Gene expression profiling in biopsied tumor tissues

p00001Obtaining gene expression profile in biopsied tumor tissues

5 Field of the Invention

p00002The present invention relates to obtaining the gene expression profile in biopsied tumor tissues. In particular, the present invention relates to sensitive methods for measuring mRNA levels in biopsied tumor tissues, including biopsy material embedded in archived paraffin. In addition, the invention provides a set of genes whose expression is important in the diagnosis and treatment of breast cancer.

p00003Oncologists have several treatment options, including different combinations of chemotherapeutic drugs that are characterized as “reference treatment,” and several drugs that do not carry a stated on the label for a particular cancer, but for which there is evidence of efficacy in that cancer The best

p00004fifteen Probability of a good outcome of treatment requires that patients be assigned to the optimal available cancer treatment, and that this assignment be made as quickly as possible after diagnosis.

p00005Currently, the diagnostic tests used in clinical practice are single analyte, and therefore do not capture the potential value of knowing the relationships between dozens of different markers. In addition, diagnostic tests are often non-quantitative, based on immunohistochemistry. This method often produces different results in different laboratories, partly because the reagents are not standardized, and partly because the interpretations are subjective and cannot easily be quantified. RNA-based tests have often not been used due to the problem of RNA degradation over time and the fact that it is difficult to obtain recent tissue samples from patients for analysis. Fabric is more readily available

p0000625 embedded in fixed paraffin and methods have been established to detect RNA in fixed tissue. However, these methods usually do not allow the study of large numbers of genes (DNA or RNA) from small amounts of material. Therefore, rarely fixed tissue has been used traditionally for other things than for immunohistochemical protein detection.

p00007Recently, several groups have published studies concerning the classification of various types of cancer by analyzing gene expression with microalignments (see, for example Golub et al., Science 286: 531-537 (1999); Bhattacharjae et al., Proc Natl. Acad Sci. USA 98: 13790-13795 (2001); Chen-Hsiang et al., Bioinformatics 17 (Suppl. 1): S316-S322 (2001); Ramaswamy et al., Proc. Natl. Acad. Sci USA 98: 15149-15154 (2001)). Certain classifications of human breast cancers have also been reported based on the

p0000835 Gene expression patterns (Martin et al., Cancer Res. 60: 2232-2238 (2000); West et al., Proc. Natl. Acad Sci. USA 98: 11462-11467 (2001); Sorlie et al., Proc Natl. Acad Sci. USA 98: 10869-10874 (2001); Yan et al., Cancer Res. 61: 8375-8380 (2001)). However, these studies focus mostly on improving and refining the already established classification of various types of cancer, including breast cancer, and generally do not provide new insights into the relationships of differentially expressed genes, and do not link the findings to treatment strategies in order to improve the clinical outcome of cancer therapy.

p00009Although modern biochemistry and molecular biology have revealed more than 100 genes whose activities influence the behavior of tumor cells, the state of their differentiation and their sensitivity or resistance to certain therapeutic drugs, with a few exceptions, the state of these genes is not has taken advantage of for the purpose of

p00010Four. Five make routine clinical decisions about pharmacological treatments. A notable exception is the use of estrogen receptor (RE) protein expression in breast carcinomas to select patients treated with antiestrogen drugs, such as tamoxifen. Another exceptional example is the use of ErbB2 (Her2) protein expression in breast carcinomas to select patients with the Her2 Herceptin® antagonist drug (Genentech, Inc., South San Francisco, CA).

p00011Despite recent advances, the challenge of cancer treatment remains to direct specific treatment regimens to pathogenically different types of tumors, and ultimately customize tumor treatment in order to maximize the outcome. Therefore, there is a need for tests that simultaneously provide predictive information about patient responses to the variety of

p0001255 treatment options This is particularly true for breast cancer, whose biology is misunderstood. It is clear that the classification of breast cancer into a few subgroups, such as the ErbB2 + subgroup and subgroups characterized by low to absent estrogen receptor gene expression (ER) and a few additional transcription factors (Perou et al., Nature 406: 747-752 (2000)) is not reflected in the cellular and molecular heterogeneity of breast cancer, and does not allow the design of treatment strategies that maximize patient response

p00013WO 01/04343 A2 (BURNHAM INST [USA]; REED JOHN C [USA] cites a method of predicting the probability of long-term survival of breast cancer without recurrence based on the determination of mRNA expression level of BAG1 (see claim 6) but the document does not provide any examples

p0001465 practical of such a method or any experimental evidence of correlation between said mRNA levels and the prognosis of breast cancer.

p00016TOWNSEND ET AL (JOURNAL OF PATHOLOGY, vol. 17, no. 1, pp. 51-59, 2002) discloses that there is no correlation between the levels of BAG1 protein expression and RNA expression.

5 Summary of the invention

p00017According to one aspect of the present invention, there is provided a method of predicting the probability of long-term survival of a patient with breast cancer without recurrence of breast cancer, after surgical removal of the primary tumor as specified in the claim. 1.

p0001810 The present invention adapts the use of archived paraffin embedded biopsy material for testing all markers in the assembly, and is therefore compatible with the most widely available type of biopsy material.

p00019fifteen In a particular embodiment, the level of expression of one or more prognostic RNA transcripts is determined, in which RNA can be obtained, for example, from a sample of breast cancer tissue embedded in wax, fixed from the patient. RNA isolation can be carried out, for example, by following any of the procedures described above or throughout the application, or by any other method known in the art.

p00020twenty The level of BAG1 RNA transcript can be quantified by RT-PCR.

p00021The expression level of the BAG1 RNA transcript, or its expression product, can be quantified by alignment. 25

Brief description of the drawings

p00022Figure 1 is a graph illustrating the workflow of the process of the invention for measuring gene expression. In the figure, FPET means "tissue embedded in fixed paraffin" and "RT-PCR" means "PCR with 30 reverse transcriptase". The RNA concentration is determined using the commercial RiboGreen ™ RNA quantification reagent and protocol.

p00023Figure 2 is a flow chart showing the steps of an RNA extraction method according to the invention together with a flow chart of a representative commercial method.

p0002435 Figure 3 is a schematic illustrating the steps of an improved method for preparing fragmented mRNA for the analysis of obtaining the expression profile.

p00025Figure 4 illustrates methods for RNA amplification before RT-PCR. 40 Figure 5 illustrates an alternative scheme for the repair and amplification of fragmented mRNA.

p00026Figure 6 shows the measurement of estrogen receptor mRNA levels in 40 FPE breast cancer samples by RT-PCR. Three 10 micrometer sections were used for each measurement. Each point of 45 data represents the average of triplicate measurements.

p00027Figure 7 shows the results of the measurement of progesterone receptor mRNA levels in 40 FPE breast cancer samples by RT-PCR performed as described in the legend of Figure 6 above.

p00028fifty Figure 8 shows results of an IVT / RT-PCR experiment.

p00029Figure 9 is a representation of the expression of 92 genes across 70 FPE breast cancer samples. The y axis shows the expression as threshold cycle times. These genes are a subset of the 55 genes listed in Table 1.

p00030Table 1 shows a list of breast cancer genes.

p00031Table 2 shows the sequences of primers and amplicons used for amplification of fragmented mRNA. 60 Table 3 shows the registration numbers and SEQ ID NO of the breast cancer genes examined.

p00032Detailed description of the preferred embodiment

p0003365 A. Definitions

p00035Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention pertains. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons

p000365 (New York, NY 1992), provide the person skilled in the art with a general guide for many of the terms used in the present application.

p00037One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. In fact, the present invention is not limited in any way to the methods and materials described. For the purposes of the present invention, the following terms are defined below.

p00038The term "microalignment" refers to an ordered arrangement of hybridizable alignment elements, preferably polynucleotide probes, on a substrate.

p00039fifteen The term "polynucleotide", when used in the singular or plural, generally refers to any polyiribonucleotide or polydeoxyribonucleotide, which may be unmodified DNA or RNA or modified DNA or RNA. Thus, for example, polynucleotides as defined herein include, without limitation, single and double stranded DNA, DNA that includes single and double stranded regions, single and double stranded RNA, and RNA that includes single and double stranded regions, hybrid molecules which comprise DNA and RNA that can be single stranded or, more usually, double stranded or include single and double stranded regions. In addition, the term "polynucleotide" as used herein refers to tricatenary regions that comprise RNA.

p00040or DNA or both RNA and DNA. The chains in such regions may be of the same molecule or of different molecules. Regions can include all of one or more of the molecules, but more usually they involve only

p0004125 a region of some of the molecules. One of the molecules of a triple helix region is often an oligonucleotide. The term "polynucleotide" specifically includes DNA and RNA that contain one or more modified bases. Therefore, DNA or RNA with major structures modified to achieve stability or for other reasons are "polynucleotides" as the term is provided herein. In addition, DNA or RNA comprising uncommon bases, such as inosine, or modified bases, such as tritiated bases, are included within the term "polynucleotides" as defined herein. In general, the term "polynucleotide" encompasses all chemically, enzymatically and / or metabolically modified forms of unmodified polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including single and complex cells.

p0004235 The term "oligonucleotide" refers to a relatively short polynucleotide, including, without limitation, single stranded deoxyribonucleotides, single or double stranded ribonucleotides, RNA hybrids: double stranded DNA and DNA. Oligonucleotides, such as single stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example using automated oligonucleotide synthesizers that are commercially available. However, oligonucleotides can be prepared by a variety of other methods, including recombinant DNA mediated techniques in vitro and by expression of DNA in cells and organisms.

p00043The terms "differentially expressed gene", "differential gene expression" and its synonyms, which are used interchangeably, refer to a gene whose expression is activated at a higher or lower level in a subject 45 suffering from a disease, specifically cancer, such as breast cancer, with respect to its expression in a normal or control subject. Expressions also include genes whose expression is activated at a higher or lower level at different stages of the same disease. It is also understood that a differentially expressed gene can either be activated or inhibited at the level of nucleic acid or protein level, or it can be subjected to alternative splicing to result in a different polypeptide product. Such differences can be evidenced by a change in mRNA levels, surface expression, secretion or other distribution of a polypeptide, for example. Differential gene expression may include a comparison of the expression between two or more genes, or a comparison of the reasons for the expression between two or more genes, or even a comparison of two products processed differently from the same gene, which differ between normal subjects and subjects suffering from a disease, specifically cancer, or between different stages of the same disease. The expression

p0004455 Differential includes both quantitative and qualitative differences in the pattern of cellular or temporal expression in a gene or its expression products between, for example, diseased and normal cells, or between cells that have experienced different disease events or stages of disease . For the purpose of this invention, "differential gene expression" is considered to be present when there is a difference of at least about twice, preferably at least about four times, more preferably at least about six times, most preferably at least about ten times between the expression of a given gene in normal and diseased subjects, or at various stages of disease development in a diseased subject.

p00045The term "gene amplification" refers to a procedure by which multiple copies of a

p0004665 gene or gene fragment in a particular cell line or cell. The duplicated region (a stretch of amplified DNA) is often called an "amplicon." Usually, the amount of messenger RNA (mRNA) produced, that is, the

p00048Gene expression level, also increases in the proportion of the number of copies produced of the particular expressed gene.

p00049The term "forecast" is used herein to refer to the prediction of the probability of

p000505 progression or death attributable to cancer, including recurrence, metastatic spread and drug resistance, of a neoplastic disease, such as breast cancer. The term "prediction" is used herein to refer to the probability that a patient responds either favorably or unfavorably to a drug or set of drugs, and also the extent of these responses. The predictive methods of the present invention can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular patient. The predictive methods of the present invention are valuable tools in prediction if a patient is likely to respond favorably to a treatment regimen, such as surgical intervention, chemotherapy with a given drug or combination of drugs, and / or radiotherapy.

p00051fifteen The term "increased resistance" to a particular drug or treatment option, when used according to the present invention, means reduction of the response with respect to a conventional dose of the drug or to a conventional treatment protocol.

p00052The term "sensitivity reduction" to a particular drug or treatment option, when used according to the present invention, means reduction of the response with respect to a conventional dose of the drug or to a conventional treatment protocol, when the reduced response may compensated (at least partially) by increasing the dose of the drug or the intensity of treatment.

p00053The "patient response" can be evaluated using any evaluation criteria that indicate a benefit to

p0005425 patient, including, without limitation, (1) inhibition, to some extent, of tumor growth, including slowing down and complete growth arrest; (2) reduction of the number of tumor cells; (3) reduction of tumor size; (4) inhibition (ie, reduction, slowdown or complete arrest) of infiltration of tumor cells in adjacent tissues and / or peripheral organs; (5) inhibition (ie reduction, slowdown or complete arrest) of metastasis; (6) potentiation of the antitumor immune response, which may, but does not have to, result in regression or rejection of the tumor; (7) relief, to some degree, of one or more symptoms associated with the tumor; (8) increase in survival duration after treatment; and / or (9) reduction of mortality at a given point of time after treatment.

p00055The term "treatment" refers to both therapeutic and prophylactic treatment or preventive measures, in which

p0005635 The object is to prevent or slow down (reduce) the disorder or pathological state selected as the target. Those who need treatment include those who already have the disorder as well as those likely to have the disorder or those in whom the disorder will be prevented. In the treatment of the tumor (for example, cancer), a therapeutic agent can directly reduce the pathology of tumor cells, or make the tumor cells more susceptible to treatment by other therapeutic agents, for example, radiation and / or chemotherapy.

p00057The term "tumor", as used herein, refers to all proliferation and growth of neoplastic cells, whether malignant or benign, and all cancerous and precancerous tissues and cells.

p00058The terms "cancer" and "cancerous (a)" refer to or describe the physiological state in mammals that are

p00059Four. Five Typically characterized by unregulated cell growth. Examples of cancer include but are not limited to breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer. , urinary tract cancer, thyroid cancer, kidney cancer, carcinoma, melanoma and brain cancer.

p00060The "pathology" of cancer includes all phenomena that compromise the patient's well-being. This includes, without limitation, abnormal or uncontrolled cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of the inflammatory or immune response, neoplasia, premalignity, malignancy, invasion of distant or surrounding organs or tissues, such as lymph nodes, etc.

p0006155 The "stringency" of the hybridization reactions can easily be determined by a person skilled in the art, and is generally an empirical calculation dependent on the length of the probe, washing temperature and salt concentration. In general, longer probes require higher temperatures for proper mating, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA to mate again when complementary strands are present in an environment below their melting temperature. The higher the degree of homology desired between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures would make them less stringent. For additional details and explanation

p0006265 for the rigor of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology. Wiley Interscience Publishers, (1995).

p00064"Rigorous conditions" or "high stringency conditions", as defined herein, typically: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride / sodium citrate 0, 0015 M / 0.1% sodium dodecyl sulfate at 50 ° C; (2) use a denaturing agent during hybridization, such as formamide, for example, 50% formamide (v / v) in 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinyl pyrrolidone, 1% / 50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 ° C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt solution, sonic salmon sperm DNA (50 μg / ml), 0.1% SDS and 10% dextran sulfate at 42 ° C, with washes at 42 ° C in 0.2 x SSC

p0006510 (sodium chloride / sodium citrate) and 50% formamide at 55 ° C, followed by a high stringency wash consisting of 0.1 x SSC containing EDTA at 55 ° C.

p00066"Moderately stringent conditions" can be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of wash solution and hybridization conditions (for example , temperature, ionic strength and% SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37 ° C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6 ), 5 x Denhardt solution, 10% dextran sulfate and denatured fragmented salmon sperm DNA 20 mg / ml, followed by washing the filters in 1 x SSC at approximately 37

p00067twenty 50 ° C The expert will recognize how to adjust the temperature, ionic strength, etc. as necessary to adapt factors such as probe length and the like.

p00068In the context of the present invention, the reference to "at least one," "at least two," "at least five," etc. of the genes listed in any particular gene set means any one or any and all 25 combinations of the genes listed.

p00069The terms "splicing" and "RNA splicing" are used interchangeably and refer to the processing of RNA that removes introns and binds exons to produce mature mRNA with continuous coding sequence that is transferred to the cytoplasm of a eukaryotic cell .

p0007030 In theory, the term "exon" refers to any segment of an interrupted gene that is represented in the mature RNA product (B. Lewin. Genes IV Cell Press, Cambridge Mass. 1990). In theory, the term "intron" refers to any segment of DNA that is transcribed but removed from the transcript by cutting and splicing together the exons on either side of it. Operationally, exon sequences occur in the sequence of

p0007135 MRNA of a gene as defined by the numbers of Ref. Seq ID. Operationally, intron sequences are the intermediate sequences within the genomic DNA of a gene, in between the exon sequences and which have consensus splicing sequences GT and AG at their 5 'and 3' ends.

p00072B. Detailed Description

p0007340 The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology and biochemistry, which are within the skill of the art. Such techniques are fully explained in the literature, such as, "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait,

p00074Four. Five ed., 1984); "Animal Cell Culture" (RI Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology", 4th edition (DM Weir & CC Blackwell, eds., Blackwell Science Inc., 1987); "Gene Transfer Vectors for Mammalian Cells" (JM Miller & MP Calos, eds., 1987); "Current Protocols in Molecular Biology" (FM Ausubel et al., Eds., 1987); and "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994).

p000751. Obtaining the gene expression profile

p00076In general, the methods of obtaining the gene expression profile can be divided into two large groups: methods based on polynucleotide hybridization analysis and methods based on the sequencing of polynucleotides. Most commonly used methods known in the art for the quantification of mRNA expression in a sample include Northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106: 247-283 (1999)); RNAse protection assays (Hod, Biotechniques 13: 852854 (1992)); and polymerase chain reaction with reverse transcription (RT-PCR) (Weis et al., Trends in Genetics 8: 263-264 (1992)). Alternatively, antibodies that can recognize duplex can be used

p0007760 specific, including DNA duplexes, RNA duplexes and hybrid DNA-RNA duplexes or protein-DNA duplexes. Representative methods for the analysis of gene expression based on sequencing include serial analysis of gene expression (SAGE), and analysis of gene expression by parallel mass signature sequencing (MPSS).

p0007865 2. PCR with reverse transcriptase (RT-PCR)

p00080Of the techniques listed above, the most sensitive and most flexible quantitative method is RT-PCR, which can be used to compare mRNA levels in different sample populations, in normal and tumor tissues, with or without drug treatment, to characterize patterns of gene expression, to discriminate between closely related mRNAs and analyze the structure of the RNA.

p000815 The first stage is mRNA isolation from a target sample. The starting material is normally total RNA isolated from human tumor cell lines or tumors, and the corresponding normal cell lines or tissues, respectively. Therefore, RNA can be isolated from a variety of primary tumors, including tumor lines and cells or breast, lung, colon, prostate, brain, liver, kidney, pancreas, spleen, thymus, testicles, ovaries, uterus, etc. ., with combined DNA from healthy donors. If the source of mRNA is a primary tumor, mRNA can be extracted, for example, from samples of frozen tissue or embedded in archived and fixed paraffin (for example fixed with formalin).

p00082General methods for mRNA extraction are well known in the art and are disclosed in books of

p00083fifteen Conventional text of molecular biology, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). Methods for the extraction of RNA from tissues embedded in paraffin are disclosed, for example, in Rupp and Locker, Lab Invest. 56: A67 (1987), and De Andrés et al., BioTechniques 18: 42044 (1995). In particular, RNA isolation can be performed using a purification kit, buffer assembly and protease from commercial manufacturers, such as Qiagen, according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using RNeasy mini-columns from Qiagen. Other commercially available RNA isolation kits include the MasterPure ™ complete RNA and DNA purification kit (EPICENTRE®, Madison, WI), and the paraffin block RNA isolation kit (Ambion, Inc.). Total RNA can be isolated from tissue samples using Stat-60 RNA (Tel-Test). RNA prepared from the tumor can be isolated, for example, by gradient centrifugation of cesium chloride density.

p0008425 Since RNA cannot serve as a template for PCR, the first stage in obtaining the gene expression profile by RT-PCR is the reverse transcription of the RNA template to give cDNA, followed by its exponential amplification in a PCR reaction. The two most commonly used reverse transcriptases are the reverse transcriptase of the avian myeloblastosis virus (VMA-RT) and the reverse transcriptase of the Moloney murine leukemia virus (VLMM-RT). The reverse transcription step is usually primed using specific primers, random hexamers, or oligo-dT primers, depending on the circumstances and the objective of obtaining the expression profile. For example, the extracted RNA can be transcribed in reverse using a GeneAmp RNA PCR kit (Perkin Elmer, CA, USA), following the manufacturer's instructions. The derived cDNA can then be used as a template in the subsequent PCR reaction.

p0008535 Although the PCR step can use a variety of thermostable DNA-dependent DNA polymerases, Taq DNA polymerase is normally employed, which has a 5'-3 'nuclease activity but lacks a 3'-5' endonuclease activity for proofreading. . Thus, TaqMan® PCR normally uses the 5 'nuclease activity of Tth or Taq polymerase to hydrolyse a hybridization probe bound to its target amplicon, but any enzyme with equivalent 5' nuclease activity can be used. Two oligonucleotide primers are used to generate a typical amplicon of a PCR reaction. A third oligonucleotide, or probe, is designed to detect the nucleotide sequence located between the two PCR primers. The probe cannot be extended by the Taq DNA polymerase enzyme, and is labeled with an indicator fluorescent dye and a quencher fluorescent dye. Any laser induced emission of the indicator dye is extinguished by the extinction dye when the two dyes are located

p00086Four. Five close to each other since they are in the probe. During the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a mold dependent manner. The resulting probe fragments dissociate in solution, and the signal of the released indicator dye is free from the extinction effect of the second fluorophore. One indicator dye molecule is released for each new synthesized molecule, and detection of the non-extinguished indicator dye provides the basis for quantitative interpretation of the data.

p00087TaqMan® RT-PCR can be performed using commercially available equipment, such as, for example, ABI PRISM 7700 ™ Sequence Detection System ™ (Perkin-Elmer-Applied Biosystems, Foster City, CA, USA), or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany). In a preferred embodiment, the 5 'nuclease process is carried out in a real-time quantitative PCR device such as ABI PRISM 7700 ™ Sequence

p0008855 Detection System ™. The system consists of a thermal cycler, a laser, a coupled charging device (CCD), a camera and a computer. The system amplifies samples in a 96-well format in a thermal cycler. During amplification, the laser-induced fluorescent signal is collected in real time through fiber optic cables for all 96 wells, and is detected in the CCD. The system includes software to operate the instrument and to analyze the data.

p00089The 5 'nuclease assay data is initially expressed as Ct, or the threshold cycle. As discussed above, fluorescence values ​​are recorded during each cycle and represent the amount of product amplified at that point in the amplification reaction. The point at which the fluorescent signal is first recorded as statistically significant is the threshold cycle (Ct).

p0009065 To minimize errors and the effect of sample-to-sample variation, RT-PCR is usually performed using

p00092An internal pattern The ideal internal pattern is expressed at a constant level between different tissues, and is not affected by experimental treatment. The most frequently used RNAs to normalize gene expression patterns are mRNAs for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin maintenance genes.

