Compositions and methods for detecting pre-cancerous conditions in cell and tissue samples using 5, 10, 15, 20-tetrakis (carboxyphenyl) porphine

IS 2 380 261 T3

DESCRIPTION

Compositions and methods for the detection of precancerous conditions in cell and tissue samples using 5, 10, 15, 20-tetrakis (carboxyphenyl) porphine

Invention sector

The present invention relates to the use of certain porphyrins to detect dysplastic, precancerous and cancerous cells from different tissue samples, both in vitro and in situ.

Background of the invention

Various scientific and academic articles are referenced in parentheses in the present application.

Pathologists, who examine disease progression and analyze tissue samples for abnormalities, such as cancer, have determined that a cellular condition called dysplasia, which refers to the abnormal formation or maturation of cells, can potentially identify cells in a precancerous condition. If left unchecked, dysplasia can progress to mild, moderate, and severe stages and eventually cancer. About one in seven moderate cases of dysplasia will develop into cancer, and up to 83% of cases with severe dysplasia have been reported to develop into cancer, depending on the types of cells involved. However, the elimination of mild and moderate dysplasias greatly reduces the development of cancer. In the lung, the elimination of dysplastic cells not only greatly reduces the formation of cancer cells, but in some cases the lung tissue will return to a normal morphology.

In general, the earlier the cancer is found, the better the prognosis for the patient's survival. If breast cancer is found early, when it is still located in a single mass, the survival rate up to five years is more than 96%. When it has spread to a distant location, the survival rate up to five years is less than 20%. For lung cancer, when detected as a single mass, survival up to 5 years is more than 46%. When it has spread, survival up to 5 years is less than 14%. For cervical cancer, further improvement in survival occurs when precancerous changes are detected and treated before developing to a more severe stage (Boring and Squires 1993, CA Cancer J Clin 43: 7-26 and Ferguson 1990 , Hematology Oncol Clin Nam 4: 1053-1168).

Lung carcinoma is currently the leading cause of cancer mortality in men and women in the United States (Wingo et al. 1995, CA J Clin Clinic 45: 8-30). In 1997, there were an estimated 160,000 deaths from lung cancer, representing 12% of cancer deaths in US men and 2% in US women (Boring & Squires 1993, supra). Lung cancer is also one of the deadliest types of cancer, as reflected in a survival rate of up to five years of only 14%. The poor prognosis of lung cancer patients, relative to other human cancers, is largely due to the lack of effective early detection methods. At the time of clinical (symptomatic) presentation, more than two-thirds of all patients have regional nodules or distant metastases, which are generally incurable. In studies of patients with localized lung cancer (Stage 0 or 1), however, survival rates up to 5 years have ranged from 40% to 70% (Boring and Squires, 1993, supra; Ferguson, 1990, supra ).

Historically, the only diagnostic tests used to detect lung cancer early on symptoms have been sputum cytology and chest radiography. As a consequence, the efficacy of these tests as population screening tools has been extensively evaluated in studies in recent decades. Both tests detect presymptomatic early-stage carcinoma, particularly squamous cell carcinoma.

Improvements in screening methods have been primarily focused around improving the utility of sputum cytology through technological advances in microscopy. Sputum cytology requires a visual examination of a sample of cells during which cell size, shape, organization, and a relationship between the size of the cell nucleus and the cytoplasm are used to determine cell morphology. . Because this assessment of cell morphology requires visual inspection and classification, the technique requires a significant amount of knowledge acquired by the clinical observer. Various investigations have been conducted with results suggesting that computer-aided high-resolution image analysis enables the detection of sub-visual changes in visually normal nuclei associated with various types of tissues (Montag et al. 1991, Anal Quant Cytol Histol 13: 159-167; Haroske and others. 1988, Arch. Geschwulstforsch, 58: 159-168 ; Hutchinson and others. 1992, Anal Quant Cytol Estol 4: 330-334). Computer-aided analysis of DNA distribution in cell samples has provided 74% correct morphological classification of nuclei without human review of the material and without the need for visually abnormal nuclei to be present when compared to standard cytological tests .

