a species-unique marker gene homolog or a foreign marker gene that has been introduced into an individual.

Furthermore, a "marker gene homologue" is to be understood to refer to a gene derived from a species other than a human, which can hybridize to the human marker gene as a probe under stringent conditions. Such stringent conditions are known to those skilled in the art who can select an appropriate condition to produce equal stringency experimentally or empirically.

A polynucleotide comprising the nucleotide sequence of a marker gene or a nucleotide sequence that is complementary to the complementary strand of the nucleotide sequence of a marker gene and has at least 15 nucleotides can be used as a probe or primer. Therefore, a "complementary strand" means a strand of a double-stranded DNA with respect to the other strand and which is composed of base pairs A: T (U for RNA) and G: C.

Furthermore, "complementary" means not only those that are completely complementary to a region of at least 15 continuous nucleotides, but also those that have a nucleotide sequence homology of at least 40% in certain cases, 50% in certain cases , 60% in certain cases, 70% in certain cases, at least 80%, 90% and 95% or more. The degree of homology between nucleotide sequences can be determined by

ES 2 425 387 T3 an algorithm, BLAST, etc.

Such polynucleotides are useful as a probe to detect a marker gene, or as a primer to amplify a marker gene. When used as a primer, the polynucleotide typically comprises from 15 bp to 100 bp, and in certain embodiments, from 15 bp to 35 bp of nucleotides. When used as a probe, a DNA comprises the complete nucleotide sequence of the marker gene (or the complementary strand thereof), or a partial sequence thereof that is at least 15 bp of nucleotides. When used as a primer, the 3 'region must be complementary to the marker gene, while the 5' region can be linked to a restriction enzyme recognition sequence or a marker.

"Polynucleotides" can be DNA or RNA. These polynucleotides can be synthetic or of natural origin. In addition, DNA used as a probe for hybridization is usually labeled. Those skilled in the art readily understand such marking procedures. Therefore, the term "oligonucleotide" means a polynucleotide with a relatively low degree of polymerization. Oligonucleotides are included in polynucleotides.

Assays for a colon cancer-related disease can be performed using hybridization techniques, eg, Northern hybridization, dot blot hybridization, or the DNA microarray technique. In addition, they may use gene amplification techniques, such as the RT-PCR procedure. Using the PCR amplification monitoring procedure during the gene amplification step in RT-PCR, a more quantitative analysis of the expression of a marker gene can be achieved.

In the PCR gene amplification monitoring procedure, the detection target (DNA or RNA reverse transcript) is hybridized with probes that are labeled with a fluorescent dye and a fluorescence-absorbing switch. When PCR is performed and the Taq polymerase degrades the probe with its 5'-3 'exonuclease activity, the fluorescent dye and the switch are separated from each other and fluorescence is detected. Fluorescence is detected in real time. By simultaneously measuring a conventional sample in which the copy number of a target is known, it is possible to determine the copy number of the target in the subject sample with the cycle number in which the PCR amplification is linear. Furthermore, one skilled in the art recognizes that the PCR amplification monitoring procedure can be carried out using any suitable procedure.

The test procedure for detecting a colon cancer-related disease can also be carried out by detecting a protein encoded by a marker gene. Hereinafter, a protein encoded by a marker gene is described as a "marker protein". For such assay procedures, for example, the Western blot procedure, the immunoprecipitation procedure and the ELISA procedure can be employed using an antibody that binds to each marker protein.

Antibodies used in detection that bind to the marker protein can be produced by any suitable technique. Furthermore, to detect a marker protein, said antibody may be appropriately labeled. Alternatively, instead of labeling the antibody, a substance that specifically binds to the antibody, eg, protein A or protein G, can be labeled to detect the label protein indirectly. More specifically, such a detection procedure can include the ELISA procedure.

A protein or a partial peptide thereof can be obtained as an antigen, for example, by inserting a marker gene or a part thereof into an expression vector, introducing the construct into an appropriate host cell to produce a transformant, culturing the transformant for expressing the recombinant protein and purifying the expressed recombinant protein from the culture or the culture supernatant. Alternatively, the amino acid sequence encoded by a gene or an oligopeptide comprising a portion of the amino acid sequence encoded by a full-length cDNA is chemically synthesized for use as an immunogen.

In addition, an assay for a colon cancer-related disease can be performed using as an index not only the level of expression of a marker gene but also the activity of a marker protein in a biological sample. The activity of a marker protein means the biological activity intrinsic to the protein. Various procedures can be used to measure the activity of each protein.

Even if a patient is not diagnosed as suffering from a colon cancer-related disease in a routine trial despite symptoms suggesting these diseases, it can be easily determined whether or not the patient has a colon cancer-related disease by performing a test. Assay according to the procedures described in this document.

More specifically, in certain embodiments, when the marker gene is one of the genes described herein, an increase or decrease in the level of expression of the marker gene in a patient whose symptoms suggest at least a susceptibility to a cancer-related disease colon indicates that the symptoms are primarily caused by a disease related to colon cancer.

Additionally, the assays are useful in determining whether a colon cancer-related disease is improving in a patient. In other words, the procedures described herein can be used to consider the therapeutic effect of a treatment for a disease related to colon cancer. What's more,

ES 2 425 387 T3 when the marker gene is one of the genes described herein, an increase or decrease in the expression level of the marker gene in a patient, who has been diagnosed as having a cancer-related disease colon, implies that the disease has progressed further.

The severity and / or susceptibility to colon cancer-related disease can also be determined based on the difference in expression levels. For example, when the marker gene is one of the genes described herein, the degree of increase in the expression level of the marker gene is correlated with the presence and / or severity of a colon cancer-related disease.