p000935 A more recent variation of the RT-PCR technique is real-time quantitative PCR, which measures the accumulation of PCR product through a double-labeled fluorogenic probe (i.e., TaqMan® probe). Real-time PCR is compatible with both quantitative competitive PCR, in which an internal competitor is used for each target sequence for normalization, and with quantitative comparative PCR using a normalization gene contained within the sample, or a gene of maintenance for RT-PCR. For additional details see, for example Held et al., Genome Research 6: 986-994 (1996).

p000943. Microalignments

p00095Differential gene expression can also be identified, or confirmed, using the microalignment technique.

p00096fifteen Therefore, the expression profile of genes associated with breast cancer in tumor tissue or embedded in paraffin or recent, can be measured using micro-alignment technology. In this method, the polynucleotide sequences of interest are plated, or aligned, on a microchip substrate. The sequences aligned with DNA probes specific to cells or tissues of interest are then hybridized. Just like in the RT-PCR method, the source of mRNA is usually total RNA isolated from human tumors or tumor cell lines, and the corresponding normal cell lines or tissues. Therefore, RNA can be isolated from a variety of primary tumors or tumor cell lines. If the source of mRNA is a primary tumor, mRNA can be extracted, for example, from frozen or embedded tissue samples in archived and fixed paraffin (for example fixed with formalin), which are routinely prepared and practically preserved. daily clinic

p0009725 In a specific embodiment of the micro-alignment technique, PCR amplified inserts of cDNA clones are applied to a substrate in a dense alignment. Preferably, at least 10,000 nucleotide sequences are applied to the substrate. The microaligned genes, immobilized in the microchip at 10,000 elements each, are suitable for hybridization under stringent conditions. Fluorescently labeled cDNA probes can be generated can be generated through the incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest. The labeled cDNA probes applied to the chip hybridize with specificity to each point of DNA in the alignment. After thorough washing to remove probes not specifically bound, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. The quantification of the hybridization of each aligned element allows the evaluation of the corresponding abundance of mRNA. With dual color fluorescence, they hybridize in pairs

p0009835 Separately labeled cDNA probes generated from two sources of RNA in alignment. Therefore, the relative abundance of transcripts is determined simultaneously from the two sources corresponding to each specified gene. The miniaturized scale of hybridization allows a rapid and convenient evaluation of the expression pattern for large numbers of genes. It has been shown that such methods have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect differences of at least about twice in expression levels (Schena et al., Proc Natl. Acad. Sci. USA 93 (2): 106-149 (1996)). Microalignment analysis can be performed using commercially available equipment, following the manufacturer's protocols, such as using Affymetrix GenChip technology, or Incyte microalignment technology.

p00099Four. Five The development of microalignment methods for large-scale analysis of gene expression makes it possible to systematically search for molecular markers of cancer classification and outcome prediction in a variety of tumor types.

p00100Four. Serial analysis of gene expression (SAGE)

p00101Serial gene expression analysis (SAGE) is a method that allows the quantitative and simultaneous analysis of a large number of gene transcripts, without the need to provide an individual hybridization probe for each transcript. First, a short sequence tag (approximately 10-14 bp) is generated that contains enough information to uniquely identify a transcript, provided the marker is obtained at

p0010255 from a unique position within each transcript. Then, many transcripts join together to form long series molecules, which can be sequenced, revealing the identity of the multiple tags simultaneously. The expression pattern of any population of transcripts can be quantitatively assessed by determining the abundance of individual markers and identifying the gene corresponding to each label. For more details see, for example Velculescu et al., Science 270: 484-487 (1995); and Velculescu et al., Cell 88: 243-51 (1997).

p001035. Analysis of gene expression by parallel sequencing of massive signatures (MPSS)

p00104This method, described by Brenner et al., Nature Biotechnology 18: 630-634 (2000), is a sequencing approach

p0010565 which combines non-gel signature sequencing with in vitro cloning of millions of molds in separate 5 µm diameter microbeads. First, a DNA mold microbead library is built

p00107by in vitro cloning. This is followed by the assembly of a flat alignment of the microbeads containing molds in a flow cell at a high density (normally greater than 3x106 microbeads / cm2). The free ends of the cloned molds in each microbead are analyzed simultaneously, using a fluorescence-based signature sequencing method that does not require separation of DNA fragments. It has been shown

p001085 This method provides simultaneously and accurately, in a single operation, hundreds of thousands of gene signature sequences from a yeast cDNA library.

p001096. General description of the methods of the invention of mRNA isolation, purification and amplification

p00110The steps of a representative protocol of the invention are illustrated in Figure 1, including isolation, purification, primer extension and mRNA amplification. As shown in Figure 1, this representative procedure begins with the cutting of approximately 10 μm thick sections of tumor tissue samples embedded in paraffin. The RNA is then extracted, and the proteins and DNA are removed, following the method of the invention described below. After the analysis of the RNA concentration,

p00111fifteen RNA repair and / or amplification steps may be included, if necessary, and the RNA is reverse transcribed using gene specific promoters followed by RT-PCR. Finally, the data is analyzed to identify the best treatment option (s) available to the patient based on the characteristic gene expression pattern identified in the tumor sample examined. The individual stages of this protocol will be discussed in more detail below.

p001127. Improved method for nucleic acid isolation from archived tissue samples

p00113As discussed above, in the first stage of the method of the invention, total RNA is extracted from the source material of interest, including samples of paraffin embedded tissue, fixed, and sufficiently purified to

p0011425 act as a substrate in an enzymatic assay. Despite the availability of commercial products, and the extensive knowledge available regarding nucleic acid isolation, such as RNA, from tissues, nucleic acid isolation (RNA) from samples of paraffin embedded tissue, fixed (FPET) is not free of difficulty.

p00115In one aspect, the present invention relates to an improved method for the isolation of nucleic acid from samples of archived tissue, for example FPET. Measured levels of mRNA species are useful for defining the physiological or pathological state of cells and tissues. RT-PCR (discussed above) is one of the most sensitive, reproducible and quantitative methods for this "obtaining gene expression profile". Formalin fixed tissue, embedded in paraffin is the most widely available material for such studies. Various

p0011635 Laboratories have shown that it is possible to successfully use fixed paraffin embedded tissue (FPET) as a source of RNA for RT-PCR (Stanta et al., Biotechniques 11: 304-308 (1991); Stanta et al., Methods Mol. Biol 86: 23-26 (1998); Jackson et al., Lancet 1: 1391 (1989); Jackson et al., J. Clin. Pathol. 43: 499-504 (1999); Finke et al., Biotechniques 14: 448-453 (1993); Goldsworthy et al., Mol. Carcinog. 25: 86-91 (1999); Stanta and Bonin, Biotechniques 24: 271-276 (1998); Godfrey et al., J. Mol. Diagnostics 2:84 (2000); Specht et al., J. Mod. Med 78: B27 (4000); Specht et al., Am. J. Pathol. 158: 419-429 (2001)). This allows the obtaining of the gene expression profile to be carried out in the most commonly available source of human biopsy samples, and therefore that new therapeutic and valuable diagnostic information is potentially created.

p00117The most widely used protocols use hazardous organic solvents, such as xylene, or octane

p00118Four. Five (Finke et al., Cited above) to remove tissue wax in paraffin blocks before nucleic acid extraction (RNA and / or DNA). They follow the elimination of the mandatory organic solvent (for example with ethanol) and rehydration stages, which need multiple manipulations, and addition of the total total time to the protocol, which can take several days. Commercial protocols and kits for RNA extraction from FPET [MasterPure ™ complete RNA and DNA purification kit (EPICENTRE®, Madison, WI); paraffin block RNA isolation kit (Ambion, Inc.) and RNeasy ™ Mini kit (Qiagen, Chatsworth, CA)] use xylene for dewaxing, in procedures that normally require multiple centrifuges and ethanol buffer changes, and incubations after incubation with xylene.

p00119The present invention provides an improved nucleic acid extraction protocol that produces acid

p0012055 nucleic, in particular RNA, sufficiently intact for gene expression measurements. The key step in the nucleic acid extraction protocol herein is the realization of the removal of the wax without the use of any organic solvent, thus eliminating the need for multiple manipulations associated with the removal of the organic solvent, and substantially reducing Total time in the protocol. According to the invention, the wax, for example paraffin, is removed from the samples of tissue embedded in wax by incubation at 65-75 ° C in a lysis buffer that solubilizes the tissue and hydrolyses the protein, after cooling to solidify the wax.

p00121Figure 2 shows a flow chart of an RNA extraction protocol of the present invention compared to a representative commercial method, using xylene to remove the wax. The times required for the individual stages in the procedures and for the procedures are shown in the diagram

p0012265 Global As shown, the commercial process requires approximately 50% more time than the process of the invention.

p00124The lysis buffer can be any known buffer for cell lysis. However, it is preferred that oligo-dT-based methods of selective purification of polyadenylated mRNA are not used to isolate the RNA for the present invention, since it is expected that the mass of the mRNA molecules will be fragmented and therefore will not have a

p001255 intact polyadenylated tail, and will not be recovered or will be available for subsequent analytical testing. Otherwise, any number of conventional nucleic acid purification schemes can be used. These include extractions with organic solvent and chaotrope, extraction using glass beads or filters, methods based on precipitation and precipitation with salts, or any of the purification methods known in the art to recover total RNA or total nucleic acid from a biological source

p00126Lysis buffers are commercially available, such as, for example, from Qiagen, Epicenter or Ambion. A preferred group of lysis buffers usually contains urea, and proteinase K or other protease. Proteinase K is very useful in the isolation of high-quality, undamaged DNA or RNA, since most mammalian DNases and RNases are rapidly inactivated by this enzyme, especially in the presence of dodecyl sulfate.

p00127fifteen 0.5-1% sodium (SDS). This is particularly important in the case of RNA, which is more susceptible to degradation than DNA. While DNases require metal ions for activity, and therefore can be easily inactivated by chelating agents, such as EDTA, there is no similar cofactor requirement for RNases.

p00128The cooling and solidification resulting from the wax allows an easy separation of the wax from the total nucleic acid, which can be conveniently precipitated, for example by isopropanol. The additional processing depends on the intended purpose. If the proposed method of RNA analysis is subjected to a bias by contaminating DNA in an extract, the RNA extract can be further treated, for example by DNase, after purification to specifically remove the DNA while the RNA is conserved. For example, if the goal is to isolate RNA from

p0012925 high quality for subsequent amplification by RT-PCR, the precipitation of the nucleic acid is followed by the elimination of DNA, usually by treatment with DNase. However, DNA can be removed at various stages of nucleic acid isolation, by DNase or other techniques well known in the art.

p00130Although the advantages of the nucleic acid extraction protocol of the invention are most evident for the isolation of RNA from samples of paraffin embedded tissue, archiving, the wax removal stage of the present invention, which does not imply the use of An organic solvent can also be included in any conventional protocol for the extraction of total nucleic acid (RNA and DNA) or DNA only. All these aspects are specifically within the scope of the invention.

p0013135 Using heat followed by cooling to remove paraffin; The process of the present invention saves valuable processing time, and eliminates a series of manipulations, thereby potentially increasing the yield of nucleic acid. In fact, the experimental tests presented in the examples below demonstrate that the method of the present invention does not compromise RNA performance.

p001328. Specific priming of multiplexed gene in 5 'of reverse transcription

p00133RT-PCR requires the reverse transcription of the test RNA population as the first stage. The most commonly used primer for reverse transcription is oligo-dT, which works well when the RNA is intact. However, this primer will not be effective when the RNA is highly fragmented as is the case in tissues.

Four. Five FPE

p00134The present invention includes the use of gene-specific primers, which are approximately 20 bases in length with an optimum Tm between approximately 58 ° C and 60 ° C. These primers will also serve as reverse primers that directs DNA amplification by PCR.

p00135Another aspect of the invention is the inclusion of multiple gene specific primers in the same reaction mixture. The number of such different primers can vary greatly and can be as much as a maximum.

p0013640,000 or more. Table 2 presents examples of reverse primers that can be used successfully to carry out the methods of the invention. Figure 9 shows the expression data obtained using this

p0013755 specific priming strategy of multiplexed gene. Specifically, Figure 9 is a representation of the expression of 92 genes (a subset of genes listed in Table 1) through 70 FPE breast cancer samples. The y axis shows the expression as threshold cycle times.

p00138An alternative approach is based on the use of random hexamers as primers for cDNA synthesis. However, it has been experimentally demonstrated that the method of using a multiplicity of gene specific primers is superior to the known approach using random hexamers.

p001399. Preparation of fragmented mRNA for expression profile assays

p0014065 It is of interest to analyze the abundance of specific mRNA species in biological samples, since this expression profile provides an index of the physiological state of that sample. MRNA is notoriously difficult to extract and

p00142keeping in its native state, consequently, the mRNA recovered from biological sources is often fragmented or somewhat degraded. This is especially true in human tissue samples that have been stored and chemically fixed for prolonged periods of time.

p001435 In one aspect, the present invention provides a means of preparing mRNA extracted from various sources, including samples of archived tissue, for obtaining the expression profile so that its relative abundance is preserved and the mRNAs of interest can be measured satisfactorily. This method is useful as a means of preparing mRNA for analysis by any of the known expression profile obtaining methods, including 5 'exonuclease-coupled RT-PCR of indicator probes (TaqMan® type assays), such

p0014410 as discussed above, flap endonuclease assays (Cleavase® and Invader® type assays), oligonucleotide hybridization alignments, cDNA hybridization alignments, oligonucleotide ligation assays, 3 'individual nucleotide extension assays and other assays designed to evaluate the abundance of specific mRNA sequences in a biological sample.

p00145fifteen According to the method of the invention, the total RNA is extracted from the source material and purified sufficiently to act as a substrate in an enzymatic assay. The extraction procedure has been discussed above, including a new and improved way of removing the wax (eg paraffin) used to embed tissue samples. It has also been indicated that it is preferred that oligo-dT-based methods of selective purification of polyadenylated mRNA are not used to isolate RNA for this invention since it is expected that the volume of the mRNA is

p00146twenty fragmented, it will not be polyadenylated and, therefore, will not be recovered and will be available for further analytical tests if an oligo-dT-based method is used.

p00147A diagram of an improved method to repair fragmented RNA is shown in Figure 3. The purified fragmented RNA is mixed from the tissue sample with DNA templates, single-stranded, gene specific or universal for each species of mRNA of interest. These templates can be full-length DNA copies of the mRNA derived from cloned gene sources, they can be fragments of the gene that represent only the segment of the gene to be tested, they can be a series of long oligonucleotides that represent or the full length gene or the specific segment (s) of interest (s). The template can represent either a unique consensus sequence or be a mixture of polymorphic variants of the gene. This DNA template, or framework, will preferably include one or more dUTP or rNTP sites in its length. This will provide a means of removing the mold before carrying out subsequent analytical steps to prevent it from acting as a substrate or target in these last analysis tests. This elimination is achieved by treating the sample with uracil-DNA glycosylase (UDG) and heating it to cause chain breaks where UDG has generated abbasic sites. In the case of rNTP, the sample can be heated in the presence of a basic buffer (pH ~ 10) to induce chain breaks

p0014835 where rNTP is located in the mold.

p00149The single-stranded DNA template is mixed with the purified RNA, the mixture is denatured and paired so that the RNA fragments complementary to the DNA template effectively become primers that can extend along the single-stranded DNA templates. DNA polymerase I requires a primer 40 for extension but either an RNA primer or a DNA primer will be used effectively. Thus, in the presence of DNA polymerase I and dNTP, the fragmented RNA can spread along the complementary DNA templates. In order to increase the effectiveness of the extension, this reaction can be thermally cycled, allowing the overlapping of the molds and the extension products to hybridize and spread until the global population of fragmented RNA is represented as double stranded DNA from of the

p00150Four. Five RNA fragment primers.

p00151After the generation of this "repaired" RNA, the sample must be treated with UDG or treated with heat in a slightly basified solution to fragment the DNA template (framework) and prevent it from participating in subsequent analytical reactions.

p00152fifty The product resulting from this enzymatic extension can then be used as a template in a conventional enzyme profile obtaining assay that includes amplification and generation of detectable signal such as fluorescent, chemiluminescent, colorimetric or other common reading from enzyme-based assays. For example, for TaqMan®-type assays, this double-stranded DNA product is added as a template in a conventional assay; and,

p0015355 For the hybridization of alignments, this product acts as a cDNA template for the cRNA labeling reaction normally used to generate labeled, single-stranded RNA for the hybridization of alignments.

p00154This method of preparing the mold has the advantage of recovering information from mRNA fragments too short to act effectively as molds in conventional cDNA generation schemes. In addition, this method works by preserving the specific locations in the mRNA sequences selected as the target by specific assay assays. For example, TaqMan® assays rely on a single contiguous sequence in a copy of mRNA cDNA to act as a PCR amplification template selected as a target by a labeled indicator probe. If mRNA chain breaks occur in this sequence, the assay will not detect that template and underestimate the amount of that mRNA in the original sample. East

p0015565 Target preparation method minimizes the effect of RNA fragmentation.

p00157The extension product formed in the RNA primer extension assay can be controlled by controlling the input amount of the single stranded DNA template and limiting the cyclization of the extension reaction. This is important in the conservation of the relative abundance of the mRNA sequences selected as the target for analysis.

p001585 This method has the added advantage of not requiring parallel preparation for each target sequence since it is easily multiplexed. It is also possible to use large combinations of random long sequence oligonucleotides or complete libraries of cloned sequences to extend the entire population of mRNA sequences in the sample extract for complete expressed genome analysis instead of specific gene analysis selected as target. .

p0015910. Amplification of mRNA species before RT-PCR

p00160Due to the limited quantity and poor quality of mRNA that can be isolated from FPET, a new

p00161fifteen A procedure that can accurately amplify mRNA of interest would be very useful, particularly for real-time quantification of gene expression (TaqMan®) and especially for a quantitatively large number (> 50) of genes> 50 to 10,000.

p00162Current protocols (for example Eberwine, Biotechniques 20: 584-91 (1996)) are optimized for mRNA amplification from a small amount of poly A + or total RNA primarily for microalignment analysis. The present invention provides an optimized protocol for the amplification of small amounts of fragmented total RNA (average size of approximately 60-150 bp), using gene-specific sequences as primers, as illustrated in Figure 4.

p0016325 The amplification process of the invention uses a very large number, usually nothing less than 100-

p00164190,000 gene specific primers (GSP) in a round of reverse transcription. Each GSP contains an RNA polymerase promoter, for example a T7 DNA-dependent RNA polymerase promoter, at the 5 'end for subsequent RNA amplification. GSPs are preferred as primers due to the small size of the RNA. Current protocols use dT primers, which would not adequately represent all reverse mRNA transcripts due to the small size of the FPET RNA. GSP can be designed by optimizing usual parameters, such as length, Tm, etc. For example, GSP can be designed using Primer Express® (Applied Biosystems), or the Primer 3 (MIT) software program. Normally at least 3 sets per gene are designed, and those that provide the lowest Ct in the FPET RNA (those that work best) are selected.

p0016535 Second-strand cDNA synthesis is performed by conventional procedures (see Figure 4, method 1), or by GSPf and Taq pol primers under PCR conditions (e.g., 95 ° C, 10 min. (Taq activation) then 60 ° C, 45 s). The advantages of the latter methods are that the second gene-specific primer, SGFf adds additional specificity (and potentially more efficient second-chain synthesis) and the option to perform several PCR cycles, if more starting DNA is necessary for amplification. of RNA by T7 RNA polymerase. RNA amplification is then performed under conventional conditions to generate multiple copies of cRNA, which is then used in a conventional TaqMan® reaction.

p00166Although this procedure is illustrated using T7-based RNA amplification, one skilled in the art will understand that other polymeric RNA promoters that do not require a primer, such as

p00167Four. Five T3 or Sp6, and are within the scope of the invention.

p00168eleven. Fragmented RNA elongation method and subsequent amplification

p00169This method, which combines and modifies the inventions described in sections 9 and 10 above, is illustrated in Figure 5. The procedure begins with the elongation of fragmented mRNA. This occurs as described above except that the framework DNAs are labeled with the T7 RNA polymerase promoter sequence at their 5 'ends, leading to extended double stranded DNA from RNA fragments. Mold sequences need to be removed after in vitro transcription. These templates may include dUTP or rNTP nucleotides, which allow enzymatic removal of the molds as described in section 9, or the templates may

p0017055 removed by treatment with DNase I.

p00171The template DNA can be a population that represents different mRNAs of any number. A source of highly complex sequence DNA templates (frameworks) can be generated by grouping RNA from a variety of cells or tissues. In one embodiment, these RNAs are converted to double stranded DNA and cloned into phagemids. The single-stranded DNA can then be rescued by growth of phagemids and isolation of single-stranded DNA from purified phagemids.

p00172This invention is useful because it increases the signals of the gene expression profile in two different ways: both by increasing the length of the polynucleotide sequence of the test mRNA and by amplification by in vitro transcription. An additional advantage is that it eliminates the need to perform reverse transcription optimization with gene-specific primers labeled with the RNA polymerase promoter sequence.

p00174of T7 and, therefore, is comparatively fast and economical.

p00175This invention can be used with a variety of different methods to obtain the gene expression profile, for example RT-PCR or a variety of DNA alignment methods. Just as in the previous protocol, this approach is illustrated using a T7 promoter but the invention is not limited in that way. One skilled in the art will appreciate, however, that other RNA polymerase promoters, such as T3 or Sp6, can also be used.

p0017612. Breast cancer gene set, gene subsets tested and clinical application of gene expression data

p00177An important aspect of the present invention is to use the measured expression of certain genes by breast cancer tissue to assign patients to the best drugs or drug combinations, and to provide prognostic information. For this purpose it is necessary to correct (normalize) both the differences in the amount of RNA tested and the variability in the quality of the RNA used. Therefore, the assay measures and incorporates the expression of certain normalization genes, including well-known maintenance genes, such as GAPDH and Cyp1. Alternatively, normalization can be based on the mean or median signal (Ct) of all genes tested or a large subset of them (global normalization approach). On a gene-to-gene basis, the measured normalized amount of a patient's tumor mRNA is compared with the amount found in a reference set of breast cancer tissue. The number (N) of breast cancer tissues in this reference set must be high enough to ensure that different reference sets (as a whole) behave in essentially the same way. If this condition is met, the identity of the individual breast cancer tissues present in a particular set will not have any significant impact on the relative amounts of the genes tested. Usually, the breast cancer tissue reference set consists of at least about 30, preferably at least about 40

p0017825 Different samples of FPE breast cancer tissue. Unless otherwise indicated, normalized expression levels for each mRNA / tumor tested / patient will be expressed as a percentage of the level of expression measured in the reference set. More specifically, the reference set of a sufficiently high number (for example 40) tumors produces a distribution of normalized levels of each mRNA species. The level measured in a particular tumor sample to be analyzed is in some percentile within this range, which can be determined by methods well known in the art. Then, unless otherwise indicated, the reference to expression levels of a gene assumes normalized expression in relation to the reference set although this is not always explicitly stated.

p00179The set of breast cancer genes is shown in Table 1. The registration numbers of the genes, and the SEQ

p0018035 NO ID for the direct primer, reverse primer and amplicon sequences that can be used for gene amplification, are listed in Table 2. The basis for the inclusion of markers, as well as the clinical significance of mRNA level variations with respect to to the reference set, indicated below. Genes are grouped into subsets based on the type of clinical significance indicated by their expression levels: A. Prediction of the patient's response to drugs used in the treatment of breast cancer, or to drugs that are approved for other indications and could be used in a manner not indicated in the treatment of breast cancer. B. Prognosis for cancer survival or recurrence.