IS 2 380 261 T3

Morphological evaluation of cytological samples has also improved due to advances in understanding lung tumor pathology. Much of this work has focused on the identification of biomarkers. Biomarkers refer to a wide range of progressive phenotypic and genetic abnormalities of the respiratory mucosa that can be used to determine the potential of the bronchial epithelium to completely transform into a malignant tumor. Markers have been widely classified as morphological changes, immuno / histochemical markers of differentially expressed proteins, markers of genomic instability, markers of epigenetic change (eg, abnormal methylation), and genetic mutations (Hirsch et al. 1997, Lung Cancer 17: 163-174).

The expression levels of these markers are being evaluated in dysplastic and neoplastic cyto / histological tissue samples collected from high-risk populations. Among the samples that are currently undergoing exploratory marker analysis is sputum. Interest in sputum samples for biomarker research has grown out of the long-held belief that exfoliated cells recovered in sputum may be the earliest possible indication of early carcinoma, as lung cancer is develops most often in the bronchial epithelium. Through the application of sophisticated molecular genetic techniques (eg PCR-based assays), studies are providing evidence that selected biomarkers can be detected in sputum (Mao et al. 1994, Cancer Res. 54: 16341637; Mao et al. 1994, Proc Natl Acad Sci USA. 91: 9871-9875; Sidransky 1995, J Natl Cancer Inst 87: 1201-1202; Tockman et al. 1988, J. Clin Oncol, 11: 1685-1693; Tockman et al. 1994, Chest, 106: 385s390s).

Commercially available cancer screening or detection services are based on cytomorphological diagnostic assays by trained clinicians who look at each sample and determine the extent and identity of abnormal cell types. This process is not only expensive and time consuming, but it also introduces human judgment and therefore procedural error. Recently, a method has been developed for the detection of lung cancer cells through the use of 5, 10, 15, 20-tetrakis (carboxyphenyl) porphine (TCPP) (US Patent No. 5,162,231 of Cole et al.) This method is based on the propensity of cancer cells to accumulate TCPP from their environment in a greater amount than non-cancer cells. After incubation of a cell sample for 6-24 hours with 200 pg / ml TCPP, TCPP enters the cells and binds to the perinuclear membrane and mitochondria of neoplastic cells. TCPP fluoresces under ultraviolet light, and thus cancer cells can be diagnosed solely by fluorescence intensity, without reference to morphology. US Patent No. 6,190,877 to Adair et al. Discloses a similar method. The extension of the use of this compound for the identification of precancerous conditions of the tissue (for example, dysplastic cells) would allow the screening in high-risk populations to identify those individuals whose tissues are progressing towards invasive cancer conditions and, in this way, facilitate the identification of cancer or dysplasia in the most treatable stage. The desirable characteristics of such a screening method would be a procedure that is fast, inexpensive, and requires a minimum of technical knowledge.

For the above reasons, there is a need for a technique and methodology for the detection of dysplastic cells in their early stages. Furthermore, there is a need for a technique that can provide highly reliable diagnostic results and is not based on subjective analysis by the clinician making the diagnosis.

Characteristics of the invention

The present invention stems from the discovery that TCPP can be used to detect dysplastic and precancerous cells as well as cancer cells, in conjunction with a new and more efficient method of TCPP solubilization, improved staining procedures, and a variety of screening strategies for cells. TCPP is a fluorescent compound that has now been discovered to bind to the components of living or fixed precancerous cells, as well as cancer cells, in a way that allows classifying the state of cells and tissue from which they originate in a progression continuous disease. This method of detecting precancerous tissues is well suited to in vitro diagnosis of tissue or cell samples, as well as in situ diagnosis.