Animal models

In another aspect, provided herein are animal models for a colon cancer-related disease, wherein the level of expression of one or more marker genes or a gene functionally equivalent to the marker gene has been elevated in the animal model. A "functionally equivalent gene" as used herein generally is a gene encoding a protein that has activity similar to a known activity of a protein encoded by the marker gene. A representative example of a functionally equivalent gene includes a homologue of a marker gene from an animal subject, which is intrinsic to the animal.

The animal model for a colon cancer-related disease is useful for detecting physiological changes due to a colon cancer-related disease. In certain embodiments, animal models are useful for revealing additional functions of marker genes and for evaluating drugs targeted by marker genes.

In one embodiment, an animal model for a colon cancer-related disease can be created by monitoring the level of expression of a homologous gene or by administering a homologous gene. The method may include creating an animal model for a colon cancer-related disease by monitoring the level of expression of a gene selected from the group of genes described herein. In another embodiment, the method may include creating an animal model for a colon cancer-related disease by administering the protein encoded by a gene described herein, or administering an antibody against the protein. It should also be understood that in certain other embodiments the marker may be overexpressed so that the marker can then be measured using appropriate procedures.

In another embodiment, an animal model for a colon cancer-related disease can be created by introducing a gene selected from said groups of genes, or by administering a protein encoded by said gene.

In another embodiment, a colon cancer-related disease can be induced by suppressing the expression of a gene selected from said groups of genes or the activity of a protein encoded by said gene. An antisense nucleic acid, ribozyme, or RNAi can be used to suppress expression. The activity of a protein can be effectively controlled by administering a substance that inhibits activity, such as an antibody.

The animal model is useful for elucidating the mechanism underlying a colon cancer-related disease and also for testing the safety of screening compounds. For example, when an animal model develops the symptoms of colon cancer-related disease, or when a measured value implicated in a certain colon cancer-related disease is altered in the animal, a screening system can be constructed to screen for compounds that have activity. to alleviate the disease.

As used herein, the term "an increase in the level of expression" refers to any one of the following: when a marker gene introduced as a foreign gene is artificially expressed; when the transcription of a marker gene intrinsic to the animal subject and its translation into the protein are enhanced; or when hydrolysis of the protein, which is the translation product, is suppressed. As used herein, the term "a reduction in expression level" refers to the state in which the transition of a marker gene of the animal subject and the translation thereof into the protein are inhibited, or the state in that the hydrolysis of the protein, which is the translation product, is enhanced. The level of expression of a gene can be determined, for example, by a difference in signal intensity on a DNA chip. Furthermore, the activity of the translation product, the protein, can be determined by comparing with that of the normal state.

It is also within the scope of contemplation that the animal model may include transgenic animals, including, for example, animals into which a marker gene has been introduced and artificially expressed; marker gene knockout animals; and knock-in animals in which another gene has replaced a marker gene. A transgenic animal, in which an antisense nucleic acid of a marker gene, a ribozyme, a polynucleotide that has an RNAi effect, or a DNA that acts as a decoy nucleic acid or the like, such as the transgenic animal, can be used. . Such transgenic animals also include, for example, animals in which the activity of a marker protein has been enhanced or suppressed by introducing a mutation or mutations in the coding region of the gene, or the amino acid sequence has been modified to become resistant or susceptible to hydrolysis. Mutations in an amino acid sequence include

ES 2 425 387 T3 substitutions, deletions, insertions and additions.

Furthermore, the expression of a marker gene itself can be controlled by introducing a mutation or mutations in the transcriptional regulatory region of the gene. Those skilled in the art understand such amino acid substitutions. Furthermore, the number of amino acids that are mutated is not particularly restricted, as long as the activity is maintained. Typically, it is within 50 amino acids, in certain non-limiting embodiments, within 30 amino acids, within 10 amino acids, or within 3 amino acids. The mutation site can be anywhere, as long as the activity continues.

In yet another aspect, screening methods for candidate compounds for therapeutic agents for treating colon cancer-related disease are provided herein. One or more marker genes are selected from the group of genes described herein. A therapeutic agent for a colon cancer-related disease can be obtained by selecting a compound capable of increasing or decreasing the level of expression of the marker gene (s).

It is to be understood that the term "a compound that increases the expression level of a gene" refers to a compound that promotes any one of the steps of gene transcription, gene translation or expression of a protein activity. On the other hand, the term "a compound that reduces the expression level of a gene", as used herein, refers to a compound that inhibits any one of these steps.

In particular aspects, the method of screening for a therapeutic agent for a colon cancer-related disease can be carried out in vivo or in vitro. This screening procedure can be performed, for example, (1) administering a candidate compound to an animal subject; (2) measuring the level of expression of a marker gene or genes in a biological sample from the animal subject; or (3) selecting a compound that increases or decreases the level of expression of a marker gene (s) compared to that of a control with which the candidate compound has not been contacted.

In yet another aspect, provided herein is a method for evaluating the efficacy of a candidate compound for a pharmaceutical agent at the level of expression of a marker gene or genes by contacting an animal subject with the candidate compound and monitoring the effect. of the compound at the expression level of the marker gene (s) in a biological sample derived from the animal subject. Variation in the level of expression of the marker gene (s) in a biological sample derived from the animal subject can be monitored using the same technique as used in the assay procedure described above. Furthermore, based on the evaluation, a candidate compound for a pharmaceutical agent can be selected by screening.

The procedures and reagents described herein are representative of preferred embodiments, are exemplary, and are not intended to be limitations on the scope of the invention. Those skilled in the art will find modifications of these and other uses. It will also be readily apparent to one skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

It should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may be resorted to by those skilled in the art, and that such modifications and variations are considered within the scope of the present invention as defined by the appended claims.

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ES 2 425 387 T3

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