p00181C. Prediction of the patient's response to therapeutic drugs

p00182Four. Five 1. Molecules that specifically influence cellular sensitivity to drugs

p00183Table 1 lists 74 genes (shown in italics) that specifically influence cellular sensitivity to potent drugs, which are also listed. Most of the drugs shown are approved and are already used to treat breast cancer (for example, anthracyclines; cyclophosphamide; methotrexate; 5-FU and the like). Several of the drugs are used to treat breast cancer in a non-indicated manner or are in the clinical development phase (for example, bisphosphonates and anti-VEGF AcM). Several of the drugs have not been widely used to treat breast cancer but are used in other cancers in which the indicated target is expressed (for example, Celebrex is used to treat familial colon cancer; cisplatin is used to treat ovarian cancer and others).

p0018455 The patient's response to 5FU is indicated if the amount of normalized thymidylate synthase mRNA is at or below the 15th percentile, or the sum of the expression of thymidylate synthase plus dihydropyrimidine phosphorylase is at or below the 25th percentile, or the sum of the expression of these mRNA plus thymidine phosphorylase is at or below the 20th percentile. Patients with dihydropyrimidine dehydrogenase below the 5th percentile are at risk of adverse response to 5FU, or analogues such as Xeloda.

p00185When the levels of thymidylate synthase and dihydropyrimidine dehydrogenase are within the acceptable range as defined in the previous paragraph, the amplification of c-myc mRNA in the upper 15%, against a background of wild-type p53 [as defined below] predicts a beneficial response to 5FU (see D. Arango et al., Cancer Res. 61: 4910-4915 (2001)). In the presence of normal levels of thymidylate synthase and dihydropyrimidine

p0018665 dehydrogenase, the levels of NFκB and cIAP2 in the top 10% indicate resistance of breast tumors to the chemotherapeutic drug 5FU.

p00188Patient resistance to anthracyclines is indicated if the level of standardized topoisomerase IIα mRNA is below the 10th percentile, or if the level of standardized topoisomerase IIβ mRNA is below the 10th percentile or if the combined standardized topoisomerase IIα and IIβ signals They are below the 10th percentile.

p001895 The patient's sensitivity to methotrexate is compromised if DHFR levels are more than ten times higher than the average reference set level for this mRNA species, or if reduced folate carrier levels are below the 10th percentile.

p0019010 Patients whose tumors express CYP1B1 in the top 10% have a reduced chance of responding to docetaxol.

p00191The sum of signals for aldehyde dehydrogenase 1A1 and 1A3, when more than ten times higher than the average of the reference set, indicates reduced probability of response to cyclophosphamide.

p00192fifteen Currently, estrogen and progesterone receptor expression as measured by immunohistochemistry is used to select patients for antiestrogen therapy. RT-PCR assays have been demonstrated for levels of estrogen and progesterone receptor mRNA that predict the levels of these proteins as determined by conventional clinical diagnostic tests, with a high degree of

p00193twenty concordance (figures 6 and 7).

p00194Patients whose tumors express mRNA of REα or RP in the upper 70% are likely to respond to tamoxifen or other antiestrogens (therefore, operationally, the lower levels of REα that these will define tumors negative for REα). However, when the signal for microsomal epoxide hydrolase is 10%

p0019525 higher or when mRNAs for pS2 / clover factor, GATA3 or human chorionic gonadotropin are at or below the average levels found in tumors negative for REα, antiestrogen therapy will not be beneficial.

p00196The absence of XIST signal compromises the probability of response to taxanes, as well as the elevation of the

p0019730 GST-π signal or prolyl endopeptidase [PREP] in the top 10%. The elevation of PLAG1 in the top 10% decreases the sensitivity to taxanes.

p00198ERCC1 mRNA expression in the top 10% indicates significant risk of resistance to cisplatin or the like.

p0019935 An RT-PCR assay of Her2 mRNA expression predicts Her2 overexpression as measured by a conventional diagnostic test, with high degree of concordance (data not shown). Patients whose tumors express Her2 (normalized to cyp.1) in the top 10% have an increased likelihood of beneficial response to treatment with Herceptin or other ErbB2 antagonists. The measurement of the

p0020040 Grb7 mRNA expression serves as a test for HER2 gene amplification, because the Grb7 gene is closely linked to Her2. When the expression of Her2 is high as defined earlier in this paragraph, Grb7 similarly raised indicates gene amplification of Her2. Overexpression of IGF1R and IGF1

p00201or IGF2 decreases the likelihood of beneficial response to Herceptin and also to EGFR antagonists.

p00202Four. Five Patients whose tumors express mutant Ha-Ras, and also express farnesyl pyrophosphate synthetase mRNA

p00203or geranyl pyrophosphonate synthetase at levels above the tenth percentile comprise a group that is especially likely to have a beneficial response to bisphosphonate drugs.

p00204Cox2 is a key control enzyme in the synthesis of prostaglandins. It is frequently expressed at levels

p00205fifty elevated in subsets of various types of carcinomas including breast carcinoma. The expression of this gene is controlled at the level of transcription, so that RT-PCR serves as a valid indicator of cellular enzymatic activity. Non-clinical research has shown that cox2 promotes tumor angiogenesis, suggesting that this enzyme is a promising pharmacological target in solid tumors. Several Cox2 antagonists are products marketed for use in anti-inflammatory conditions. The treatment of patients with polyposis

p0020655 Adenomatous family with the cox2 inhibitor Celebrex significantly decreased the number and size of neoplastic polyps. No cox2 inhibitor has yet been approved for the treatment of breast cancer, but generally this class of drugs is safe and could be prescribed in an unspecified manner in breast cancers in which cox2 is overexpressed. Tumors that express COX2 at levels in the top ten percentile have an increased chance of beneficial response to Celebrex or other cyclooxygenase 2 inhibitors.

p0020760 Tyrosine kinases ErbB1 [EGFR], ErbB3 and ErbB4 [Her4]; also TGFalfa, anfiregulin ligands, EGF-like growth factor binding to heparin and epiregulin; also BRK, a non-receptor kinase. Several drugs in clinical development block the EGF receptor. ErbB2-4, the indicated ligands and BRK also increase the activity of the EGFR pathway. Breast cancer patients whose tumors express high levels of

p0020865 EGFR or EGFR and abnormally high levels of the other triggers indicated in the EGFR pathway are candidates

p00210Potential for treatment with an EGFR antagonist.

p00211Patients whose tumors express less than 10% of the average level of EGFR mRNA observed in the reference panel are relatively less likely to respond to EGFR antagonists [such as Iressa, or ImClone

p002125 225]. In cases where the EGFR is above this low range, the additional presence of epiregulin, TGFα, anfiregulin, or ErbB3, or BRK, CD9, MMP9, or Lot1 at levels above the 90th percentile predisposes to the response to antagonists from EGFR. Epiregulin gene expression, in particular, is a good substitute marker for EGFR activation, and can be used not only to predict the response to EGFR antagonists, but also to monitor the response to EGFR antagonists [taking fine needle biopsies to provide tumor tissue during treatment]. CD82 levels above the 90th percentile suggest worse efficacy of EGFR antagonists.

p00213Tyrosine kinases abl, c-kit, PDGFRalfa, PDGFbeta and ARG; also, the ligands that transmit the c-kit, PDGFA, B, C and D ligand signal. The tyrosine kinases listed are all targets of the Gleevec ™ drug (imatinib mesylate, 15 Novartis), and the listed ligands stimulate one or more of the Tyrosine kinases listed. In the two indications for which Gleevec ™ is approved, the tyrosine kinase targets (bcr-abl and ckit) are overexpressed and also contain activating mutations. A finding that one of the target tyrosine kinase targets of Gleevec ™ is expressed in breast cancer tissue will result in a second phase of analysis in which the gene will be sequenced to determine if it is mutated. That a mutation found is an activating mutation can be demonstrated by methods known in the art, such as, for example, by measuring the kinase enzymatic activity or by measuring the phosphorylation state of the particular kinase, relative to the corresponding wild-type kinase. Breast cancer patients whose tumors express high levels of mRNA encoding Gleevec ™ target tyrosine kinases, specifically, in the top ten percentile, or mRNA for Gleevec ™ target tyrosine kinases in the average range and mRNA for their stimulant ligands related growth

p0021425 in the top ten percentile, they are particularly good candidates for treatment with Gleevec ™.

p00215VEGF is a potent and pathologically important angiogenic factor. (See below in Forecast Indicators). When VEGF mRNA levels are in the top ten percentile, aggressive treatment is warranted. Such levels particularly suggest the value of treatment with antiangiogenic drugs, including VEGF antagonists, such as anti-VEGF antibodies. Additionally, a level of KDR or CD31 mRNA in the upper 20th percentile further increases the likelihood of benefit of VEGF antagonists.

p00216Farnesyl pyrophosphate synthetase and geranyl geranyl pyrophosphate synthetase. These enzymes are targets of marketed bisphosphonate drugs, which were originally developed for the treatment of osteoporosis but that

p0021735 They have recently begun to be prescribed in an unspecified manner in breast cancer. High levels of mRNA encoding these enzymes in breast cancer tissue, by enzyme of the 90th percentile, suggest the use of bisphosphonates as a treatment option.

p00218two. Multiple drug resistance factors

p00219These factors include 10 genes: gamma glutamyl cysteine ​​synthetase [GCS]; GST-α; GST-π; MDR-1; MRP1-4; breast cancer resistance protein [BCRP]; lung resistance protein [MVP]; SXR; YB-1

p00220GCS and both GST-α and GST-π regulate glutathione levels, which decreases cellular sensitivity to drugs

p00221Four. Five chemotherapeutic agents and other toxins through reductive derivatization. Glutathione is a necessary cofactor for multi-drug resistant pumps, MDR-1 and MRP. MDR1 and MRP work by actively transporting several important chemotherapeutic drugs used in breast cancer out of cells.

p00222GST, MDR-1 and MRP-1 have been studied extensively to determine if they have a possible predictive or prognostic significance in human cancer. However, there is much disagreement in the literature regarding these issues. Recently, new members of the MRP family have been identified: MRP-2, MRP-3, MRP-4, BCRP and lung resistance protein [main Vault protein]. These have substrate specificities that overlap with those of MDR-1 and MRP-1. The incorporation of all these relevant members of the ABC family as well as synthetic glutathione enzymes in the present invention captures the contribution of this family to the

p0022355 drug resistance, in a way that they cannot do single or double analyte assays.

p00224MRP-1, the gene that codes for multi-drug resistance protein.

p00225P-glycoprotein is not primarily regulated at the level of transcription. However, p-glycoprotein stimulates the transcription of PTP1b. An embodiment of the present invention is the use of the mRNA level for PTP1b phosphatase as a substitute measure of the activity of MRP-1 / p-glycoprotein.

p00226The SXR gene is also a multi-drug resistance activator, as it stimulates the transcription of certain multi-drug resistance factors.

p0022765 The impact of multiple drug resistance factors with respect to the chemotherapeutic agents used in the

p00229Breast cancer is as follows. The beneficial response to doxorubicin is compromised when mRNA levels of either MDR1, GSTα, GSTπ, SXR, BCRP YB-1 or LRP / MVP are in the top four percentile. The beneficial response to methotrexate is inhibited if mRNA levels of either MRP1, MRP2, MRP3 or MRP4 or gamma-glutamyl cysteine ​​synthetase are in the upper four percentile.

p002303. Eukaryotic translation initiation factor 4E [EIF4E]

p00231EIF4E mRNA levels provide evidence of protein expression and thereby expand the ability of RT-PCR to indicate variation in gene expression. Therefore, a claim of the present invention is the use of EIF4E as an added indicator of the gene expression of certain genes [eg, cyclinD1, mdm2, VEGF and others]. For example, in two tissue samples that contain the same amount of normalized VEGF mRNA, it is likely that the tissue containing the higher standardized level of EIF4E has the highest level of VEGF gene expression.

p00232fifteen The background is as follows. A key point in the regulation of mRNA translation is the selection of mRNA by the EIF4G complex to bind to the 43S ribosomal subunit. The EIF4E protein [the m7G CAP binding protein] is often limiting because there are more copies of mRNA than EIF4E in cells. UTRs in 5 'highly structured or highly rich in GC are inefficiently translated, and these often code for genes that perform functions relevant to cancer [eg, cyclinD1, mdm2 and VEGF]. EIF4E regulates itself at the level of transcription / mRNA. Therefore, the expression of EIF4E provides an added indication of increased activity of several proteins.

p00233It is also noteworthy that the overexpression of EIF4E transforms cultured cells, and is therefore an oncogene. Overexpression of EIF4E occurs in several different types of carcinomas but is particularly significant in

p0023425 breast cancer EIF4E is normally expressed at very low levels in normal breast tissue.

p00235D. Forecast indicators

p002361. DNA repair enzymes

p00237Loss of BRCA1 or BRCA2 activity by mutation represents the critical oncogenic stage in the most common / common type (s) of family breast cancer. The mRNA levels of these important enzymes are abnormal in subsets of sporadic breast cancer as well. Loss of signals from anyone [up to the lowest ten percentile] accentuates the risk of short survival.

p00238two. Cell cycle regulators

p00239Cell cycle regulators include 14 genes: c-MYC; c-Src; Cyclin D1; You will do; mdm2; p14ARF; p21WAF1 / CIP; p16INK4a / p14; p23; p27; p53; PI3K; PKC-epsilon; PKC-delta.

p00240The gene for p53 [TP53] is mutated in a large fraction of breast cancers. Frequently, p53 levels rise when the loss of function mutation occurs. When the mutation is dominant-negative, it creates a survival value for the cancer cell because growth is promoted and apoptosis is inhibited. Thousands of different p53 mutations have been found in breast cancer, and the functional consequences

p00241Four. Five Many of them are not clear. A large group of academic literature addresses the predictive and prognostic significance of mutated p53 and the results are highly contradictory. The present invention provides a functional genomic measure of p53 activity, as follows. The activated wild-type p53 molecule triggers the transcription of the p21 cell cycle inhibitor. Therefore, the ratio of p53 to p21 should be low when p53 is of the natural type and is activated. When p53 is detectable and the ratio of p53 to p21 is high in tumors in relation to the normal breast, it means non-functional or dominant negative p53 p53. The cancer literature provides evidence of this as confirmed by a poor prognosis.

p00242Mdm2 is an important regulator of p53. Activated natural type p53 stimulates the transcription of mdm2. The mdm2 protein binds to p53 and promotes its proteolytic destruction. Therefore, abnormally low levels of

p0024355 mdm2 in the presence of normal or higher levels of p53 indicate that p53 is mutated or inactivated.

p00244An aspect of the present invention is the use of ratios of p53: p21 and p53: mdm2 mRNA levels to provide an image of the state of p53. Tests of p53 negative dominant mutation (as indicated by mRNA ratios p53: high p21 and / or p53: high mdm2, specifically in the top ten percentile) presage a higher risk of recurrence in breast cancer and therefore induce a decision to use chemotherapy in breast cancer after surgery with a negative node.

p00245Another important regulator of the cell cycle is p27, which in the activated form blocks the progression of the cell cycle at the level of cdk4. The protein is mainly regulated by phosphorylation / dephosphorylation, rather than at the level of

p0024665 transcription. However, p27 mRNA levels vary. Therefore, a p27 mRNA level in the top ten percentile indicates reduced risk of breast cancer recurrence after surgery.

p00248Cyclin D 1 is a major positive regulator of the S-phase entry of the cell cycle. The cyclin D1 gene is amplified in approximately 20% of breast cancer patients, and therefore promotes tumor growth in those cases. An aspect of the present invention is the use of cyclin D1 mRNA levels for

p002495 diagnostic purposes in breast cancer. A level of cyclin D1 mRNA in the top ten percentile suggests high risk of recurrence in breast cancer after surgery and suggests a particular benefit of adjuvant chemotherapy.

p002503. Other tumor suppressors and related proteins

p00251These include APC and E-cadherin. It has long been known that the APC tumor suppressor is lost in approximately 50% of colon cancers, with regulation by increasing the concomitant transcription of E-cadherin, an important cell adhesion molecule and growth suppressor. Recently, it has been found that the APC gene is silenced in 15-40% of breast cancers. Likewise, the Ecadherina gene is silenced [by methylation of CpG islands] in approximately 30% of cancers of

p00252fifteen mom. An abnormally low level of APC mRNA and / or E-cadherin in the lower 5th percentile suggests a high risk of recurrence in breast cancer and an increased risk of shortened survival.

p00253Four. Apoptosis regulators

p00254These include members of the BCl / BAX family BCl2, Bcl-xl, Bak, Bax and related factors, NFκ-B and related factors, and also p53BP1 / ASPP1 and p53BP2 / ASPP2.

p00255Bax and Bak are proapoptotic and BCl2 and Bcl-xl are antiapoptotic. Therefore, the reasons for these factors influence the resistance or sensitivity of a cell to toxic (proapoptotic) drugs. In breast cancer, unlike

p0025625 of other cancers, a high level of BCl2 (in the top ten percentile) correlates with good outcome. This reflects the fact that BCl2 has growth inhibitory activity as well as antiapoptotic activity, and in breast cancer the significance of the first activity has more weight than the significance of the latter. The impact of BC12 depends in turn on the state of the transcription factor that stimulates c-MYC growth. The gene for c-MYC is amplified in approximately 20% of breast cancers. When c-MYC messenger levels are abnormally high relative to BC12 (so this ratio is in the top ten percentile), then the high level of BC12 mRNA mRNA is no longer a positive indicator.

p00257NFκ-B is another important antiapoptotic factor. Originally recognized as a proinflammatory transcription factor, it is now clear that it prevents programmed cell death in response to several extracellular toxic factors [such as tumor necrosis factor]. The activity of this transcription factor is mainly regulated by phosphorylation / dephosphorylation events. However, NFκ-B levels vary, however, from cell to cell, and elevated levels must be correlated with increased apoptosis resistance. Importantly for the present purposes, NFκ-B exerts its antiapoptotic activity largely through its stimulation of mRNA transcription encoding certain members of the IAP [apoptosis inhibitor] family of proteins, specifically cIAP1, cIAP2 , XIAP and survivin. Thus, abnormally high levels of mRNA for these IAPs and for any NFκ-B in the top 5 percentile mean activation of the NFκ-B antiapoptotic pathway. This suggests a high risk of recurrence in breast cancer after chemotherapy and therefore a poor prognosis. An embodiment of the present invention is the inclusion in the set of genes of the previous apoptotic regulators, and the use explained in a summary manner above of

p00258Four. Five combinations and reasons of the levels of their mRNA for breast cancer prognosis.

p00259The p53BP1 and 2 proteins bind to p53 and promote the transcriptional activation of proapoptotic genes. The levels of p53BP1 and 2 are suppressed in a significant fraction of breast cancers, correlating with a poor prognosis. When any one is expressed in the lower tenth percentile, a poor prognosis is indicated.

p002605. Factors that control angiogenesis and cell invasion

p00261These include uPA, PAI1, cathepsins B, G and L, dispersion factor [HGF], c-met, KDR, VEGF and CD31. The uPA plasminogen activator and its PAI1 serpine regulator promote the decomposition of extracellular matrices and the

p0026255 invasion of tumor cells. Abnormally high levels of both mRNAs in malignant breast tumors (in the upper twentieth percentile) mean an increased risk of shortened survival, increased recurrence in breast cancer patients after surgery, and increased importance of receiving adjuvant chemotherapy. On the other hand, patients with a negative node whose tumors do not express high levels of these mRNA species are less likely to have recurrence of this cancer and could be considered more seriously if the benefits of conventional chemotherapy justify the associated toxicity.

p00263Cathepsins B or L, when expressed in the top ten percentile, predict poor overall and disease-free survival. In particular, cathepsin L predicts a short survival in patients with a positive node.

p0026465 The dispersion factor and its related c-met receptor promote cell invasion and motility, growth

p00266Cellular and angiogenesis. In breast cancer, elevated levels of mRNA encoding these factors should lead to aggressive treatment with chemotherapeutic drugs, when the expression of either, or the combination, is above the 90th percentile.

p002675 VEGF is a central positive regulator of angiogenesis, and elevated levels in solid tumors predict short survival [note many references showing that an elevated level of VEGF predicts short survival]. Therefore, VEGF inhibitors slow the growth of solid tumors in animals and humans. VEGF activity is monitored at the level of transcription. VEGF mRNA levels in the top ten percentile indicate a significantly worse prognosis than average. Other vascularization markers, CD31 [PECAM] and KDR indicate high vessel density in tumors that the tumor will be particularly malignant and aggressive, and therefore an aggressive therapeutic strategy is warranted.

p002686. Markers for immune and inflammatory processes and cells

p00269fifteen These markers include the genes for the λ light chain of immunoglobulins CD18, CD3, CD68, Fas [CD95] and Fas ligand.

p00270Several sets of tests suggest that the mechanism of action of certain drugs used in breast cancer involves the activation of the host's immune / inflammatory response (for example, Herceptin®). An aspect of the present invention is the inclusion in the set of marker genes for inflammatory and immune cells, and markers that predict tumor resistance against immune surveillance. The lambda immunoglobulin light chain is a marker for cells that produce immunoglobulins. CD18 is a marker for all white blood cells. CD3 is a marker for T cells. CD68 is a marker for macrophages.

p0027125 CD95 and Fas ligand are a receptor: a pair of ligands that mediate one of the two main pathways by which cytotoxic T cells and NK cells destroy selected target cells. Decreased expression of CD95 and increased expression of the Fas ligand indicates poor prognosis in breast cancer. Both CD95 and Fas ligand are transmembrane proteins, and need to be anchored to the membrane to trigger cell death. Certain tumor cells produce a truncated soluble variant of CD95, created as a result of the alternative splicing of the CD95 mRNA. This blocks the destruction of tumor cells mediated by the FAS ligand of cytotoxic T cells and NK cells. The presence of soluble CD95 correlates with poor survival in breast cancer. The gene set includes both soluble and full-length variants of CD95.

p0027235 7. Cell proliferation markers

p00273The gene set includes Ki67 / MiB1 cell proliferation markers, PCNA, Pin1 and thymidine kinase. High levels of proliferation marker expression are associated with a high histological grade, and short survival. High levels of thymidine kinase in the top ten percentile suggest an increased risk of short survival. Pin1 is a prolyl isomerase that stimulates cell growth, partly through transcriptional activation of the cyclin D1 gene, and levels in the top ten percentile contribute to a negative prognosis profile.

p002748. Other receptors and growth factors

p00275Four. Five This set of genes includes IGF1, IGF2, IGFBP3, IGF1R, FGF2, FGFR1, CSF-1R / fms, CSF-1, IL6 and IL8. All these proteins are expressed in breast cancer. Most stimulate tumor growth. However, the expression of the growth factor FGF2 correlates with good outcome. Some have antiapoptotic activity, mainly IGF1. The activation of the IGF1 axis by IGF1, IGF1R or elevated IGFBP3 (as indicated by the sum of these signals in the top ten percentile) inhibits the death of tumor cells and greatly contributes to a poor prognosis profile.