One aspect of the present invention is a method for detecting precancerous cells, which in its simplest form comprises incubating live or fixed (i.e. dead) cells in a TCPp solution for a time sufficient to bind to cellular components and detect bound TCPP by fluorimetry. This method has many variants. In a variant, the cells are fixed on a surface, preferably a microscope slide, and more preferably on a monolayer. In another variant, cells are treated with formalin or other suitable fixative solution, kept in suspension, and treated with TCPP, cells are separated from unbound TCPP, and then analyzed and sorted by flow cytometry.

Preferred embodiments of the incubation step include the use of a TCPP solution with approximately 4 pg / ml to 400 pg / ml TCPP, a temperature between approximately 23 ° C and approximately 42 ° C, and a time between approximately 0.2 minutes. and 2 hours. Unbound TCPP is removed and the remaining TCPP is detected fluorimetrically. In a preferred embodiment, TCPP is detected between approximately 1 and 24 hours after the assay is performed.

IS 2 380 261 T3

In another embodiment of the present invention, the percentage of fluorescent cells in a sample of cells is calculated. Preferred embodiments comprise analysis of fluorescent cells to determine their fluorescence intensity and other cytomorphological characteristics. In a particularly preferred embodiment, the fluorescent cells are classified according to a set of predetermined cytomorphological and fluorescence intensity characteristics, This facilitates the characterization of cells in a continuous range from normal to metaplastic to dysplastic (mild to severe) to carcinomic (mild to severe) and increases the efficiency and reliability of diagnoses and prognoses made using the methods of the present invention. Other embodiments of the present invention comprise separating normal or metaplastic cells in a sample of dysplastic or carcinomic cells, using fluorescence intensity criteria (eg, by fluorometric flow cytometry).

In order to facilitate the practice of the detection method mentioned above, another aspect of the present invention provides a method for preparing a TCPP solution which comprises dissolving TCPP in about 50% to about 90% alcohol at a higher pH. at about pH 8.5 and lower at about pH 12.5. In a preferred embodiment, the alcohol is isopropanol and in another preferred embodiment, the pH of the solution is adjusted with sodium bicarbonate or ammonium hydroxide.

Another aspect of the present invention is a composition comprising TCPP in about 50% to about 90% alcohol at a pH greater than about pH 8.5 and less than about pH 12. In a preferred embodiment, the alcohol is isopropanol and in another preferred embodiment, the pH of the solution is adjusted with sodium bicarbonate or ammonium hydroxide. In one embodiment, the composition is prepared by the method of preparing a TCPP solution. In a related embodiment, the TCPP solution used in the detection method is diluted.

In another aspect of the present invention, the cells are in situ within a mammalian patient. The cells are exposed to the TCPP solution and fluorescence is detected using endoscopic techniques.

Another aspect of the present invention is a kit for the detection of precancerous cells, which comprises the composition of the present invention in a container. In another embodiment, the kit comprises one or more additional components, such as instructions and reagents for carrying out the detection method of the present invention, or positive and negative controls.

Other features and advantages of the present invention will be better understood with reference to the drawings, detailed description and examples that follow.

Detailed description of the invention

The present invention encompasses compositions and methods for detecting precancerous conditions in human cells using 5, 10, 15, 20-tetrakis (carboxyphenyl) porphine (TCPP). The present invention stems from the discovery that TCPP specifically binds to precancerous abnormal cells in addition to cancer cells, but does not bind to normal (non-cancer) cells. Furthermore, this differential binding is observed in fixed cells as well as living cells. In addition to this new and useful feature of TCPP, the present invention further incorporates an improved method of solubilizing TCPP that retains its activity to a greater degree, as well as several novel aspects that make it the most suitable method for screening and automation. Using the methods of the present invention, less time is required for cells to bind to TCPP when compared to the method described in US Pat. No. 5,162,231 (eg 0.2 min to 2 hours vs. 24 hours) and a lower TCPP concentration is also required (eg 40 pg / ml vs. 200 pg / ml). In addition, a cell monolayer can be used instead of a cell solution, although a cell solution can also be used.