p002769. Gene expression markers that define breast cancer subclasses

p00277These include: oncogene GRO1 alpha, Grb7, cytokeratins 5 and 17, retinal binding protein 4, nuclear factor of

p0027855 hepatocytes 3, integrin alfa 7 and lipoprotein lipase. These markers divide breast cancer into different cell types that are phenotypically different at the level of gene expression. Tumors that express signals for Bcl2, nuclear hepatocyte factor 3, LIV1 and RE above the average have the best prognosis for overall and disease-free survival after surgical removal of the cancer. Another category of breast cancer tumor type, characterized by elevated expression of lipoprotein lipase, retinol-binding protein 4 and α7 integrin, carry an intermediate prognosis. Tumors that express either high levels of cytokeratins 5 and 17, GRO oncogene at levels four times or higher above average, or ErbB2 and Grb7 at levels ten times or more above average, have the worst prognosis.

p00279Although throughout the present description, including the examples below, various

p0028065 Aspects of the invention with reference to gene expression studies, the disclosure can be carried out in a similar manner, and similar results can be achieved by applying proteomics techniques that are well known in

p00282The technique. The proteome is the totality of the proteins present in a sample (for example, tissue, organism or cell culture) at a certain point of time. Proteomics includes, among other things, the study of global changes in protein expression in a sample (also called "expression proteomics"). Proteomics normally includes the following steps: (1) separation of individual proteins in a sample

p002835 by two-dimensional gel electrophoresis (2-D PAGE); (2) identification of the individual proteins recovered from the gel, for example by mass spectrometry and / or N-terminal sequencing, and (3) analysis of the data using bioinformatics. Proteomic methods are valuable complements to other methods of obtaining the gene expression profile and can be used alone or in combination with other methods of the present disclosure, to detect the products of the gene markers of the present invention.

p0028410 Additional details of the invention will be described in the following non-limiting examples.

p00285Example 1

p00286fifteen RNA isolation from samples of paraffin-embedded, formalin-fixed tissue (FPET)

p00287A. Protocols

p00288I. EPICENTRE® Xylene Protocol

p00289twenty RNA isolation

p00290(1) Cut 1-6 sections (thickness of 10 μm each) of paraffin embedded tissue per sample using a

p00291Clean microtome blade and place them in a 1.5 ml eppendorf tube. 25

<dl><dt>(2)</dt><dd> To extract the paraffin, add 1 ml of xylene and invert the tubes for 10 minutes shaking in a nutador. </dd></dl>

<dl><dt>(3)</dt><dd> Sediment the sections by centrifugation for 10 minutes at 14,000 xg in a microcentrifuge </dd></dl>

p00292eppendorf. 30

p00293(4) Remove xylene, leaving something at the bottom to avoid displacing sediment.

p00294(5) Repeat steps 2-4. 35 (6) Add 1 ml of 100% ethanol and invert for 3 minutes shaking in the nutador.

p00295(7) Sediment the residues by centrifugation for 10 minutes at 14,000 xg in an eppendorf microcentrifuge.

p0029640 (8) Remove ethanol, leaving something at the bottom to avoid sediment.

p00297(9) Repeat steps 6-8 twice.

p00298(10) Remove all remaining ethanol. Four. Five

<dl><dt>(11)</dt><dd> For each sample, add 2 μl of proteinase K 50 μg / μl to 300 μl of cell and tissue lysis solution. </dd></dl>

<dl><dt>(12)</dt><dd> Add 300 μl of cell and tissue lysis solution containing proteinase K to each sample and mix </dd></dl>

p00299conscientiously. fifty

p00300(13) Incubate at 65 ° C for 90 minutes (mixed with vortex every 5 minutes). Visually monitor the remaining tissue fragment. If it is still visible after 30 minutes, add an additional 2 μl of proteinase K 50 μg / μl and continue incubating at 65 ° C until the fragment dissolves.

p0030155 (14) Place the samples on ice for 3-5 minutes and proceed with the removal of proteins and precipitation of the total nucleic acid.

p00302Protein removal and precipitation of total nucleic acid

p0030360 (1) Add 150 μl of MPC protein precipitation reagent to each lysed sample and vortex vigorously for 10 seconds.

p00304(2) Sediment the residues by centrifugation for 10 minutes at 14,000 xg in a microcentrifuge

p00305eppendorf. 65

<dl><dt>(3)</dt><dd> Transfer the supernatant to clean eppendorf tubes and discard the sediment. </dd></dl>

<dl><dt>(4)</dt><dd> Add 500 μl of isopropanol to the recovered supernatant and mix thoroughly shaking in the nutador </dd></dl>

p00307for 3 minutes 5

<dl><dt>(5)</dt><dd> Sediment the RNA / DNA by centrifugation at 4 ° C for 10 minutes at 14,000 xg in an eppendorf microcentrifuge. </dd></dl>

<dl><dt>(6)</dt><dd> Remove all isopropanol with a pipette, being careful not to displace the sediment. Elimination of contaminating DNA from RNA preparations </dd></dl>

<dl><dt>(1)</dt><dd> Prepare 200 μl of DNase I solution for each sample by adding 5 μl of DNase I without RNAse (1 </dd></dl>

p00308U / μl) at 195 μl of 1X DNase buffer. fifteen

<dl><dt>(2)</dt><dd> Resuspend completely the sedimented RNA in 200 μl of DNase I solution by vortexing. </dd></dl>

<dl><dt>(3)</dt><dd> Incubate the samples at 37 ° C for 60 minutes. </dd></dl>

<dl><dt>(4)</dt><dd> Add 200 μl of 2X T and lysis solution C to each sample and vortex for 5 seconds. </dd></dl>

<dl><dt>(5)</dt><dd> Add 200 μl of MPC protein precipitation reagent, mix with vortexing for 10 seconds and </dd></dl>

p00309place on ice for 3-5 minutes. 25

<dl><dt>(6)</dt><dd> Sediment the residues by centrifugation for 10 minutes at 14,000 xg in an eppendorf microcentrifuge. </dd></dl>

<dl><dt>(7)</dt><dd> Transfer the supernatant containing the RNA to clean the eppendorf tubes and discard the sediment. (Be careful to avoid transferring the sediment).</dd></dl>

<dl><dt>(8)</dt><dd> Add 500 μl of isopropanol to each supernatant and shake the samples in the nutador for 3 minutes. </dd></dl>

<dl><dt>(9)</dt><dd> Sediment the RNA by centrifugation at 4 ° C for 10 minutes at 14,000 xg in an eppendorf microcentrifuge. </dd></dl>

<dl><dt>(10)</dt><dd> Remove isopropanol, leaving something at the bottom to avoid displacing the sediment. </dd></dl>

<dl><dt>(11)</dt><dd> Rinse twice with 1 ml of 75% ethanol. Briefly centrifuge if the RNA sediment moves.</dd></dl>

<dl><dt>(12)</dt><dd> Remove ethanol carefully. </dd></dl>

<dl><dt>(13)</dt><dd> Put under an extraction hood for approximately 3 minutes to remove residual ethanol. 45 (14) Resuspend the RNA in 30 μl of TE buffer and store at -30 ° C.</dd></dl>

p00310II. Hot Wax / Urea Protocol of the Invention RNA isolation

<dl><dt>(1)</dt><dd> Cut 3 sections (thickness of 10 μm each) of paraffin embedded tissue using a clean microtome blade and place them in a 1.5 ml eppendorf tube. </dd></dl>

<dl><dt>(2)</dt><dd> Add 300 μl of lysis buffer (10 mM Tris 7.5, 0.5% sodium lauroylsarcosine, 0.1 mM EDTA pH 7.5, urea 4 </dd></dl>

p0031155 M) containing proteinase K 330 μg / ml (recently added from a stock solution of 50 μg / μl) and vortexing briefly.

<dl><dt>(3)</dt><dd> Incubate at 65 ° C for 90 minutes (mixed with vortex every 5 minutes). Visually monitor the tissue fragment. If it is still visible after 30 minutes, add an additional 2 μl of proteinase K 50 μg / μl and continue incubating at 65 ° C until the fragment dissolves.</dd></dl>

<dl><dt>(4)</dt><dd> Centrifuge for 5 minutes at 14,000 xg and transfer the upper aqueous phase to a new tube, being careful not to break the paraffin seal. </dd></dl>

p0031265 (5) Place the samples on ice for 3-5 minutes and proceed with protein removal and

p00314total nucleic acid precipitation.

p00315Protein removal and precipitation of total nucleic acid

p003165 (1) Add 150 μl of 7.5 M NH4OAc to each lysed sample and vortex vigorously for 10 seconds.

<dl><dt>(2)</dt><dd> Sediment the residues by centrifugation for 10 minutes at 14,000 xg in an eppendorf microcentrifuge. </dd></dl>

<dl><dt>(3)</dt><dd> Transfer the supernatant to clean eppendorf tubes and discard the sediment. </dd></dl>

<dl><dt>(4)</dt><dd> Add 500 μl of isopropanol to the recovered supernatant and mix thoroughly shaking in the nutador for 3 minutes. </dd></dl>

p00317fifteen (5) Sediment the RNA / DNA by centrifugation at 4 ° C for 10 minutes at 14,000 xg in an eppendorf microcentrifuge.

<dl><dt>(6)</dt><dd> Remove all isopropanol with a pipette, being careful not to displace the sediment. </dd></dl>

Elimination of contaminating DNA from RNA preparations

<dl><dt>(1)</dt><dd> Add 45 μl of 1X DNase I buffer (10 mM Tris-Cl, pH 7.5, 2.5 mM MgCl2, 0.1 mM CaCl2) and 5 μl of DNase free DNase I (2 U / μl, Ambion) to each sample. </dd></dl>

p0031825 (2) Incubate the samples at 37 ° C for 60 minutes.

p00319Inactivate DNase I by heating at 70 ° C for 5 minutes.

p00320B. Results

p00321Experimental tests show that the hot RNA extraction protocol of the invention does not compromise RNA performance. Using 19 FPE breast cancer samples, extracting RNA from three adjacent sections in the same samples, RNA yields were measured by capillary electrophoresis with fluorescence detection (Agilent bioanalyzer). The average RNA yields in nanograms and the

p0032235 Standard deviations with the invented and commercial methods, respectively, were: 139 +/- 21 versus 141 +/- 34.

p00323In addition, it was found that the EPICENTRE® T&amp;C lysis buffer can be replaced by the urea-containing lysis buffer of the present invention, and the EPICENTRE® MPC protein precipitation solution can be replaced by the 7.5 M NH4OAc reagent used for the protein precipitation according to the present invention without any significant compromise of RNA performance or the efficacy of TaqMan®.

p00324Example 2

p00325Amplification of mRNA species before RT-PCR

p00326Four. Five The method described in section 10 above was used with RNA isolated from fixed paraffin embedded breast cancer tissue. TaqMan® analyzes were performed with first strand cDNA generated with primer T7-GSP ((T7-GSPr) not amplified), amplified T7 RNA ((T7-GSPr) amplified). RNA was amplified according to step 2 of Figure 4. As a control, TaqMan® was also performed with cDNA generated with an unmodified GSPr ((GSPr) amplified). An equivalent amount of initial mold (1 ng / well) was used in each TaqMan® reaction.

p00327The results are shown in Figure 8. In vitro transcription increased the intensity of the RT-PCR signal by more than 10 times, and for certain genes more than 100 times in relation to controls in which the RT- primers PCR were the same primers used in method 2 for the generation of double stranded DNA for the

p0032855 in vitro transcription (GSP-T7r and GSPf). Also shown in Figure 8 are RT-PCR data generated when conventional optimized RT-PCR primers were used (i.e., lacking T7 tails). As shown, in comparison to this control, the new method produced substantial increases in the RT-PCR signal (from 4 to 64 times in this experiment).

p00329The new method requires that each T7-GSP sequence be optimized so that the increase in the RT-PCR signal is the same for each gene, relative to conventional optimized RT-PCR (with T7 tailless primers).

p00330Example 3

p0033165 A study of gene expression in premalignant and malignant breast tumors

p00333A study of gene expression was designed and conducted with the primary objective of molecularly characterizing gene expression in fixed tissue samples, embedded in paraffin from invasive breast ductal carcinoma, and exploring the correlation between such molecular profiles and free survival. of disease An additional objective

p003345 The study was to compare the molecular profiles in samples of invasive breast cancer tissue with the molecular profiles obtained in ductal carcinoma in situ. The study was further designed to obtain data on molecular profiles in lobular carcinoma in situ and in fixed tissue samples embedded in paraffin of invasive lobular carcinoma.

p0033510 Molecular assays were performed on primary mammary tumor tissues fixed with formalin, embedded in paraffin obtained from 202 individual patients who were diagnosed with breast cancer. All patients underwent surgery with a diagnosis of invasive ductal carcinoma of the breast, pure ductal carcinoma in situ (DCIS), lobular carcinoma of the breast or pure lobular carcinoma in situ (LCIS). Patients were included in the study only if the histopathological evaluation, performed as described in the Materials and methods section, indicated

p00336fifteen adequate amounts of tumor tissue and homogeneous pathology.

p00337The individuals participating in the study were divided into the following groups:

p00338Group 1: Pure ductal carcinoma in situ (DCIS); n = 18

p00339twenty Group 2: Invasive ductal carcinoma n = 130

p00340Group 3: Pure lobular carcinoma in situ (LCIS); n = 7

p0034125 Group 4: Invasive lobular carcinoma n = 16

p00342Materials and methods

p00343Each representative tumor block was characterized by conventional histopathology to determine the

p0034430 diagnosis, semiquantitative evaluation of the amount of tumor and the grade of the tumor. A total of 6 sections (10 micrometers thick each) were prepared and placed in two Costar brand microcentrifuge tubes (polypropylene, 1.7 ml tubes, transparent, 3 sections in each tube). If the tumor constituted less than 30% of the total sample area, the pathologist may have rudimentary dissected the tumor tissue, using macroscopic microdissection, placing the tumor tissue directly into the Costar tube.

p0034535 If more than one tumor block was obtained as part of the surgical procedure, all tumor blocks were subjected to the same characterization, as described above, and the most representative block of the pathology was used for analysis.

p0034640 Gene Expression Analysis

p00347The mRNA was extracted and purified from samples of paraffin embedded tissue, fixed and prepared for gene expression analysis as described in chapters 7-11 above.

p00348Four. Five Molecular quantitative gene expression assays were performed by RT-PCR, using the ABI PRISM 7900 ™ Sequence Detection System ™ (Perkin-Elmer-Applied Biosystems, Foster City, CA, USA). ABI PRISM 7900 ™ consists of a thermal cycler, a laser, a coupled charging device (CCD), a camera and a computer. The system amplifies samples in a 384-well format in a thermal cycler. During amplification, the laser-induced fluorescent signal is collected in real time through fiber optic cables for all 384 wells,

p00349fifty and is detected in the CCD. The system includes software to operate the instrument and to analyze the data.

p00350Analysis and results

p00351Tumor tissue was analyzed for 185 cancer-related genes and 7 reference genes. The normalized

p0035255 Threshold cycle values ​​(CT) for each patient based on the median of all genes for that particular patient. Clinical outcome data were available for all patients from a review of registration data and diagrams of selected patients.

p00353The outcomes were classified as:

p0035460 0 dead due to breast cancer or unknown cause or alive with breast cancer recurrence;

p003551 living without recurrence of breast cancer or dead due to a different cause of breast cancer.

p0035665 The analysis was performed by:

<dl><dt>1.</dt><dd> Analysis of the relationship between normalized gene expression and binary outcomes of 0 or 1. </dd></dl>

<dl><dt>2.</dt><dd> Analysis of the relationship between normalized gene expression and the time to outcome (0 or 1 as defined above) in which patients who were alive without recurrence of breast cancer were censored or </dd></dl>

p003585 who died due to a cause other than breast cancer. This approach was used to assess the prognostic impact of individual genes and also sets of multiple genes.

p00359Analysis of 147 patients with invasive breast carcinoma using the binary approach

p0036010 In the first (binary) approach, the analysis was performed in all 146 patients with invasive breast carcinoma. A t test was performed in the group of patients classified as 0 or 1 and p values ​​were calculated for differences between groups for each gene.

p00361The following table 4 lists the 45 genes for which the p-value for the differences between the groups was &lt;0.05. 15 Table 4

<dl><dt>Gen / SEQ ID NO: </dt><dd>CT half alive Half dead CT T value Degrees of freedom p </dd></dl>

<dl><dt>FOXM1 </dt><dd> 33,66 32,52 3,92 144 0,0001 </dd></dl>

<dl><dt>PRAME </dt><dd> 35,45 33,84 3,71 144 0,0003 </dd></dl>

<dl><dt>Bcl2 </dt><dd> 28,52 29,32 -3,53 144 0,0006 </dd></dl>

<dl><dt>STK15 </dt><dd> 30,82 30,10 3,49 144 0,0006 </dd></dl>

<dl><dt>CEGP1 </dt><dd> 29,12 30,86 -3,39 144 0,0009 </dd></dl>

<dl><dt>Ki-67 </dt><dd> 30,57 29,62 3,34 144 0,0011 </dd></dl>

<dl><dt>GSTM1 </dt><dd> 30,62 31,63 -3,27 144 0,0014 </dd></dl>

<dl><dt>CA9 </dt><dd> 34,96 33,54 3,18 144 0,0018 </dd></dl>

<dl><dt>PR </dt><dd> 29,56 31,22 -3,16 144 0,0019 </dd></dl>

<dl><dt>BBC3 </dt><dd> 31,54 32,10 -3,10 144 0,0023 </dd></dl>

<dl><dt>NME1 </dt><dd> 27,31 26,68 3,04 144 0,0028 </dd></dl>

<dl><dt>SURV </dt><dd> 31,64 30,68 2,92 144 0,0041 </dd></dl>

<dl><dt>CAT3 </dt><dd> 26,06 26,99 -2,91 144 0,0042 </dd></dl>

<dl><dt>TFRC </dt><dd> 28,96 28,48 2,87 144 0,0047 </dd></dl>

<dl><dt>YB-1 </dt><dd> 26,72 26,41 2,79 144 0,0060 </dd></dl>

<dl><dt>DPYD </dt><dd> 28,51 28,84 -2,67 144 0,0084 </dd></dl>

<dl><dt>GSTM3 </dt><dd> 28,21 29,03 -2,63 144 0,0095 </dd></dl>

<dl><dt>RPS6KB </dt><dd> 31,18 30,61 2,61 144 0,0099 </dd></dl>

<dl><dt>Src </dt><dd> 27,97 27,69 2,59 144 0,0105 </dd></dl>

<dl><dt>Chk1 </dt><dd> 32,63 31,99 2,57 144 0,0113 </dd></dl>

<dl><dt>ID1 </dt><dd> 28,73 29,13 -2,48 144 0,0141 </dd></dl>

<dl><dt>EstR1 </dt><dd> 24,22 25,40 -2,44 144 0,0160 </dd></dl>

<dl><dt>p27 </dt><dd> 27,15 27,51 -2,41 144 0,0174 </dd></dl>

<dl><dt>CCNB1 </dt><dd> 31,63 30,87 2,40 144 0,0176 </dd></dl>

<dl><dt>XIAP </dt><dd> 30,27 30,51 -2,40 144 0,0178 </dd></dl>

<dl><dt>Chk2 </dt><dd> 31,48 31,11 2,39 144 0,0179 </dd></dl>

<dl><dt>CDC25B </dt><dd> 29,75 29,39 2,37 144 0,0193 </dd></dl>

<dl><dt>IGF1R </dt><dd> 28,85 29,44 -2,34 144 0,0209 </dd></dl>

<dl><dt>AK055699 </dt><dd> 33,23 34,11 -2,28 144 0,0242 </dd></dl>

<dl><dt>PI3KC2A </dt><dd> 31,07 31,42 -2,25 144 0,0257 </dd></dl>

<dl><dt>TGFB3 </dt><dd> 28,42 28,85 -2,25 144 0,0258 </dd></dl>

<dl><dt>BAGI1 </dt><dd> 28,40 28,75 -2,24 144 0,0269 </dd></dl>

<dl><dt>CYP3A4 </dt><dd> 35,70 35,32 2,17 144 0,0317 </dd></dl>

<dl><dt>Epcam </dt><dd> 28,73 28,34 2,16 144 0,0321 </dd></dl>

<dl><dt>VEGFC </dt><dd> 32,28 31,82 2,16 144 0,0326 </dd></dl>

<dl><dt>pS2 </dt><dd> 28,96 30,60 -2,14 144 0,0341 </dd></dl>

<dl><dt>hENT1 </dt><dd> 27,19 26,91 2,12 144 0,0357 </dd></dl>

<dl><dt>WISP1 </dt><dd> 31,20 31,64 -2,10 144 0,0377 </dd></dl>

<dl><dt>HNF3A </dt><dd> 27,89 28,64 -2,09 144 0,0384 </dd></dl>

<dl><dt>NFKBp65 </dt><dd> 33,22 33,80 -2,08 144 0,0396 </dd></dl>

<dl><dt>BRCA2 </dt><dd> 33,06 32,62 2,08 144 0,0397 </dd></dl>

<dl><dt>EGFR </dt><dd> 30,68 30,13 2,06 144 0,0414 </dd></dl>

<dl><dt>TK1 </dt><dd> 32,27 31,72 2,02 144 0,0453 </dd></dl>

<dl><dt>VDR </dt><dd> 30,08 29,73 1,99 144 0,0488 </dd></dl>

p00363In Table 4 above, lower (negative) t values ​​indicate higher expression (or lower CT), associated with better outcomes and, conversely, higher (positive) t values ​​indicate higher expression (lower CT) associated with worse outcomes. Therefore, for example, the elevated expression of the FOXM1 gene (value of t = 3.92, CT

p003645 live medium> CT half dead) indicates a reduced probability of disease-free survival. Similarly, high expression of the CEGP 1 gene (t value = -3.39; mean live CT <half dead CT) indicates an increased probability of disease-free survival.

p00365Based on the data presented in Table 4, overexpression of any of the following genes in cancer

p0036610 of breast indicates a reduced probability of survival without cancer recurrence after surgery: FOXM1; PRAME; SKT15, Ki-67; CA9; NME1; SURV; TFRC; YB-1; RPS6KB1; Src; Chk1; CCNB1; Chk2; CDC25B; CYP3A4; EpCAM; VEGFC; hENT1; BRCA2; EGFR; TK1; VDR

p00367Based on the data presented in Table 4, overexpression of any of the following genes in cancer

p00368fifteen Breast indicates a better prognosis for survival without cancer recurrence after surgery: Blc12; CEGP1; GSTM1; PR; BBC3; GATA3; DPYD; GSTM3; ID1; EstR1; p27; XIAP; IGF1R; AK055699; P13KC2A; TGFB3; BAGI1; pS2; WISP1; HNF3A; NFKBp65.

p00369Analysis of 108 patients positive for RE through the binary approach

p00370twenty 108 patients with standardized CT for the estrogen receptor (RE) &lt;25.2 (ie patients positive for RE) were subjected to separate analysis. A t test was performed on the groups of patients classified as 0 or 1 and p values ​​were calculated for the differences between the groups for each gene. The following table 5 lists the 12 genes in which the p-value for the differences between the groups was &lt;0.05.

p0037125 Table 5

<dl><dt>Gen / SEQ ID NO: </dt><dd>CT half alive Half dead CT T value Degrees of freedom p </dd></dl>

<dl><dt>PRAME </dt><dd> 35,54 33,88 3,03 106 0,0031 </dd></dl>

<dl><dt>Bcl2 </dt><dd> 28,24 28,87 -2,70 106 0,0082 </dd></dl>

<dl><dt>FOXM1 </dt><dd> 33,82 32,85 2,66 106 0,089 </dd></dl>

<dl><dt>DEVIL </dt><dd> 30,33 30,71 -2,47 106 0,0153 </dd></dl>

<dl><dt>EPHX1 </dt><dd> 28,62 28,03 2,44 106 0,0163 </dd></dl>

<dl><dt>HIF1A </dt><dd> 29,37 28,88 2,40 106 0,0180 </dd></dl>

<dl><dt>VEGFC </dt><dd> 32,39 31,69 2,39 106 0,0187 </dd></dl>

<dl><dt>Ki-67 </dt><dd> 30,73 29,82 2,38 106 0,0191 </dd></dl>

<dl><dt>IGF1R </dt><dd> 28,60 29,18 -2,37 106 0,0194 </dd></dl>

<dl><dt>VDR </dt><dd> 30,14 29,60 2,17 106 0,0322 </dd></dl>

<dl><dt>NME1 </dt><dd> 27,34 26,80 2,03 106 0,0452 </dd></dl>

<dl><dt>GSTM3 </dt><dd> 28,08 28,92 -2,00 106 0,0485 </dd></dl>

p00373For each gene, a classification algorithm was used to identify the best threshold value (CT) to use each gene only in the prediction of the clinical outcome.

p003745 Based on the data presented in Table 5, overexpression of the following genes in ER-positive cancer is indicative of a reduced probability of survival without cancer recurrence after surgery: PRAME; FOXM1; EPHX1; HIF1A; VEGFC; Ki-67; VDR; NME1. Some of these genes (PRAME; FOXM1; VEGFC; Ki-67; VDR; and NME1) were also identified as indicators of poor prognosis in the previous analyzes, not limited to breast cancer positive for RE. Overexpression of the remaining genes (EPHX1 and HIF1A) seems to be a

p0037510 Negative indicator of disease-free survival in breast cancer positive for RE alone. Based on the data presented in Table 5, overexpression of the following genes in ER-positive cancer is indicative of a better prognosis for survival without cancer recurrence after surgery: Bcl-2; DEVIL; IGF1R; GSTM3. Of these last genes, Bcl-2; IGFR1; and GSTM3 have also been identified as indicators of good prognosis in the previous analysis, not limited to breast cancer positive for RE. Overexpression of DEVIL seems to be a

p00376fifteen positive indicator of disease-free survival in breast cancer positive for RE alone.

p00377Multiple gene analysis and outcome indicators

p00378Two approaches were adopted in order to determine whether the use of multiple genes could provide a better discrimination between outcomes.

p00379First, a discrimination analysis was conducted using a direct gradual approach. Models were generated that classified the outcome with greater discrimination than that obtained with any single gene alone.

p0038025 According to a second approach (time to event approach), a Cox proportional hazards model was defined for each gene (see, for example, Cox, DR, and Oakes, D. (1984), Analysis of Survival Data, Chapman and Hall , London, New York) with time until recurrence or death as a dependent variable, and the level of gene expression as an independent variable. Genes that have a value of p &lt;0.05 in the Cox model were identified. For each gene, the Cox model provides the relative risk (RR) of recurrence or death for a

p0038130 unit change in gene expression. It may be chosen to divide the patients into subgroups at any threshold value of the measured expression (on the CT scale), when all patients with expression values ​​above the threshold are at higher risk, and all patients with expression values ​​below Thresholds have lower risk, or vice versa, depending on whether the gene is an indicator of good (RR> 1.01) or poor (RR <1.01) prognosis. Therefore, any threshold value will define subgroups of patients with respectively increase or decrease in

p0038235 risk. The results are summarized in the following tables 6 and 7.

p00383Table 6

p00384Results of the Cox model for 146 patients with invasive breast cancer 40

<dl><dt>Gen </dt><dd>Relative Risk (RR) SE relative risk P value </dd></dl>

<dl><dt>FOXM1 </dt><dd> 0,58 0,15 0,0002 </dd></dl>

<dl><dt>STK15 </dt><dd> 0,51 0,20 0,0006 </dd></dl>

<dl><dt>PRAME </dt><dd> 0,78 0,07 0,0007 </dd></dl>

<dl><dt>Bcl2 </dt><dd> 1,66 0,15 0,0009 </dd></dl>

<dl><dt>CEGP1 </dt><dd> 1,25 0,07 0,0014 </dd></dl>

<dl><dt>GSTM1 </dt><dd> 1,40 0,11 0,0014 </dd></dl>

<dl><dt>Ki67 </dt><dd> 0,62 0,15 0,0016 </dd></dl>

<dl><dt>PR </dt><dd> 1,23 0,07 0,0017 </dd></dl>

<dl><dt>Contig51037 </dt><dd> 0,81 0,07 0,0022 </dd></dl>

<dl><dt>NME1 </dt><dd> 0,64 0,15 0,0023 </dd></dl>

<dl><dt>YB-1 </dt><dd> 0,39 0,32 0,0033 </dd></dl>

<dl><dt>TFRC </dt><dd> 0,53 0,21 0,0035 </dd></dl>

<dl><dt>BBC3 </dt><dd> 1,72 0,19 0,0036 </dd></dl>

<dl><dt>CAT3 </dt><dd> 1,32 0,10 0,0039 </dd></dl>

<dl><dt>CA9 </dt><dd> 0,81 0,07 0,0049 </dd></dl>

<dl><dt>SURV </dt><dd> 0,69 0,13 0,0049 </dd></dl>

<dl><dt>DPYD </dt><dd> 2,58 0,34 0,0052 </dd></dl>

<dl><dt>RPS6KB1 </dt><dd> 0,60 0,18 0,0055 </dd></dl>

<dl><dt>GSTM3 </dt><dd> 1,36 0,12 0,0078 </dd></dl>

<dl><dt>Src.2 </dt><dd> 0,39 0,36 0,0094 </dd></dl>

<dl><dt>TGFB3 </dt><dd> 1,61 0,19 0,0109 </dd></dl>

<dl><dt>CDC25B </dt><dd> 0,54 0,25 0,0122 </dd></dl>

<dl><dt>XIAP </dt><dd> 3,20 0,47 0,0126 </dd></dl>

<dl><dt>CCNB1 </dt><dd> 0,68 0,16 0,0151 </dd></dl>

<dl><dt>IGF1R </dt><dd> 1,42 0,15 0,0153 </dd></dl>

<dl><dt>Chk1 </dt><dd> 0,68 0,16 0,0155 </dd></dl>

<dl><dt>ID1 </dt><dd> 1,80 0,25 0,0164 </dd></dl>

<dl><dt>p27 </dt><dd> 1,69 0,22 0,0168 </dd></dl>

<dl><dt>Chk2 </dt><dd> 0,52 0,27 0,0175 </dd></dl>

<dl><dt>EstR1 </dt><dd> 1,17 0,07 0,0196 </dd></dl>

<dl><dt>HNF3A </dt><dd> 1,21 0,08 0,206 </dd></dl>

<dl><dt>pS2 </dt><dd> 1,12 0,05 0,0230 </dd></dl>

<dl><dt>BAGI1 </dt><dd> 1,88 0,29 0,0266 </dd></dl>

<dl><dt>AK055699 </dt><dd> 1,24 0,10 0,0276 </dd></dl>

<dl><dt>PENT1 </dt><dd> 0,51 0,31 0,0293 </dd></dl>

<dl><dt>Epcam </dt><dd> 0,62 0,22 0,0310 </dd></dl>

<dl><dt>WISP1 </dt><dd> 1,39 0,16 0,0338 </dd></dl>

<dl><dt>VEGFC </dt><dd> 0,62 0,23 0,0364 </dd></dl>

<dl><dt>TK1 </dt><dd> 0,73 0,15 0,0382 </dd></dl>

<dl><dt>NFKBp65 </dt><dd> 1,32 0,14 0,0384 </dd></dl>

<dl><dt>BRCA2 </dt><dd> 0,66 0,20 0,0404 </dd></dl>

<dl><dt>CYP3A4 </dt><dd> 0,60 0,25 0,0417 </dd></dl>

<dl><dt>EGFR </dt><dd> 0,72 0,16 0,0436 </dd></dl>

p00386Table 7

p00388Results of the Cox model for 108 patients with RE + invasive breast cancer

<dl><dt>Gen </dt><dd>Relative Risk (RR) SE relative risk P value </dd></dl>

<dl><dt>PRAME </dt><dd> 0,75 0,10 0,0045 </dd></dl>

<dl><dt>Contig51037 </dt><dd> 0,75 0,11 0,0060 </dd></dl>

<dl><dt>Blc2 </dt><dd> 2,11 0,28 0,0075 </dd></dl>

<dl><dt>HIF1A </dt><dd> 0,42 0,34 0,0117 </dd></dl>

<dl><dt>IGF1R </dt><dd> 1,92 0,26 0,0117 </dd></dl>

<dl><dt>FOXM1 </dt><dd> 0,54 0,24 0,0119 </dd></dl>

<dl><dt>EPHX1 </dt><dd> 0,43 0,33 0,0120 </dd></dl>

<dl><dt>Ki67 </dt><dd> 0,60 0,21 0,0160 </dd></dl>

<dl><dt>CDC25B </dt><dd> 0,41 0,38 0,0200 </dd></dl>

<dl><dt>VEGFC </dt><dd> 0,45 0,37 0,0288 </dd></dl>

<dl><dt>CTSB </dt><dd> 0,32 0,53 0,0328 </dd></dl>

<dl><dt>DEVIL </dt><dd> 2,91 0,50 0,0328 </dd></dl>

<dl><dt>p27 </dt><dd> 1,83 0,28 0,0341 </dd></dl>

<dl><dt>CDH1 </dt><dd> 0,57 0,27 0,0352 </dd></dl>

<dl><dt>IGFBP3 </dt><dd> 0,45 0,40 0,0499 </dd></dl>

p003895 Binary and time-to-event analyzes, with few exceptions, identified the same genes as prognostic markers. For example, the comparison of Tables 4 and 6 shows that, with the exception of a single gene, the two analyzes generated the same list of 15 main markers (as defined by the lowest p values). In addition, when both analyzes identified the same gene, they agreed with respect to the direction (positive or negative sign) of the correlation with survival / recurrence. Globally, these results

p0039010 reinforce the conclusion that the markers identified have a significant prognostic value.

p00391For Cox models that comprise more than two genes (multivariate models), a gradual entry of each individual gene into the model is made, when the first gene introduced is preselected from among the genes that have significant univariate p values, and the gene selected for entry into the model at each stage

p00392fifteen Later is the gene that improves the fit of the model to the data. This analysis can be performed with any total number of genes. In the analysis whose results are shown below, gradual entry is made for up to 10 genes.

p00393Multivariate analysis is performed using the following equation:

p00394twenty RR = exp [coef (gene A) x Ct (gene A) + coef (gene B) x Ct (gene B) + coef (gene C) x Ct (gene C) + ...]

p00395In this equation, the coefficients for genes that are prognostic factors of beneficial outcome are positive numbers and the coefficients for genes that are prognostic factors of unfavorable outcome are negative numbers. The "Ct" values ​​in the equation are ΔCt, that is, they reflect the difference between the average normalized Ct value for a population and the normalized Ct measured for the patient in question. The convention used in the present analysis has been that ΔCt lower and above the average of the population have positive and negative signs, respectively (reflecting a greater or lesser abundance of mRNA). The relative risk (RR) calculated by solving this equation will indicate whether the patient has an increase or reduction in the chances of

p0039630 Long-term survival without cancer recurrence.

p00397Multivariate gene analysis of 147 patients with invasive breast carcinoma

p00398(a) A multivariate gradual analysis was performed, using Cox proportional hazards model, on the data

p0039935 of gene expression obtained for the 147 patients with invasive breast carcinoma. CEGP1, FOXM1, STK15 and PRAME genes were excluded from this analysis. It has been identified by this analysis that the following sets of ten genes have a particularly strong predictive value of the survival of patients without cancer recurrence after surgical removal of the primary tumor.

p004011. Bc12, cyclin G1, NFKBp65, NME1, EPHX1, TOP2B, DR5, TERC, Src, DEVIL;

p00402two. Ki67, XIAP, hENT1, TS, CD9, p27, cyclin G1, pS2, NFKBp65, CYP3A4; 5

<dl><dt>3.</dt><dd> GSTM1, XIAP, Ki67, TS, cyclin G1, p27, CYP3A4, pS2, NFKBp65, ErbB3; </dd></dl>

<dl><dt>4.</dt><dd> PR, NME1, XIAP, upa, cyclin G1, Contig51037, TERC, EPHX1, ALDH1A3, CTSL; </dd></dl>

p0040310 5. CA9, NME1, TERC, cyclin G1, EPHX1, DPYD, Src, TOP2B, NFKBp65, VEGFC;

p004046. TFRC, XIAP, Ki67, TS, cyclin G1, p27, CYP3A4, pS2, ErbB3, NFKBp65.

p00405(b) A multivariate gradual analysis, using Cox proportional hazards model, was performed on the data

p00406fifteen of gene expression obtained for the 147 patients with invasive breast carcinoma, using an interrogation set that included a small number of genes. It has been identified by this analysis that the following sets of ten genes have a particularly strong predictive value of the survival of patients without cancer recurrence after surgical removal of the primary tumor.

p00407twenty 1. Bc12, PRAME, cyclin G1, FOXM1, NFKBp65, TS, XIAP, Ki67, CYP3A4, p27;

p00408two. FOXM1, cyclin G1, XIAP, Contig51037, PRAME, TS, Ki67, PDGFRa, p27, NFKBp65;

p004093. PRAME, FOXM1, cyclin G1, XIAP, Contig51037, TS, Ki6, PDGFRa, p27, NFKBp65; 25

<dl><dt>4.</dt><dd> Ki67, XIAP, PRAME, HENTI, contig51037, TS, CD9, p27, ErbB3, cyclinG1; </dd></dl>

<dl><dt>5.</dt><dd> STK15, XIAP, PRAME, PLAUR, p27, CTSL, CD18, PREP, p53, RPS6KB1; </dd></dl>

p0041030 6. GSTM1, XIAP, PRAME, p27, Contig51037, ErbB3, GSTp, EREG, IDI, PLAUR;

p004117. PR, PRAME, NME1, XIAP, PLAUR, cyclin G1, Contig51037, TERC, EPHX1, DR5;

p004128. CA9, FOXM1, cyclin G1, XIAP, TS, Ki67, NFKBp65, CYP3A4, GSTM3, p27; 35

<dl><dt>9.</dt><dd> TFRC, XIAP, PRAME, p27, Contig51037, ErbB3, DPYD, TERC, NME1, VEGFC; </dd></dl>

<dl><dt>10.</dt><dd> CEGP1, PRAME, hENT1, XIAP, Contig51037, ErbB3, DPYD, NFKBp65, ID1, TS. </dd></dl>

p0041340 Multivariate analysis of patients with invasive breast carcinoma positive for RE

p00414A multivariate gradual analysis was performed, using Cox proportional hazards model, on gene expression data obtained for patients with invasive breast carcinoma positive for ER. It has been identified by this analysis that the following sets of ten genes have a particularly strong predictive value

p00415Four. Five of the survival of patients without cancer recurrence after surgical removal of the primary tumor.

p004161. PRAME, p27, IGFBP2, HIF1A, TIMP2, ILT2, CYP3A4, ID1, EstR1, DEVIL;

p00417two. Contig51037, EPHX1, Ki67, TIMP2, cyclinG1, DPYD, CYP3A4, TP, AIB1, CYP2C8; fifty

<dl><dt>3.</dt><dd> Bc12, hENT1, FOXM1, Contig51037, cyclinG1, Contig46653, PTEN, CYP3A4, TIMP2, AREG; </dd></dl>

<dl><dt>4.</dt><dd> HIF1A, PRAME, p27, IGFBP2, TIMP2, ILT2, CYP3A4, ID1, EstR1, DEVIL; </dd></dl>

55 5. IGF1R, PRAME, EPHX1, Contig51037, cyclinG1, Bc12, NME1, PTEN, TBP, TIMP2;

p004186. FOXM1, Contig51037, VEGFC, TBP, HIF1A, DPYD, RAD51C, DCR3, cyclin G1, BAG1;

<dl><dt>7.</dt><dd> EPHX1, Contig51037, Ki67, TIMP2, cyclin G1, DPYD, CYP3A4, TP, AIB1, CYP2C8; 60</dd></dl>

8. Ki67, VEGFC, VDR, GSTM3, p27, upa, ITGA7, rhoC, TERC, Pin1;

<dl><dt>9.</dt><dd> CDC25B, Contig51037, hENT1, Bc12, HLAG, TERC, NME1, upa, ID1, CYP; 65 10. VEGFC, Ki67, VDR, GSTM3, p27, upa, ITGA7, rhoC, TERC, Pin1;</dd></dl>

<dl><dt>11.</dt><dd> CTSB, PRAME, p27, IGFBP2, EPHX1, CTSL, BAD, DR5, DCR3, XIAP; </dd></dl>

<dl><dt>12.</dt><dd> DEVIL, Ki67, hENT1, TIMP2, ID1, p27, KRT19, IGFBP2, TS, PDGFB; </dd></dl>

p004205 13. p27, PRAME, IGFBP2, HIF1A, TIMP2, ILT2, CYP3A4, ID1, EstR1, DEVIL;

p0042114. CDH1; PRAME, VEGFC; HIF1A; DPYD, TIMP2, CYP3A4, EstR1, RBP4, p27;

<dl><dt>15.</dt><dd> IGFBP3, PRAME, p27, Bcl2, XIAP, EstR1, Ki67, TS, Src, VEGF; 10</dd></dl>

16. GSTM3, PRAME, p27, IGFBP3, XIAP, FGF2, hENT1, PTEN, EstR1, APC;

<dl><dt>17.</dt><dd> hENT1, Bcl2, FOXM1, Contig51037, CiclinaG1, Contig46653, PTEN, CYP3A4, TIMP2, AREG; 18. 18. STK15, VEGFC, PRAME, p27, GCLC, hENT1, ID1, TIMP2, EstR1, MCP1;</dd></dl>

p0042219. NME1, PRAM, p27, IGFBP3, XIAP, PTEN, hENT1, Bcl2, CYP3A4, HLAG;

<dl><dt>20.</dt><dd> VDR, Bcl2, p27, hENT1, p53, PI3KC2A, EIF4E, TFRC, MCM3, ID1; twenty</dd></dl>

twenty-one. EIF4E, Contig51037, EPHX1, cyclin G1, Bcl2, DR5, TBP, PTEN, NME1, HER2;

<dl><dt>22. </dt><dd>CCNB1, PRAME, VEGFC, HIF1A, hENT1, GCLC, TIMP2, ID1, p27, upa; 23. 23. ID1, PRAME, DEVIL, hENT1, p27, PDGFRa, NME1, BIN1, BRCA1, TP;</dd></dl>

p0042324. FBXO5, PRAME, IGFBP3, p27, GSTM3, hENT1, XIAP, FGF2, TS, PTEN;

p0042425. GUS, HIA1A, VEGFC, GSTM3, DPYD, hENT1, FBXO5, CA9, CYP, KRT18; 30

p0042526. Bclx, Bcl2, hENT1, Contig51037, HLAG, CD9, ID1, BRCA1, BIN1, HBEGF.

p00426It should be noted that many of the previous gene sets include genes that alone did not have enough predictive value to qualify as prognostic markers under the standards discussed above, but in combination with other genes, their presence provides valuable information on the probability of survival. of the long-term patient without cancer recurrence.

TABLE 1

<dl><dt>1. </dt><dd>ADD3 42. c-myc 77. Gamma-GCS 115. KDR 156. Pin1 (adduct 3 (glutamyl cysteine ​​gamma) * synthetase) </dd></dl>

<dl><dt>2.</dt><dd> AKT1 / Protein 43. cN-1 78. GATA3 ^ 116. Ki-67 / MIB1 157. PKC-ε kinase B </dd></dl>

<dl><dt>3.</dt><dd> AKT 2 44. Cryptochrome1 * 79. Geranyl geranyl 117. Lipoprotein 158. Pkc-δ pyrophosphate synthetase lipase ^ </dd></dl>

<dl><dt>4.</dt><dd> AKT 3 45. c-Src 80. G-CSF 118. LIV1 159. PLAG1 (pleiomorphic adenoma 1) * </dd></dl>

<dl><dt>5.</dt><dd> Aldehyde 46. Cyclin D1 81. GPC3 119. Protein 160. PREP prolyl dehydrogenase endopeptidase resistance * PEP 1A1 pulmonary / MVP </dd></dl>

<dl><dt>6. Aldehyde </dt><dd>47. CYP1B1 82. gravina * [AK 120. Lot1 161. Receiver of </dd></dl>

<dl><dt>dehydrogenase </dt><dd>AP258] progesterone </dd></dl>

<dl><dt>1A3 </dt><dd /></dl>

<dl><dt>7. Anfirregulina </dt><dd>48. CYP2C9 * 83. GRO1 oncogene 121. Maspin 162. pS2 / clover factor </dd></dl>

<dl><dt>alpha ^ </dt><dd> 1 </dd></dl>

<dl><dt>8. APC </dt><dd>49. Cytokeratin 5 ^ 84. Grb7 ^ 122. MCM2 163. PTEN </dd></dl>

<dl><dt>9. ARG </dt><dd>50. Cytokeratin 17 ^ 85. GST-alpha 123. MCM3 164. PTP1b </dd></dl>

<dl><dt>10. ATM </dt><dd>51. Cytokeratin 18 ^ 86. GST-pi ^ 124. MCM7 165. RAR-alpha </dd></dl>

<dl><dt>eleven. Bak </dt><dd>52. DAP-Kinase-1 87. Ha-Ras 125. MCP-1 166. RAR-beta2 </dd></dl>

<dl><dt>12. Bax </dt><dd>53. DHFR 88. HB-EGF 126. Protein 4 167. CPR </dd></dl>

<dl><dt>associated with </dt><dd /></dl>

<dl><dt>microtubules </dt><dd /></dl>

<dl><dt>13. Bcl2 </dt><dd>54. DEVIL 89. HE4- Homologue 127. MCI 168. Folate carrier </dd></dl>

<dl><dt>of inhibitor of </dt><dd>reduced </dd></dl>

<dl><dt>14. Bcl-xl 15. BRK 16. BCRP 17. BRCA-1 18. BRCA-2 19. Caspasa-3 </dt><dd>55. Dihydropyrimidine dehydrogenase 56. EGF 57. ECadherin / CDH1 ^ 58. ELF.3 * 59. Endothelin 60. Epirregulin </dd></dl>

<dl><dt>twenty. Cathepsin B </dt><dd>61. ER-alpha ^ </dd></dl>

<dl><dt>twenty-one. Cathepsin G </dt><dd>62. ErbB-1 </dd></dl>

<dl><dt>22 Cathepsin L </dt><dd>63. ErbR-2 ^ </dd></dl>

<dl><dt>2. 3. CD3 </dt><dd>64. ErbB-3</dd></dl>

<dl><dt>24. CD9 </dt><dd>65. ErbB-4 </dd></dl>

<dl><dt>25. CD18 </dt><dd>66. ER-Beta </dd></dl>

<dl><dt>26. CD31 27. CD44 ^ 28. CD68 29. CD82 / KAI-1 30. Cdc25A 31. Cdc25B 32. CGA </dt><dd>67. Eukaryotic translation initiation factor 4B * (EIF4B) 68. EIF4E 69. Farnesyl pyrophosphate synthetase 70. FAS (CD95) 71. FasL 72. FGFR1 * 73. FGF2 [bFGF] </dd></dl>

<dl><dt>33. COX2 34. CSF-1 35. CSF-1R / fms 36. cIAP1 37. cIAP2 38. c-abl </dt><dd>74. 53BP1 75. 53BP2 76. GALC (galactosylceramidase) * </dd></dl>

<dl><dt>39. c-kit 40. c-kit L </dt><dd /></dl>

p0042941. c-met

p00430Gen Registration No.

p00431ABCB1 NM_000927 ABCC1 NM_004996 ABCC2 NM_000392 ABCC3 NM_003786 ABCC4 NM_005845 ABL1 NM_005157 ABL2 NM_005158 ACTB NM_001101

p00432extracellular proteinase *

<dl><dt>90.</dt><dd> Hepatocyte nuclear factor 3 ^ </dd></dl>

<dl><dt>91.</dt><dd> HER-2 </dd></dl>

<dl><dt>92.</dt><dd> HGF / Dispersion Factor </dd></dl>

<dl><dt>93.</dt><dd> hIAP1 </dd></dl>

<dl><dt>94.</dt><dd> hIAP2 </dd></dl>

<dl><dt>95.</dt><dd> HIF-1 </dd></dl>

<dl><dt>96.</dt><dd> Human Calicrein 10 </dd></dl>

<dl><dt>97.</dt><dd> MLH1 </dd></dl>

<dl><dt>98.</dt><dd> hsp 27 </dd></dl>

<dl><dt>99. </dt><dd>Human Chorionic Gonadotropin / CGA </dd></dl>

<dl><dt>100.</dt><dd> S1-5 human extracellular protein </dd></dl>

<dl><dt>101.</dt><dd> Id-1 </dd></dl>

<dl><dt>102. </dt><dd>ld-2 </dd></dl>

<dl><dt>103.</dt><dd> Id-3 </dd></dl>

<dl><dt>104.</dt><dd> IGF-1 </dd></dl>

<dl><dt>105.</dt><dd> IGF2 </dd></dl>

<dl><dt>106.</dt><dd> IGF1R </dd></dl>

<dl><dt>107.</dt><dd> IGFBP3 </dd></dl>

<dl><dt>108.</dt><dd> Interstitial Integrin Alpha 7 </dd></dl>

<dl><dt>109.</dt><dd> IL6 </dd></dl>

<dl><dt>110.</dt><dd> IL8 </dd></dl>

<dl><dt>111.</dt><dd> IRF-2 * </dd></dl>

<dl><dt>112.</dt><dd> IRF9 protein </dd></dl>

<dl><dt>113.</dt><dd> Calicrein 5 </dd></dl>

<dl><dt>114.</dt><dd> Calicrein 6 </dd></dl>

TABLE 2

p00434Direct primer SEQ ID NO. 1 4 7 10 13 16 19 22

<dl><dt>128. mdm2 </dt><dd>169. Binding protein </dd></dl>

<dl><dt>to retinol 4 ^ </dt><dd /></dl>

<dl><dt> 129. MDR-1 </dt><dd>170. STK15 / BTAK </dd></dl>

<dl><dt>130. Epoxy </dt><dd>171. Survivina </dd></dl>

<dl><dt>microsomal hydrolase </dt><dd /></dl>

<dl><dt>131. MMP9 </dt><dd>172. SXR </dd></dl>

<dl><dt>132. MRP1 </dt><dd>173. Syk </dd></dl>

<dl><dt>133 MRP2 </dt><dd>174. TGD (thymine-DNA </dd></dl>

<dl><dt>glycosylase) * </dt><dd /></dl>

<dl><dt>134 MRP3 </dt><dd> 175. TGFalfa </dd></dl>

<dl><dt>135 MRP4 </dt><dd>176. Thymidine kinase </dd></dl>

<dl><dt>136. MSN (Moesina) * </dt><dd>177. Thymidine </dd></dl>

<dl><dt>phosphorylase </dt><dd /></dl>

<dl><dt>137. mTOR. </dt><dd>178. Thymidylate synthase </dd></dl>

<dl><dt>138. Muc1 / CA 15-3 </dt><dd>179. Topoisomerase II</dd></dl>

<dl><dt>α </dt><dd /></dl>

<dl><dt>139. NF-KB </dt><dd>180. Topoisomerase II</dd></dl>

<dl><dt>β </dt><dd /></dl>

<dl><dt>140. P14ARF </dt><dd>181. TRAMP </dd></dl>

<dl><dt>182. UPA </dt><dd /></dl>

<dl><dt>183 VEGF </dt><dd /></dl>

<dl><dt>141. P16INK4a / p14 </dt><dd>184. Vimentin </dd></dl>

<dl><dt>142. p21wAF1 / CIP1 </dt><dd>185. WTH3 </dd></dl>

<dl><dt>186. XAF1 </dt><dd /></dl>

<dl><dt>143. p23 </dt><dd>187. XIAT </dd></dl>

<dl><dt>144. p27 </dt><dd>188. XIST </dd></dl>

<dl><dt>145. p311 * </dt><dd>189. XPA </dd></dl>

<dl><dt>146. p53 </dt><dd>190. YB-1 </dd></dl>

<dl><dt>147. PAI1 </dt><dd /></dl>

<dl><dt>148. PCNA </dt><dd>* Farm sensitivity. </dd></dl>

<dl><dt>149. PDGF-A </dt><dd>NCI 60 / Marker </dd></dl>

<dl><dt>resistance </dt><dd /></dl>

<dl><dt>150 PDGF-B </dt><dd /></dl>

<dl><dt>151. PDGF-C </dt><dd>^ Tumor subclass </dd></dl>

<dl><dt>152. PDGF-D </dt><dd>of definition in </dd></dl>

<dl><dt>grouping </dt><dd /></dl>

<dl><dt>153. PDGPR-α </dt><dd /></dl>

<dl><dt>154 PDGFR-β </dt><dd> 19/1/02 </dd></dl>

155. PI3K

p00436Amplicon reverse primer SEQ ID NO. SEQ ID NO. 2 3 5 6 8 9 11 12 14 15 17 18 20 21 23 24

p00438AKT1 NM_005163 25 26 27 AKT3 NM_005465 28 29 30 ALDH1 NM_000689 31 32 33 ALDH1A3 NM_000693 34 35 36 APC NM_000038 37 38 39 AREG NM_001657 40 41 42 B2M NM_004048 43 44 45 BAK1 NM_001188 46 47 48 BAX NM_004322 534 542 534 NM4004322 534 524CL2 BCL2L1 NM_001191 55 56 57 BIRC3 NM_001165 58 59 60 BIRC4 NM_001167 61 62 63 BIRC5 NM_001168 64 65 66 BRCA1 NM_007295 67 68 69 BRCA2 NM_000059 70 71 72 CCND1 NM_001758 73 74 75 CD3Z NM_000700 76 77 78 CD79 8317 8317 CDC 7979 8317 8317 CDC 83 791 797 CDC 83 791 797 CDC 83 84 CDH1 NM_004360 85 86 87 CDKN1A NM_000389 88 89 90 CDKN1B NM_004064 91 92 93 CDKN2A NM_000077 94 95 96 CYP1B1 NM_000104 97 98 99 DHFR NM_000791 100 101 102 DPYD NM_000110 103 104 105 ECGF1 NM_001953 106 107 108 EGF NM 1101 112 107 114 112 112 NMF 112 107 112 114 112 112 NMR 112 107 ERBB2 NM_004448 115 116 117 ERBB3 NM_001982 118 119 120 ESR1 NM_000125 121 122 123 ESR2 NM_001437 124 125 126 GAPD NM_002046 127 128 129 GATA3 NM_002051 130 131 132 GRB7 NM_005310 133 134 135 GRO1 NM_001511 136 137 138 GSTP1 NM_000852 139 140 141 GUSB NM_000181 142 143 144 hHGF M29145 145 146 147 HNF3A NM_004496 148 149 150 ID2 NM_002166 151 152 153 IGF1 NM_000618 154 155 156 IGFBP3 NM_000598 157 158 159 ITGA7 NM_002206 160 161 162 NR162 165 161 167 NR16 165 K 165 160 167 165 NM 165 KIT NM_000222 169 170 171 KITLG NM_000899 172 173 174 KRT17 NM_000422 175 176 177 KRT5 NM_000424 178 179 180

p00440LPL NM_000237 181 182 183 MET NM_000245 184 185 186 MKI67 NM_002417 187 188 189 MVP NM_017458 190 191 192 MYC NM_002467 193 194 195 PDGFA NM_002607 196 197 198 PDGFB NM_002608 199 200 201 PDGFC NM_016205 202 203 204 PDGFRA NM_006206 205 206 207 PDGFRB NM_002609 208 209 210 PGK1 NM_000291 211 212 213 PGR NM_000926 214 215 216 PIN1 NM_006221 217 218 219 PLAU NM_002658 220 221 222 PPIH NM_006347 223 224 225 PTEN NM_000314 226 227 228 PTGS2 NM_000963 229 230 231 RBP4 23 233 RBP4 233 233 RBP4 233 RELA NM_021975 235 236 237 RPL19 NM_000981 238 239 240 RPLPO NM_001002 241 242 243 SCDGF-B NM_025208 244 245 246 SERPINE1 NM_000602 247 248 249 SLC19A1 NM_003056 250 251 252 TBP NM_003251 258 251 258 251 258 251 258 251 251 251 251 251 251 251 251 251 258 251 251 251 251 251 251 251 258 251 251 258 251 251 258 251 258 251 251 258 251 258 251 256 263 264 TNFRSF6 NM_000043 265 266 267 TNFSF6 NM_000639 268 269 270 TOP2A NM_001067 271 272 273 TOP2B NM_001068 274 275 276 TP53 NM_000546 277 278 279 TYMS NM_001071 280 281 282 VEGF NM_00335 283 283 283 283 283 283

TABLE 3

<dl><dt>GEN </dt><dd>REGISTRATION NO. SEQ ID NO: </dd></dl>

<dl><dt>AK055699 </dt><dd /><dt>AK055699 </dt><dd> 286 </dd></dl>

<dl><dt>BAG1 </dt><dd>NM_004323 287 </dd></dl>

<dl><dt>BBC3 </dt><dd>NM_014417 288 </dd></dl>

<dl><dt>Bcl2 </dt><dd>NM_000633 289 </dd></dl>

<dl><dt>BRCA2 </dt><dd>NM_000059 290 </dd></dl>

<dl><dt>CA9 </dt><dd>NM_001216 291 </dd></dl>

<dl><dt>CCNB1 </dt><dd>NM_031966 292 </dd></dl>

<dl><dt>CDC25B </dt><dd>NM_021874 293 </dd></dl>

<dl><dt>CEGP1 </dt><dd>NM_020974 294 </dd></dl>

<dl><dt>Chk1 </dt><dd>NM_001274 295 </dd></dl>

<dl><dt>Chk2 </dt><dd>NM_007194 296 </dd></dl>

<dl><dt>CYP3A4 </dt><dd>NM_017460 297 </dd></dl>

<dl><dt>DIABLO </dt><dd>NM_019887 298 </dd></dl>

<dl><dt>DPYD </dt><dd>NM_000110 299 </dd></dl>

<dl><dt>EGFR </dt><dd>NM_005228 300 </dd></dl>

<dl><dt>EpCAM </dt><dd>NM_002354 301 </dd></dl>

<dl><dt>EPHX1 </dt><dd>NM_000120 302 </dd></dl>

<dl><dt>EstR1 </dt><dd>NM_000125 303 </dd></dl>

<dl><dt>FOXM1 </dt><dd>NM_021953 304 </dd></dl>

<dl><dt>GATA3 </dt><dd>NM_002051 305 </dd></dl>

<dl><dt>GSTM1 </dt><dd>NM_000561 306 </dd></dl>

<dl><dt>GSTM3 </dt><dd>NM_000849 307 </dd></dl>

<dl><dt>hENT1 </dt><dd>NM_004955 308 </dd></dl>

<dl><dt>HIF1A </dt><dd>NM_001530 309 </dd></dl>

<dl><dt>HNF3A </dt><dd>NM_004496 310 </dd></dl>

<dl><dt>ID1 </dt><dd>NM_002165 311 </dd></dl>

<dl><dt>IGF1R </dt><dd>NM_000875 312 </dd></dl>

<dl><dt>Ki-67 </dt><dd>NM_002417 313 </dd></dl>

<dl><dt>NFKBp65 </dt><dd>NM_021975 314 </dd></dl>

<dl><dt>NME1 </dt><dd>NM_000269 315 </dd></dl>

<dl><dt>p27 </dt><dd>NM_004064 316 </dd></dl>

<dl><dt>P13KC2A </dt><dd>NM_002645 317 </dd></dl>

<dl><dt>PR </dt><dd>NM_000926 318 </dd></dl>

<dl><dt>PRAME </dt><dd>NM_006115 319 </dd></dl>

<dl><dt>pS2 </dt><dd>NM_003225 320 </dd></dl>

<dl><dt>RPS6KB1 </dt><dd>NM_003161 321 </dd></dl>

<dl><dt>Src </dt><dd>NM_004383 322 </dd></dl>

<dl><dt>STK15 </dt><dd>NM_003600 323 </dd></dl>

<dl><dt>SURV </dt><dd>NM_001168 324 </dd></dl>

<dl><dt>TFRC </dt><dd>NM_003234 325 </dd></dl>

<dl><dt>TGFB3 </dt><dd>NM_003239 326 </dd></dl>

<dl><dt>TK1 </dt><dd>NM_003258 327 </dd></dl>

<dl><dt>VDR </dt><dd>NM_000376 328 </dd></dl>

<dl><dt>VEGFC </dt><dd>NM_005429 329 </dd></dl>

<dl><dt>WISP1 </dt><dd>NM_003882 330 </dd></dl>

<dl><dt>XIAP </dt><dd>NM_001167 331 </dd></dl>

<dl><dt>YB-1 </dt><dd>NM_004559 332 </dd></dl>

<dl><dt>ITGA7 </dt><dd>NM_002206 333 </dd></dl>

<dl><dt>PDGFB </dt><dd>NM_002608 334 </dd></dl>

<dl><dt>Upa </dt><dd>NM_002658 335 </dd></dl>

<dl><dt>TBP </dt><dd>NM_003194 336 </dd></dl>

<dl><dt>PDGFRa </dt><dd>NM_006206 337 </dd></dl>

<dl><dt>Pin1 </dt><dd>NM_006221 338 </dd></dl>

<dl><dt>CYP </dt><dd>NM_006347 339 </dd></dl>

<dl><dt>RBP4 </dt><dd>NM_006744 340 </dd></dl>

<dl><dt>BRCA1 </dt><dd>NM_007295 341 </dd></dl>

<dl><dt>APC </dt><dd>NM_000038 342 </dd></dl>

<dl><dt>GUS </dt><dd>NM_000161 343 </dd></dl>

<dl><dt>CD18 </dt><dd>NM_000211 344 </dd></dl>

<dl><dt>PTEN </dt><dd>NM_000314 345 </dd></dl>

<dl><dt>P53 </dt><dd>NM_000546 346 </dd></dl>

<dl><dt>ALDH1A3 </dt><dd>NM_000693 347 </dd></dl>

<dl><dt>GSTp </dt><dd>NM_000852 348 </dd></dl>

<dl><dt>TOP2B </dt><dd>NM_001068 349 </dd></dl>

<dl><dt>TS </dt><dd>NM_001071 350 </dd></dl>

<dl><dt>Bclx </dt><dd>NM_001191 351 </dd></dl>

<dl><dt>AREG </dt><dd>NM_001657 352 </dd></dl>

<dl><dt>TP </dt><dd>NM_001953 353 </dd></dl>

<dl><dt>EIF4E </dt><dd>NM_001968 354 </dd></dl>

<dl><dt>ErbB3 </dt><dd>NM_001982 355 </dd></dl>

<dl><dt>EREG </dt><dd>NM_001432 356 </dd></dl>

<dl><dt>GCLC </dt><dd>NM_001498 357 </dd></dl>

<dl><dt>CD9 </dt><dd>NM_001769 358 </dd></dl>

<dl><dt>HB-EGF </dt><dd>NM_001945 359 </dd></dl>

<dl><dt>IGFBP2 </dt><dd>NM_000597 360 </dd></dl>

<dl><dt>CTSL </dt><dd>NM_001912 361 </dd></dl>

<dl><dt>PREP </dt><dd>NM_002726 362 </dd></dl>

<dl><dt>CYP3A4 </dt><dd>NM_017460 363 </dd></dl>

<dl><dt>ILT-2 </dt><dd>NM_006669 364 </dd></dl>

<dl><dt>MCM3 </dt><dd>NM_002388 365 </dd></dl>

<dl><dt>KRT19 </dt><dd>NM_002276 366 </dd></dl>

<dl><dt>KRT18 </dt><dd>NM_000224 367 </dd></dl>

<dl><dt>TIMP2 </dt><dd>NM_003255 368 </dd></dl>

<dl><dt>BAD </dt><dd>NM_004322 369 </dd></dl>

<dl><dt>CYP2C8 </dt><dd>NM_030878 370 </dd></dl>

<dl><dt>DCR3 </dt><dd>NM_016434 371 </dd></dl>

<dl><dt>PLAUR </dt><dd>NM_002859 372 </dd></dl>

<dl><dt>PI3KC2A </dt><dd>NM_002645 373 </dd></dl>

<dl><dt>FGF2 </dt><dd>NM_002006 374 </dd></dl>

<dl><dt>HLA-G </dt><dd>NM_002127 375 </dd></dl>

<dl><dt>AIB1 </dt><dd>NM_005534 376 </dd></dl>

<dl><dt>MCP1 </dt><dd>NM_002982 377 </dd></dl>

<dl><dt>Contig46653 </dt><dd /><dt>Contig46653 </dt><dd> 378 </dd></dl>

<dl><dt>RhoC </dt><dd>NM_005167 379 </dd></dl>

<dl><dt>DR5 </dt><dd>NM_003842 380 </dd></dl>

<dl><dt>RAD51C </dt><dd>NM_058216 381 </dd></dl>

<dl><dt>BIN1 </dt><dd>NM_004305 382 </dd></dl>

<dl><dt>VDR </dt><dd>NM_000376 383 </dd></dl>

<dl><dt>TERC </dt><dd>U86046 384 </dd></dl>

p00443Lista de secuencias

p00444<110> GENOMIC HEALTH

p00445Baker, Joffre B. Cronin, Maureen T. Kiefer, Michael C. Shak, Steve Walker, Michael Graham 5

p00446<120> OBTENCIÓN DE PERFIL DE EXPRESIÓN GÉNICA EN TEJIDOS TUMORALES BIOPSIADOS

p00447<130> H64199PCEPT1

p00448<140> por asignar

p00449<141>

p00450<150> US 60/412.049

p00451<151> 15

p00452<150> US 60/364.890

p00453<151>

p00454<160> 384

p00455<170> FastSEQ para Windows versión 4.0

p00456<210> 1

p00457<211> 18 25 <212> ADN

p00458<213> Homo sapiens

p00459<400> 1

p00460<210> 2

p00461<211> 19

p00462<212> ADN

p00463<213> Homo sapiens

p00464<400> 2

p00465<210> 3

p00466<211> 68

p00467<212> ADN

p00468<213> Homo sapiens

p00469<400> 3

p00470<211> 18

p00471<212> ADN

p00472<213> Homo sapiens

p00473<400>4

p00474<210> 5

p00475<211> 23 55 <212> ADN

p00476<213> Homo sapiens

p00477<400> 5

p00478<210> 6

p00479<211> 79

p00480<212> ADN

p00481<213> Homo sapiens

p00483<400> 6

p00484<210> 7

p00485<211> 20

p00486<212> ADN

p00487<213> Homo sapiens

p00488<211> 20

p00489<212> ADN

p00490<213> Homo sapiens

p00491<400> 8

p00492<210> 9 20 <211> 65

p00493<212> ADN

p00494<213> Homo sapiens

p00495<210> 10

p00496<211> 22

p00497<212> ADN 30 <213> Homo sapiens

p00498<400>10

p00499<210> 11 35 <211> 20

p00500<212> ADN

p00501<213> Homo sapiens

p00502<211> 91

p00503<212> ADN

p00504<213> Homo sapiens

p00505<400> 12

p00506<210> 13

p00507<211> 20 50 <212> ADN

p00508<213> Homo sapiens

p00509<400> 13

55 <210> 14

p00510<211> 20

p00511<212> ADN

p00512<213> Homo sapiens

p0051360 <400> 14

p00515<210> 15

p00516<211> 66

p00517<212> ADN

p00518<213> Homo sapiens

p00519<400> 15

p00520<210> 16 10 <211> 24

p00521<212> ADN

p00522<213> Homo sapiens

p00523<211> 22

p00524<212> ADN

p00525<213> Homo sapiens

p00526<400> 17

p00527<210> 18

p00528<211> 94 25 <212> ADN

p00529<213> Homo sapiens

p00530<400> 18

p0053130 <210> 19

p00532<211> 21

p00533<212> ADN

p00534<213> Homo sapiens

p0053535 <400> 19

p00536<210> 20

p00537<211> 21 40 <212> ADN

p00538<213> Homo sapiens

p00539<400> 20

p0054045 <210> 21

p00541<211> 80

p00542<212> ADN

p00543<213> Homo sapiens

p0054450 <400> 21

p00545<210> 22

p00546<211> 21

p00547<212> ADN 55 <213> Homo sapiens

p00548<400> 22

p00550<210> 23

p00551<211> 18

p00552<212> ADN

p00553<213> Homo sapiens

p00554<400> 23

p00555<210> 24 10 <211> 66

p00556<212> ADN

p00557<213> Homo sapiens

p00558<210> 25

p00559<211> 20

p00560<212> ADN 20 <213> Homo sapiens

p00561<400> 25

p00562<210> 26 25 <211> 20

p00563<212> ADN

p00564<213> Homo sapiens

p00565<210> 27

p00566<211> 71

p00567<212> ADN 35 <213> Homo sapiens

p00568<400> 27

p0056940 <210> 28

p00570<211> 25

p00571<212> ADN

p00572<213> Homo sapiens

p0057345 <400> 28

p00574<210> 29

p00575<211> 24 50 <212> ADN

p00576<213> Homo sapiens

p00577<400> 29

<dl><dt>55 </dt><dd /></dl>

<dl><dt><210> 30 </dt><dd /></dl>

<dl><dt><211> 75 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>60 </dt><dd /></dl>

<dl><dt><400> 30 </dt><dd /></dl>

p00579<210> 31

p00580<211> 25

p00581<212> ADN

p00582<213> Homo sapiens

p00583<400> 31

p00584<210> 32 10 <211> 18

p00585<212> ADN

p00586<213> Homo sapiens

p00587<211> 74

p00588<212> ADN

p00589<213> Homo sapiens

p00590<400> 33

p00591<210> 34

p00592<211> 21 25 <212> ADN

p00593<213> Homo sapiens

p00594<400> 34

p0059530 <210> 35

p00596<211> 22

p00597<212> ADN

p00598<213> Homo sapiens

p0059935 <400> 35

p00600<210> 36

p00601<211> 80

p00602<212> ADN 40 <213> Homo sapiens

p00603<400> 36

p00604<210> 37 45 <211> 20

p00605<212> ADN

p00606<213> Homo sapiens

p00607<210> 38

p00608<211> 20

p00609<212> ADN 55 <213> Homo sapiens

p00610<400> 38

p00611<210> 39

p00613<211> 69

p00614<212> ADN

p00615<213> Homo sapiens

p00616<400> 39

p00617<210> 40

p00618<211> 27 10 <212> ADN

p00619<213> Homo sapiens

p00620<400> 40

p0062115 <210> 41

p00622<211> 27

p00623<212> ADN

p00624<213> Homo sapiens

p0062520 <400> 41

p00626<210> 42

p00627<211> 82

p00628<212> ADN 25 <213> Homo sapiens

p00629<400> 42

p00630<210> 43 30 <211> 19

p00631<212> ADN

p00632<213> Homo sapiens

p00633<210> 44

p00634<211> 24

p00635<212> ADN 40 <213> Homo sapiens

p00636<400> 44

p0063745 <210> 45

p00638<211> 93

p00639<212> ADN

p00640<213> Homo sapiens

p0064150 <400> 45

p00642<210> 46

p00643<211> 20 55 <212> ADN

p00644<213> Homo sapiens

p00645<400> 46

p00647<210> 47

p00648<211> 20

p00649<212> ADN

p00650<213> Homo sapiens

p00651<400> 47

p00652<210> 48

<dl><dt><211></dt><dd> 66 10 <212> ADN </dd></dl>

<213> Homo sapiens

<400> 48

<210> 50

<dl><dt><211></dt><dd> 21 25 <212> ADN </dd></dl>

<dl><dt>15 </dt><dd /></dl>

<dl><dt><210> 49 </dt><dd /></dl>

<dl><dt><211> 18 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>20 </dt><dd /></dl>

<dl><dt><400> 49 </dt><dd /></dl>

p00653<213> Homo sapiens

p00654<400> 50

p0065530 <210> 51

p00656<211> 70

p00657<212> ADN

p00658<213> Homo sapiens

p0065935 <400> 51

p00660<210> 52

p00661<211> 25 40 <212> ADN

p00662<213> Homo sapiens

p00663<400> 52

p0066445 <210> 53

p00665<211> 24

p00666<212> ADN

p00667<213> Homo sapiens

p0066850 <400> 53

p00669<210> 54

p00670<211> 73

p00671<212> ADN 55 <213> Homo sapiens

p00672<400> 54

p0067360 <210> 55

p00675<211> 24

p00676<212> ADN

p00677<213> Homo sapiens

p00678<400> 55

p00679<210> 56

p00680<211> 19

p00681<212> ADN 10 <213> Homo sapiens

p00682<400> 56

p00683<210> 57 15 <211> 70

p00684<212> ADN

p00685<213> Homo sapiens

p00686<211> 24

p00687<212> ADN

p00688<213> Homo sapiens

p00689<400> 58

p00690<210> 59

p00691<211> 25 30 <212> ADN

p00692<213> Homo sapiens

p00693<400> 59

p0069435 <210> 60

p00695<211> 86

p00696<212> ADN

p00697<213> Homo sapiens

p0069840 <400> 60

p00699<210> 61

p00700<211> 23

p00701<212> ADN 45 <213> Homo sapiens

p00702<400> 61

p00703<210> 62 50 <211> 21

p00704<212> ADN

p00705<213> Homo sapiens

p00706<210> 63

p00707<211> 77

p00708<212> ADN

p00709<213> Homo sapiens

p00711<400> 63

p00712<210> 64

p00713<211> 20

p00714<212> ADN

p00715<213> Homo sapiens

p00716<211> 24

p00717<212> ADN

p00718<213> Homo sapiens

p00719<400> 65

p00720<210> 66

<dl><dt><211></dt><dd> 80 20 <212> ADN </dd></dl>

<213> Homo sapiens

<400> 66

<210> 68

<dl><dt><211></dt><dd> 20 35 <212> ADN </dd></dl>

<dl><dt>25 </dt><dd /></dl>

<dl><dt><210> 67 </dt><dd /></dl>

<dl><dt><211> 20 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>30 </dt><dd /></dl>

<dl><dt><400> 67 </dt><dd /></dl>

p00721<213> Homo sapiens

p00722<400> 68

p0072340 <210> 69

p00724<211> 65

p00725<212> ADN

p00726<213> Homo sapiens

p0072745 <400> 69

p00728<210> 70

p00729<211> 20

p00730<212> ADN 50 <213> Homo sapiens

p00731<400> 70

p00732<210> 71 55 <211> 20

p00733<212> ADN

p00734<213> Homo sapiens

p00735<400> 71

p00737<210> 72

p00738<211> 70

p00739<212> ADN

p00740<213> Homo sapiens

p00741<400> 72

<dl><dt>10 </dt><dd /></dl>

<dl><dt><210> 73 </dt><dd /></dl>

<dl><dt><211> 21 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>15 </dt><dd /></dl>

<dl><dt><400> 73 </dt><dd /></dl>

p00742<210> 74

<dl><dt><211></dt><dd> 22 20 <212> ADN </dd></dl>

<213> Homo sapiens

<400> 74

<210> 76

<dl><dt><211></dt><dd> 20 35 <212> ADN </dd></dl>

<dl><dt>25 </dt><dd /></dl>

<dl><dt><210> 75 </dt><dd /></dl>

<dl><dt><211> 69 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>30 </dt><dd /></dl>

<dl><dt><400> 75 </dt><dd /></dl>

p00743<213> Homo sapiens

p00744<400> 76

p0074540 <210> 77

p00746<211> 21

p00747<212> ADN

p00748<213> Homo sapiens

p0074945 <400> 77

p00750<210> 78

p00751<211> 65 50 <212> ADN

p00752<213> Homo sapiens

p00753<400> 78

p0075455 <210> 79

p00755<211> 18

p00756<212> ADN

p00757<213> Homo sapiens

p00759<400> 79

p00760<210> 80

p00761<211> 19

p00762<212> ADN

p00763<213> Homo sapiens

p00764<400> 80

p0076510 <210> 81

p00766<211> 74

p00767<212> ADN

p00768<213> Homo sapiens

p0076915 <400> 81

p00770<210> 82

p00771<211> 20 20 <212> ADN

p00772<213> Homo sapiens

p00773<400> 82

p0077425 <210> 83

p00775<211> 21

p00776<212> ADN

p00777<213> Homo sapiens

p0077830 <400> 83

p00779<210> 84

p00780<211> 71

p00781<212> ADN 35 <213> Homo sapiens

p00782<400> 84

p00783<210> 85 40 <211> 21

p00784<212> ADN

p00785<213> Homo sapiens

p00786<211> 21

p00787<212> ADN

p00788<213> Homo sapiens

p00789<400> 86

p00790<210> 87

p00791<211> 81 55 <212> ADN

p00792<213> Homo sapiens

p00793<400> 87

p00795<210> 88

p00796<211> 21

p00797<212> ADN

p00798<213> Homo sapiens

p00799<400> 88

p00800<210> 89

p00801<211> 22 10 <212> ADN

p00802<213> Homo sapiens

p00803<400> 89

p0080415 <210> 90

p00805<211> 65

p00806<212> ADN

p00807<213> Homo sapiens

p0080820 <400> 90

p00809<210> 91

p00810<211> 21

p00811<212> ADN 25 <213> Homo sapiens

p00812<400> 91

p0081330 <210> 92

p00814<211> 19

p00815<212> ADN

p00816<213> Homo sapiens

p0081735 <400> 92

p00818<210> 93

p00819<211> 66 40 <212> ADN

p00820<213> Homo sapiens

p00821<400> 93

p0082245 <210> 94

p00823<211> 19

p00824<212> ADN

p00825<213> Homo sapiens

p0082650 <400> 94

p00827<210> 95

p00828<211> 23

p00829<212> ADN 55 <213> Homo sapiens

p00830<400> 95

p00831<210> 96 60 <211> 70

p00833<212> ADN

p00834<213> Homo sapiens

p00835<211> 22

p00836<212> ADN

p00837<213> Homo sapiens

p00838<400> 97

p00839<210> 98

p00840<211> 20 15 <212> ADN

p00841<213> Homo sapiens

p00842<400> 98

p0084320 <210> 99

p00844<211> 71

p00845<212> ADN

p00846<213> Homo sapiens

p0084725 <400> 99

p00848<210> 100

p00849<211> 27

p00850<212> ADN 30 <213> Homo sapiens

p00851<400> 100

p00852<210> 101 35 <211> 22

p00853<212> ADN

p00854<213> Homo sapiens

p00855<400> 101

p00856<211> 73

p00857<212> ADN

p00858<213> Homo sapiens

p00859<400> 102

p00860<210> 103

p00861<211> 19 50 <212> ADN

p00862<213> Homo sapiens

p00863<400> 103

55 <210> 104

p00864<211> 21

p00865<212> ADN

p00866<213> Homo sapiens

p0086760 <400> 104

p00869<210> 105

p00870<211> 87

p00871<212> ADN

p00872<213> Homo sapiens

p00873<400> 105

p0087410 <210> 106

p00875<211> 24

p00876<212> ADN

p00877<213> Homo sapiens

p0087815 <400> 106

p00879<210> 107

p00880<211> 24

p00881<212> ADN 20 <213> Homo sapiens

p00882<400> 107

p0088325 <210> 108

p00884<211> 82

p00885<212> ADN

p00886<213> Homo sapiens

p0088730 <400> 108

p00888<210> 109

p00889<211> 20

p00890<212> ADN 35 <213> Homo sapiens

p00891<400> 109

<dl><dt><210></dt><dd> 110 40 <211> 19 </dd></dl>

<212> ADN

<213> Homo sapiens

<211> 62

<212> ADN

<213> Homo sapiens

<400> 111

<dl><dt><210></dt><dd> 112 55 <211> 23 </dd></dl>

p00892<212> ADN

p00893<213> Homo sapiens

p00895<400> 112

p00896<210> 113

p00897<211> 25

p00898<212> ADN

p00899<213> Homo sapiens

p00900<210> 114

p00901<211> 82

p00902<212> ADN 15 <213> Homo sapiens

p00903<400> 114

p0090420 <210> 115

p00905<211> 20

p00906<212> ADN

p00907<213> Homo sapiens

p0090825 <400> 115

p00909<210> 116

p00910<211> 29 30 <212> ADN

p00911<213> Homo sapiens

p00912<400> 116

<dl><dt>35 </dt><dd /></dl>

<dl><dt><210> 117 </dt><dd /></dl>

<dl><dt><211> 70 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>40 </dt><dd /></dl>

<dl><dt><400> 117 </dt><dd /></dl>

p00913<210> 118 45 <211> 23

p00914<212> ADN

p00915<213> Homo sapiens

p00916<210> 119

p00917<211> 24

p00918<212> ADN 55 <213> Homo sapiens

p00919<400> 119

p0092060 <210> 120

p00922<211> 81

p00923<212> ADN

p00924<213> Homo sapiens

p00925<400> 120

p00926<210> 121

p00927<211> 19 10 <212> ADN

p00928<213> Homo sapiens

p00929<400> 121

<dl><dt>15 </dt><dd /></dl>

<dl><dt><210> 122 </dt><dd /></dl>

<dl><dt><211> 19 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>20 </dt><dd /></dl>

<dl><dt><400> 122 </dt><dd /></dl>

p00930<210> 123 25 <211> 68

p00931<212> ADN

p00932<213> Homo sapiens

p00933<400> 123

p00934<210> 124

p00935<211> 20

p00936<212> ADN 35 <213> Homo sapiens

p00937<400> 124

p0093840 <210> 125

p00939<211> 23

p00940<212> ADN

p00941<213> Homo sapiens

p0094245 <400> 125

p00943<210> 126

p00944<211> 76 50 <212> ADN

p00945<213> Homo sapiens

p00946<400> 126

p00947<210> 127

p00948<211> 24

p00949<212> ADN

p00950<213> Homo sapiens

p00952<400> 127

p009535 <210> 128

p00954<211> 21

p00955<212> ADN

p00956<213> Homo sapiens

p0095710 <400> 128

p00958<210> 129

p00959<211> 74 15 <212> ADN

p00960<213> Homo sapiens

p00961<400> 129

<dl><dt>20 </dt><dd /></dl>

<dl><dt><210> 130 </dt><dd /></dl>

<dl><dt><211> 23 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>25 </dt><dd /></dl>

<dl><dt><400> 130 </dt><dd /></dl>

p00962<210> 131 30 <211> 26

p00963<212> ADN

p00964<213> Homo sapiens

p00965<210> 132

p00966<211> 75

p00967<212> ADN 40 <213> Homo sapiens

p00968<400> 132

p0096945 <210> 133

p00970<211> 20

p00971<212> ADN

p00972<213> Homo sapiens

p0097350 <400> 133

p00974<210> 134

p00975<211> 20 55 <212> ADN

p00976<213> Homo sapiens

p00977<400> 134

p00979<210> 135

p00980<211> 67

p00981<212> ADN

p00982<213> Homo sapiens

p00983<400> 135

p00984<210> 136 10 <211> 23

p00985<212> ADN

p00986<213> Homo sapiens

p00987<210> 137

p00988<211> 20

p00989<212> ADN 20 <213> Homo sapiens

p00990<400> 137

p0099125 <210> 138

p00992<211> 73

p00993<212> ADN

p00994<213> Homo sapiens

p0099530 <400> 138

p00996<210> 139

p00997<211> 20 35 <212> ADN

p00998<213> Homo sapiens

p00999<400> 139

<dl><dt>40 </dt><dd /></dl>

<dl><dt><210> 140 </dt><dd /></dl>

<dl><dt><211> 23 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>45 </dt><dd /></dl>

<dl><dt><400> 140 </dt><dd /></dl>

p01000<210> 141 50 <211> 76

p01001<212> ADN

p01002<213> Homo sapiens

p01003<210> 142

p01004<211> 20

p01006<212> ADN

p01007<213> Homo sapiens

p01008<210> 143

p01009<211> 20

p01010<212> ADN 10 <213> Homo sapiens

p01011<400> 143

p0101215 <210> 144

p01013<211> 73

p01014<212> ADN

p01015<213> Homo sapiens

p0101620 <400> 144

p01017<210> 145

p01018<211> 24 25 <212> ADN

p01019<213> Homo sapiens

p01020<400> 145

<dl><dt>30 </dt><dd /></dl>

<dl><dt><210> 146 </dt><dd /></dl>

<dl><dt><211> 26 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>35 </dt><dd /></dl>

<dl><dt><400> 146 </dt><dd /></dl>

p01021<210> 147 40 <211> 85

p01022<212> ADN

p01023<213> Homo sapiens

p01024<210> 148

p01025<211> 24

p01026<212> ADN 50 <213> Homo sapiens

p01027<400> 148

55 <210> 149

p01028<211> 22

p01029<212> ADN

p01030<213> Homo sapiens

p0103160 <400> 149

p01033<210> 150

p01034<211> 73

p01035<212> ADN

p01036<213> Homo sapiens

p01037<400> 150

<dl><dt>10 </dt><dd /></dl>

<dl><dt><210> 151 </dt><dd /></dl>

<dl><dt><211> 23 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>15 </dt><dd /></dl>

<dl><dt><400> 151 </dt><dd /></dl>

p01038<210> 152 20 <211> 22

p01039<212> ADN

p01040<213> Homo sapiens

p01041<210> 153

p01042<211> 76

p01043<212> ADN 30 <213> Homo sapiens

p01044<400> 153

p0104535 <210> 154

p01046<211> 21

p01047<212> ADN

p01048<213> Homo sapiens

p0104940 <400> 154

p01050<210> 155

p01051<211> 20 45 <212> ADN

p01052<213> Homo sapiens

p01053<400> 155

<dl><dt>50 </dt><dd /></dl>

<dl><dt><210> 156 </dt><dd /></dl>

<dl><dt><211> 76 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>55 </dt><dd /></dl>

<dl><dt><400> 156 </dt><dd /></dl>

p01054<210> 157

p01056<211> 17

p01057<212> ADN

p01058<213> Homo sapiens

p01059<400> 157

p01060<210> 158

p01061<211> 24 10 <212> ADN

p01062<213> Homo sapiens

p01063<400> 158

<dl><dt>15 </dt><dd /></dl>

<dl><dt><210> 159 </dt><dd /></dl>

<dl><dt><211> 68 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>20 </dt><dd /></dl>

<dl><dt><400> 159 </dt><dd /></dl>

p01064<210> 160 25 <211> 22

p01065<212> ADN

p01066<213> Homo sapiens

p01067<400> 160

p01068<210> 161

p01069<211> 21

p01070<212> ADN 35 <213> Homo sapiens

p01071<400> 161

p0107240 <210> 162

p01073<211> 68

p01074<212> ADN

p01075<213> Homo sapiens

p0107645 <400> 162

p01077<210> 163

p01078<211> 20 50 <212> ADN

p01079<213> Homo sapiens

p01080<400> 163

<dl><dt>55 </dt><dd /></dl>

<dl><dt><210> 164 </dt><dd /></dl>

<dl><dt><211> 24 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>60 </dt><dd /></dl>

p01082<400> 164

p01083<210> 165

p01084<211> 81

p01085<212> ADN

p01086<213> Homo sapiens

p01087<210> 166

p01088<211> 23

p01089<212> ADN 15 <213> Homo sapiens

p01090<400> 166

p0109120 <210> 167

p01092<211> 18

p01093<212> ADN

p01094<213> Homo sapiens

p0109525 <400> 167

p01096<210> 168

p01097<211> 88 30 <212> ADN

p01098<213> Homo sapiens

p01099<400> 168

<dl><dt>35 </dt><dd /></dl>

<dl><dt><210> 169 </dt><dd /></dl>

<dl><dt><211> 25 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>40 </dt><dd /></dl>

<dl><dt><400> 169 </dt><dd /></dl>

p01100<210> 170 45 <211> 18

p01101<212> ADN

p01102<213> Homo sapiens

p01103<400> 170

p01104<210> 171

p01105<211> 75

p01106<212> ADN 55 <213> Homo sapiens

p01107<400> 171

p01109<210> 172

p01110<211> 18

p01111<212> ADN

p01112<213> Homo sapiens

p01113<400> 172

p0111410 <210> 173

p01115<211> 25

p01116<212> ADN

p01117<213> Homo sapiens

p0111815 <400> 173

p01119<210> 174

p01120<211> 79 20 <212> ADN

p01121<213> Homo sapiens

p01122<400> 174

<dl><dt>25 </dt><dd /></dl>

<dl><dt><210> 175 </dt><dd /></dl>

<dl><dt><211> 21 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>30 </dt><dd /></dl>

<dl><dt><400> 175 </dt><dd /></dl>

p01123<210> 176 35 <211> 22

p01124<212> ADN

p01125<213> Homo sapiens

p01126<210> 177

p01127<211> 73

p01128<212> ADN 45 <213> Homo sapiens

p01129<400> 177

p0113050 <210> 178

p01131<211> 20

p01132<212> ADN

p01133<213> Homo sapiens

p0113455 <400> 178

p01135<210> 179

p01136<211> 20 60 <212> ADN

p01138<213> Homo sapiens

p01139<400> 179

<dl><dt>5 </dt><dd /></dl>

<dl><dt><210> 180 </dt><dd /></dl>

<dl><dt><211> 69 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>10 </dt><dd /></dl>

<dl><dt><400> 180 </dt><dd /></dl>

p01140<210> 181 15 <211> 26

p01141<212> ADN

p01142<213> Homo sapiens

p01143<210> 182

p01144<211> 18

p01145<212> ADN 25 <213> Homo sapiens

p01146<400> 182

p0114730 <210> 183

p01148<211> 87

p01149<212> ADN

p01150<213> Homo sapiens

p0115135 <400> 183

p01152<210> 184

p01153<211> 22 40 <212> ADN

p01154<213> Homo sapiens

p01155<400> 184

<dl><dt>45 </dt><dd /></dl>

<dl><dt><210> 185 </dt><dd /></dl>

<dl><dt><211> 20 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>50 </dt><dd /></dl>

<dl><dt><400> 185 </dt><dd /></dl>

p01156<210> 186 55 <211> 86

p01157<212> ADN

p01158<213> Homo sapiens

p01159<400> 186

p01161<210> 187

p01162<211> 24

p01163<212> ADN

p01164<213> Homo sapiens

p01165<400> 187

<dl><dt>10 </dt><dd /></dl>

<dl><dt><210> 188 </dt><dd /></dl>

<dl><dt><211> 26 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>15 </dt><dd /></dl>

<dl><dt><400> 188 </dt><dd /></dl>

p01166<210> 189 20 <211> 101

p01167<212> ADN

p01168<213> Homo sapiens

p01169<210> 190

p01170<211> 22

p01171<212> ADN 30 <213> Homo sapiens

p01172<400> 190

p0117335 <210> 191

p01174<211> 22

p01175<212> ADN

p01176<213> Homo sapiens

p0117740 <400> 191

p01178<210> 192

p01179<211> 75 45 <212> ADN

p01180<213> Homo sapiens

p01181<400> 192

<dl><dt>50 </dt><dd /></dl>

<dl><dt><210> 193 </dt><dd /></dl>

<dl><dt><211> 21 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>55 </dt><dd /></dl>

<dl><dt><400> 193 </dt><dd /></dl>

p01183<210> 194

p01184<211> 22

p01185<212> ADN

p01186<213> Homo sapiens

p01187<400> 194

p01188<210> 195 10 <211> 84

p01189<212> ADN

p01190<213> Homo sapiens

p01191<210> 196

p01192<211> 19

p01193<212> ADN 20 <213> Homo sapiens

p01194<400> 196

p0119525 <210> 197

p01196<211> 21

p01197<212> ADN

p01198<213> Homo sapiens

p0119930 <400> 197

p01200<210> 198

p01201<211> 67 35 <212> ADN

p01202<213> Homo sapiens

p01203<400> 198

<dl><dt>40 </dt><dd /></dl>

<dl><dt><210> 199 </dt><dd /></dl>

<dl><dt><211> 20 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>45 </dt><dd /></dl>

<dl><dt><400> 199 </dt><dd /></dl>

p01204<210> 200 50 <211> 20

p01205<212> ADN

p01206<213> Homo sapiens

p01207<210> 201

p01208<211> 62

p01209<212> ADN 60 <213> Homo sapiens

p01210<400> 201

p01211<210> 202

p01212<211> 28

p01213<212> ADN

p01214<213> Homo sapiens

p01215<400> 202

p01216<210> 203

p01217<211> 21

p01218<212> ADN

p01219<213> Homo sapiens

p01220<400> 203

p01221<210> 204

p01222<211> 79

p01223<212> ADN

p01224<213> Homo sapiens

p01225<400> 204

p01226<210> 205

p01227<211> 20

p01228<212> ADN

p01229<213> Homo sapiens

p01230<400> 205

p01231<210> 206

p01232<211> 20

p01233<212> ADN

p01234<213> Homo sapiens

p01235<400> 206

p01236<210> 207

p01237<211> 72

p01238<212> ADN

p01239<213> Homo sapiens

p01240<400> 207

p01241<210> 208

p01242<211> 23

p01243<212> ADN

p01244<213> Homo sapiens

p01245<400> 208 <210> 209

p01246<211> 20

p01247<212> ADN

p01248<213> Homo sapiens

p01249<400> 209

p01250<210> 210

p01251<211> 90

p01252<212> ADN

p01253<213> Homo sapiens

p01254<400> 210

p01255<210> 211

p01256<211> 24

p01257<212> ADN

p01258<213> Homo sapiens

p01259<400> 211

p01260<210> 212

p01261<211> 21

p01262<212> ADN

p01263<213> Homo sapiens

p01264<400> 212

p01265<210> 213

p01266<211> 74

p01267<212> ADN

p01268<213> Homo sapiens

p01269<400> 213

p01270<210> 214

p01271<211> 26

p01272<212> ADN

p01273<213> Homo sapiens

p01274<400> 214

p01275<210> 215

p01276<211> 19

p01277<212> ADN

p01278<213> Homo sapiens

p01279<400> 215

p01280<210> 216

p01281<211> 78

p01282<212> ADN

p01283<213> Homo sapiens

p01284<400> 216

p01285<210> 217

p01286<211> 20

p01287<212> ADN

p01288<213> Homo sapiens

p01289<400> 217

p01290<210> 218

p01291<211> 20

p01292<212> ADN

p01293<213> Homo sapiens

p01294<400> 218

p01295<210> 219

p01296<211> 68

p01297<212> ADN

p01298<213> Homo sapiens

p01299<400> 219

p01300<210> 220

p01301<211> 19

p01302<212> ADN

p01303<213> Homo sapiens

p01304<400> 220

p01305<210> 221

p01306<211> 20

p01307<212> ADN

p01308<213> Homo sapiens

p01309<400> 221

p01310<210> 222

p01311<211> 70

p01312<212> ADN

p01313<213> Homo sapiens

p01314<400> 222

p01315<210> 223

p01316<211> 27

p01317<212> ADN

p01318<213> Homo sapiens

p01319<400> 223 <210> 224

p01320<211> 22

p01321<212> ADN

p01322<213> Homo sapiens

p01323<400> 224

p01324<210> 225

p01325<211> 84

p01326<212> ADN

p01327<213> Homo sapiens

p01328<400> 225

p01329<210> 226

p01330<211> 25

p01331<212> ADN

p01332<213> Homo sapiens

p01333<400> 226

p01334<210> 227

p01335<211> 25

p01336<212> ADN

p01337<213> Homo sapiens

p01338<400> 227

p01339<210> 228

p01340<211> 81

p01341<212> ADN

p01342<213> Homo sapiens

p01343<400> 228

p01344<210> 229

p01345<211> 23

p01346<212> ADN

p01347<213> Homo sapiens

p01348<400> 229

p01349<210> 230

p01350<211> 21

p01351<212> ADN

p01352<213> Homo sapiens

p01353<400> 230

p01354<210> 231

p01355<211> 79

p01356<212> ADN

p01357<213> Homo sapiens

p01358<400> 231

p01359<210> 232

p01360<211> 24

p01361<212> ADN

p01362<213> Homo sapiens

p01363<400> 232

p01364<210> 233

p01365<211> 18

p01366<212> ADN

p01367<213> Homo sapiens

p01368<400> 233

p01369<210> 234

p01370<211> 86

p01371<212> ADN

p01372<213> Homo sapiens

p01373<400> 234

p01374<210> 235

p01375<211> 19

p01376<212> ADN

p01377<213> Homo sapiens

p01378<400> 235

p01379<210> 236

p01380<211> 22

p01381<212> ADN

p01382<213> Homo sapiens

p01383<400> 236

p01384<210> 237

p01385<211> 68

p01386<212> ADN

p01387<213> Homo sapiens

p01388<400> 237

p01389<210> 238

p01390<211> 20

p01391<212> ADN

p01392<213> Homo sapiens

p01393<400> 238 <210> 239

p01394<211> 21

p01395<212> ADN

p01396<213> Homo sapiens

p01397<400> 239

p01398<210> 240

p01399<211> 85

p01400<212> ADN

p01401<213> Homo sapiens

p01402<400> 240

p01403<210> 241

p01404<211> 24

p01405<212> ADN

p01406<213> Homo sapiens

p01407<400> 241

p01408<210> 242

p01409<211> 23

p01410<212> ADN

p01411<213> Homo sapiens

p01412<400> 242

p01413<210> 243

p01414<211> 75

p01415<212> ADN

p01416<213> Homo sapiens

p01417<400> 243

p01418<210> 244

p01419<211> 20

p01420<212> ADN

p01421<213> Homo sapiens

p01422<400> 244

p01423<210> 245

p01424<211> 20

p01425<212> ADN

p01426<213> Homo sapiens

p01427<400> 245

p01428<210> 246

p01429<211> 74

p01430<212> ADN

p01431<213> Homo sapiens

p01432<400> 246

p01433<210> 247

p01434<211> 19

p01435<212> ADN

p01436<213> Homo sapiens

p01437<400> 247

p01438<210> 248

p01439<211> 21

p01440<212> ADN

p01441<213> Homo sapiens

p01442<400> 248

p01443<210> 249

p01444<211> 81

p01445<212> ADN

p01446<213> Homo sapiens

p01447<400> 249

p01448<210> 250

p01449<211> 25

p01450<212> ADN

p01451<213> Homo sapiens

p01452<400> 250

p01453<210> 251

p01454<211> 27

p01455<212> ADN

p01456<213> Homo sapiens

p01457<400> 251

p01458<210> 252

p01459<211> 96

p01460<212> ADN

p01461<213> Homo sapiens

p01462<400> 252

p01463<210> 253

p01464<211> 19

p01465<212> ADN

p01466<213> Homo sapiens

p01467<400> 253 <210> 254

p01468<211> 23

p01469<212> ADN

p01470<213> Homo sapiens

p01471<400> 254

p01472<210> 255

p01473<211> 65

p01474<212> ADN

p01475<213> Homo sapiens

p01476<400> 255

p01477<210> 256

p01478<211> 19

p01479<212> ADN

p01480<213> Homo sapiens

p01481<400> 256

p01482<210> 257

p01483<211> 25

p01484<212> ADN

p01485<213> Homo sapiens

p01486<400> 257

p01487<210> 258

p01488<211> 86

p01489<212> ADN

p01490<213> Homo sapiens

p01491<400> 258

p01492<210> 259

p01493<211> 27

p01494<212> ADN

p01495<213> Homo sapiens

p01496<400> 259

p01497<210> 260

p01498<211> 25

p01499<212> ADN

p01500<213> Homo sapiens

p01501<400> 260

p01502<210> 261

p01503<211> 99

p01504<212> ADN

p01505<213> Homo sapiens

p01506<400> 261

p01507<210> 262

p01508<211> 22

p01509<212> ADN

p01510<213> Homo sapiens

p01511<400> 262

p01512<210> 263

p01513<211> 20

p01514<212> ADN

p01515<213> Homo sapiens

p01516<400> 263

p01517<210> 264

p01518<211> 75

p01519<212> ADN

p01520<213> Homo sapiens

p01521<400> 264

p01522<210> 265

p01523<211> 21

p01524<212> ADN

p01525<213> Homo sapiens

p01526<400> 265

p01527<210> 266

p01528<211> 24

p01529<212> ADN

p01530<213> Homo sapiens

p01531<400> 266

p01532<210> 267

p01533<211> 91

p01534<212> ADN

p01535<213> Homo sapiens

p01536<400> 267

p01537<210> 268

p01538<211> 24

p01539<212> ADN

p01540<213> Homo sapiens

p01541<400> 268 <210> 269

p01542<211> 24

p01543<212> ADN

p01544<213> Homo sapiens

p01545<400> 269

p01546<210> 270

p01547<211> 80

p01548<212> ADN

p01549<213> Homo sapiens

p01550<400> 270

p01551<210> 271

p01552<211> 20

p01553<212> ADN

p01554<213> Homo sapiens

p01555<400> 271

p01556<210> 272

p01557<211> 20

p01558<212> ADN

p01559<213> Homo sapiens

p01560<400> 272

p01561<210> 273

p01562<211> 72

p01563<212> ADN

p01564<213> Homo sapiens

p01565<400> 273

p01566<210> 274

p01567<211> 21

p01568<212> ADN

p01569<213> Homo sapiens

p01570<400> 274

p01571<210> 275

p01572<211> 18

p01573<212> ADN

p01574<213> Homo sapiens

p01575<400> 275

p01576<210> 276

p01577<211> 66

p01578<212> ADN

p01579<213> Homo sapiens

p01580<400> 276

p01581<210> 277

p01582<211> 20

p01583<212> ADN

p01584<213> Homo sapiens

p01585<400> 277

p01586<210> 278

p01587<211> 18

p01588<212> ADN

p01589<213> Homo sapiens

p01590<400> 278

p01591<210> 279

p01592<211> 68

p01593<212> ADN

p01594<213> Homo sapiens

p01595<400> 279

p01596<210> 280

p01597<211> 18

p01598<212> ADN

p01599<213> Homo sapiens

p01600<400> 280

p01601<210> 281

p01602<211> 19

p01603<212> ADN

p01604<213> Homo sapiens

p01605<400> 281

p01606<210> 282

p01607<211> 65

p01608<212> ADN

p01609<213> Homo sapiens

p01610<400> 282

p01611<210> 283

p01612<211> 20

p01613<212> ADN

p01614<213> Homo sapiens

p01615<400> 283

p01616<210> 284

p01617<211> 18

p01618<212> ADN

p01619<213> Homo sapiens

p01620<400> 284

p01621<210> 285 10 <211> 71

p01622<212> ADN

p01623<213> Homo sapiens

p01624<210> 286

p01625<211> 1947

p01626<212> ADN 20 <213> Homo sapiens

p01627<400> 286

p0162825 <210> 287

p01629<211> 1311

p01630<212> ADN

p01631<213> Homo sapiens

p0163230 <400> 287

p01633<210> 288

p01634<211> 582

p01635<212> ADN

p01636<213> Homo sapiens

p01637<400> 288

<dl><dt>10 </dt><dd /></dl>

<dl><dt><210> 289 </dt><dd /></dl>

<dl><dt><211> 6030 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>15 </dt><dd /></dl>

<dl><dt><400> 289 </dt><dd /></dl>

p01638<210> 290

p01639<211> 10987

p01640<212> ADN

p01641<213> Homo sapiens

p01642<400> 290

p01643<210> 291

p01644<211> 1552

p01645<212> ADN

p01646<213> Homo sapiens

p01647<400> 291

<dl><dt>10 </dt><dd /></dl>

<dl><dt><210> 292 </dt><dd /></dl>

<dl><dt><211> 1578 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>15 </dt><dd /></dl>

<dl><dt><400> 292 </dt><dd /></dl>

p01648<210> 293

p01649<211> 3195

p01650<212> ADN

p01651<213> Homo sapiens

p01652<400> 293

p01653<210> 294

p01654<211> 3737

p01655<212> ADN

p01656<213> Homo sapiens

p01657<400> 294

p01658<210> 295

p01659<211> 2042

p01660<212> ADN

p01661<213> Homo sapiens

p01662<400> 295

<dl><dt><210></dt><dd> 296 10 <211> 2547 </dd></dl>

<212> ADN

<213> Homo sapiens

<400> 296

<210> 297

<211> 2768

<212> ADN

<213> Homo sapiens

<400> 297

<210> 298

<211> 1358

<212> ADN

<213> Homo sapiens

<400> 298

<210> 299

<211> 4407

<212> ADN

<213> Homo sapiens

<400> 299

<210> 300

<211> 5532

<212> ADN

<213> Homo sapiens

<400> 300

<210> 301

<211> 1528

<212> ADN

<213> Homo sapiens

<400> 301

<210> 303

<211> 6450

<212> ADN

<213> Homo sapiens

<400> 303

<210> 305

<211> 2365

<212> ADN

<213> Homo sapiens

<400> 305

<210> 306

<211> 1117

<212> ADN

<213> Homo sapiens

<400> 306

<210> 307

<211> 1266

<212> ADN

<213> Homo sapiens

<400> 307

<dl><dt><210></dt><dd> 308 10 <211> 2162 </dd></dl>

<dl><dt>10 </dt><dd /></dl>

<dl><dt><210> 302 </dt><dd /></dl>

<dl><dt><211> 1856 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>15 </dt><dd /></dl>

<dl><dt><400> 302 </dt><dd /></dl>

<dl><dt><210> 304 </dt><dd /></dl>

<dl><dt><211> 3336 </dt><dd /></dl>

<dl><dt><212> ADN </dt><dd /></dl>

<dl><dt><213> Homo sapiens </dt><dd /></dl>

<dl><dt>5 </dt><dd /></dl>

<dl><dt><220> </dt><dd /></dl>

<dl><dt><221> no seguro </dt><dd /></dl>

<dl><dt><222> (0)...(0) </dt><dd /></dl>

<dl><dt><223> n = A, T, C o G </dt><dd /></dl>

<dl><dt>10 </dt><dd /></dl>

<dl><dt><400> 304 </dt><dd /></dl>

p01663<212> ADN

p01664<213> Homo sapiens

p01665<400> 308

p01666<210> 309

p01667<211> 3933

p01668<212> ADN

p01669<213> Homo sapiens

p01670<400> 309

p01671<210> 310

p01672<211> 2872

p01673<212> ADN

p01674<213> Homo sapiens

p01675<400> 310

p01676<210> 311

p01677<211> 926

p01678<212> ADN

p01679<213> Homo sapiens

p01680<400> 311

p01681<210> 312

p01682<211> 4989

p01683<212> ADN

p01684<213> Homo sapiens

p01685<400> 312

p01686<210> 313

p01687<211> 12515

p01688<212> ADN

p01689<213> Homo sapiens

p01690<400> 313

p01691<210> 314

p01692<211> 2444

p01693<212> ADN

p01694<213> Homo sapiens

p01695<400> 314

p01696<210> 315

p01697<211> 732

p01698<212> ADN

p01699<213> Homo sapiens

p01700<210> 316

p01701<211> 2422

p01702<212> ADN

p01703<213> Homo sapiens

p01704<400> 316

p01705<210> 317

p01706<211> 5061

p01707<212> ADN 10 <213> Homo sapiens

p01708<400> 317

p01709<210> 318

p01710<211> 3014

p01711<212> ADN

p01712<213> Homo sapiens

p01713<400> 318

p01714<210> 319

p01715<211> 2148 10 <212> ADN

p01716<213> Homo sapiens

p01717<400> 319

p01718<210> 320

p01719<211> 540

p01720<212> ADN

p01721<213> Homo sapiens

p01722<400> 320

p0172310 <210> 321

p01724<211> 2346

p01725<212> ADN

p01726<213> Homo sapiens

p0172715 <400> 321

p01728<210> 322

p01729<211> 2420

p01730<212> ADN

p01731<213> Homo sapiens

p01732<400> 322

p01733<210> 323

p01734<211> 2253

p01735<212> ADN

p01736<213> Homo sapiens

p01737<400> 323

p01738<210> 324

p01739<211> 1619

p01740<212> ADN

p01741<213> Homo sapiens

p01742<400> 324

p01743<210> 325

p01744<211> 5010

p01745<212> ADN

p01746<213> Homo sapiens

p01747<400> 325

p01748<210> 326

p01749<211> 2574

p01750<212> ADN

p01751<213> Homo sapiens

p01752<400> 326

p01753<210> 327

p01754<211> 1421

p01755<212> ADN

p01756<213> Homo sapiens

p01757<400> 327

p01758<210> 328

p01759<211> 4604

p01760<212> ADN

p01761<213> Homo sapiens

p01762<400> 328

p01763<210> 329

p01764<211> 2076

p01765<212> ADN

p01766<213> Homo sapiens

p01767<400> 329

p01768<210> 330 10 <211> 2819

p01769<212> ADN

p01770<213> Homo sapiens

p01771<400> 330

p01772<210> 331

p01773<211> 2540

p01774<212> ADN

p01775<213> Homo sapiens

p01776<400> 331

p01777<210> 332

p01778<211> 1474

p01779<212> ADN

p01780<213> Homo sapiens

p01781<400> 332

p01782<210> 333

p01783<211> 4079

p01784<212> ADN

p01785<213> Homo sapiens

p01786<400> 333

p01787<210> 334

p01788<211> 3373

p01789<212> ADN

p01790<213> Homo sapiens

p01791<400> 334

p01792<210> 335

p01793<211> 2304

p01794<212> ADN

p01795<213> Homo sapiens

p01796<400> 335

p01797<210> 336

p01798<211> 1876

p01799<212> ADN

p01800<213> Homo sapiens

p01801<400> 336

p01802<210> 337

p01803<211> 6633

p01804<212> ADN

p01805<213> Homo sapiens

p01806<400> 337

p01807<210> 338

p01808<211> 994

p01809<212> ADN

p01810<213> Homo sapiens

p01811<400> 338

p01812<210> 339

p01813<211> 772

p01814<212> ADN

p01815<213> Homo sapiens

p01816<400> 339

p0181710 <210> 340

p01818<211> 919

p01819<212> ADN

p01820<213> Homo sapiens

p0182115 <400> 340

p01822<210> 341

p01823<211> 7365

<dl><dt><212></dt><dd> ADN 20 <213> Homo sapiens </dd></dl>

<400> 341

<210> 342

<211> 10386

<dl><dt><212></dt><dd> ADN 5 <213> Homo sapiens </dd></dl>

p01824<220>

p01825<221> no seguro

p01826<222> (0)...(0) 10 <223> n = a, t, c, o g

p01827<400> 342

p01828<210> 343

p01829<211> 2191

p01830<212> ADN

p01831<213> Homo sapiens

p01832<400> 343

p01833<210> 344

p01834<211> 2776

p01835<212> ADN

p01836<213> Homo sapiens

p01837<400> 344

p01838<210> 345

p01839<211> 3160

p01840<212> ADN

p01841<213> Homo sapiens

p01842<400> 345

p01843<210> 346

p01844<211> 2629

p01845<212> ADN

p01846<213> Homo sapiens

p01847<400> 346

p01848<210> 347

p01849<211> 3442

p01850<212> ADN

p01851<213> Homo sapiens

p01852<400> 347

p01853<210> 348

p01854<211> 737

p01855<212> ADN

p01856<213> Homo sapiens

p01857<400> 348

p01858<210> 349

p01859<211> 5189

p01860<212> ADN

p01861<213> Homo sapiens

p01862<210> 350

p01863<211> 1536

p01864<212> ADN

p01865<213> Homo sapiens

p01866<400> 350

p01867<210> 351 10 <211> 2386

p01868<212> ADN

p01869<213> Homo sapiens

p01870<400> 351

p01871<210> 352

p01872<211> 1270

p01873<212> ADN

p01874<213> Homo sapiens

p01875<400> 352

p01876<210> 353

p01877<211> 1600

p01878<212> ADN

p01879<213> Homo sapiens

p01880<400> 353

p0188110 <210> 354

p01882<211> 1842

p01883<212> ADN

p01884<213> Homo sapiens

p0188515 <400> 354

p01886<210> 355

p01887<211> 4975

p01888<212> ADN

p01889<213> Homo sapiens

p01890<400> 355

p01891<210> 356

p01892<211> 4627

p01893<212> ADN

p01894<213> Homo sapiens

p01895<400> 356

p01896<210> 357

p01897<211> 2634

p01898<212> ADN

p01899<213> Homo sapiens

p01900<400> 357

p01901<210> 358

p01902<211> 1246

p01903<212> ADN

p01904<213> Homo sapiens

p01905<400> 358

p01906<210> 359

p01907<211> 2360

p01908<212> ADN

p01909<213> Homo sapiens

p01910<400> 359

p01911<210> 360

p01912<211> 1433

p01913<212> ADN

p01914<213> Homo sapiens

p01915<400> 360

<dl><dt><210></dt><dd> 361 10 <211> 1632 </dd></dl>

<212> ADN

<213> Homo sapiens

<400> 361

<210> 362

<211> 2756

<212> ADN

<213> Homo sapiens

<400> 362

<210> 363

<211> 2768

<212> ADN

<213> Homo sapiens

<400> 363

<210> 364

<211> 2984

<212> ADN

<213> Homo sapiens

<400> 364

<210> 365

<211> 3061

<212> ADN

<213> Homo sapiens

<400> 365

<210> 366

<211> 1360

<212> ADN

<213> Homo sapiens

<400> 366

<210> 367

<211> 1412

<212> ADN

<213> Homo sapiens

<400> 367

<dl><dt><210></dt><dd> 368 10 <211> 1075 </dd></dl>

p01916<212> ADN

p01917<213> Homo sapiens

p01918<400> 368

p01919<210> 369

p01920<211> 1127

p01921<212> ADN

p01922<213> Homo sapiens

p01923<400> 369

p0192410 <210> 370

p01925<211> 1890

p01926<212> ADN

p01927<213> Homo sapiens

15 <400> 370

p01928<210> 371

p01929<211> 4946

p01930<212> ADN

p01931<213> Homo sapiens

p01932<400> 371

p01933<210> 372

p01934<211> 1743

p01935<212> ADN

p01936<213> Homo sapiens

p01937<400> 372

<dl><dt><210></dt><dd> 373 10 <211> 5061 </dd></dl>

<212> ADN

<213> Homo sapiens

<400> 373

<210> 374

<211> 6802

<212> ADN

<213> Homo sapiens

<400> 374

<210> 375

<211> 1840

<212> ADN

<213> Homo sapiens

<400> 375

<210> 376

<211> 6754

<212> ADN

<213> Homo sapiens

<400> 376

<210> 377

<211> 757

<212> ADN

<213> Homo sapiens

<400> 377

<dl><dt><210></dt><dd> 378 10 <211> 476 </dd></dl>

p01938<212> ADN

p01939<213> Homo sapiens

p01940<210> 379

p01941<211> 2518

p01942<212> ADN

p01943<213> Homo sapiens

p01944<400> 379

p01945<210> 380

p01946<211> 4160

p01947<212> ADN

p01948<213> Homo sapiens

p01949<400> 380

p01950<210> 381

p01951<211> 1295

p01952<212> ADN

p01953<213> Homo sapiens

p01954<400> 381

p01955<210> 382

p01956<211> 2210

p01957<212> ADN

p01958<213> Homo sapiens

p01959<400> 382

p01960<210> 383

p01961<211> 4604

p01962<212> ADN

p01963<213> Homo sapiens

p01964<400> 383

p01965<210> 384

p01966<211> 545

p01967<212> ADN

p01968<213> Homo sapiens

p01969<400> 384