The key to the convenience and efficiency of the detection method is the new TCPP solubilization method. The previous methods used 1 M NaOH to dissolve the porphyrin. This method requires titration with 1 M HCl, which is inaccurate and requires each solution to be analyzed for undissolved TCPP. Furthermore, the NaOH method places the porphyrin in an oxidizing environment with a pH as high as 13.0, thus exposing the porphyrin to a high risk of degradation. The solubilization method of the present invention uses a pH of 9.1, in conjunction with the novel addition of 90% alcohol to achieve a more complete and reliable solubilization. Finally, the method of the present invention uses a buffer to stabilize the pH of the final TCPP working solution in the preferred range of 5.8 to 6.8.

The present invention involves the detection of precancerous and cancerous cells in human tissue samples using the unique propensity of these cells to bind to TCPP in greater amounts than healthy cells. As used herein, the terms precancerous or abnormal precancerous refer to cells exhibiting mild to severe dysplasia and the term cancerous refers to cells exhibiting mild to severe carcinoma. These cytological states are defined morphologically herein by the criteria used to determine cell morphology using Papanicalou staining cytology (PAP staining).

IS 2 380 261 T3

They can also be defined by other indicators commonly used in the art for a particular cell or tissue (eg, indicators of pulmonary inflammation in the lung or sputum samples). From normal to severe carcinomic, the stages of a cell are classified herein as (1) normal (no significant abnormalities), (2) metaplasia (squamous metaplasia), (3) mild dysplasia (squamous atypia), (4) moderate dysplasia (squamous atypia), (5) severe dysplasia (marked squamous atypia), (6) squamous cell carcinoma in situ (CIS, non-invasive) (also known as mild to moderate carcinoma) and (7) squamous cell carcinoma (well differentiated keratinizing invasive type) (also known as moderate to severe carcinoma). According to the present invention, it has been determined that after exposure to TCPP, dysplastic and carcinomic cells show TCPP fluorescence, while normal cells show little or no TCPP fluorescence. Some metaplastic cells may show low to moderate TCPP fluorescence, but in many cases they do not, so TCPP fluorescence is not as reliable as an indicator of metaplasia, as it is for dysplasia and carcinoma.

The method comprises (1) incubating a sample of live or fixed cells with TCPP for a time sufficient to allow TCPP to bind to cellular components of precancerous or abnormal cancerous cells, if present in the sample, (2) removing the Unincorporated TCPP, (3) determine by fluorimetry the amount of TCPP remaining in the sample, if any; and, optionally, depending on the results of step (3), (4) evaluating the fluorescent TCPP cells to determine their divergence status towards cancer from the normal (or abnormal metaplastic, but not dysplastic) state. Specifically, as described above, the method of the present invention allows a determination that a cell sample contains cells that are dysplastic (mild to severe) or carcinomic (mild to severe).

In an exemplary but not limiting embodiment, the detection method comprises the following steps:

1. fix the cells in a monolayer on a microscope slide;

2. exposing cells to a TCPP solution at about 40 pg / ml in a buffered solution at a pH of about 6.1 (for example, by immersing the slide in the solution or by placing drops of solution on the slides) about at 36 ° C for a specified time, as described below;

3. wash the slides with a buffered solution at a pH of approximately 6.1;

Four. wait at least 1 hour but not more than 24 hours, and

5. quantify the fluorescence of cells at approximately 610-740 nm when excited with light at approximately 380-450 nm.

Variations of this exemplary method are explained in more detail below.

When used herein to describe the components of the test mixtures or other parameters of the present invention, the term "approximately" means within a commonly acceptable margin of error, for the determination made, using standard methods.

The first step, incubation of the cells with fixative, is optional, but preferred, as it has been found to reduce the time required for incubation, as well as the TCPP concentration in the working solution, in this exemplary embodiment. and in others.

The following sections set out a variety of other embodiments of the present invention.

The methods of the present invention can be used on a variety of cell types as described below and are further applicable to veterinary as well as human diagnostic and prognostic applications. Accordingly, the term patient or individual, as used herein, is understood to apply to a human or an animal.

The detection method can be used to detect precancerous and cancerous cells in cell samples in vitro. Cell samples can be acquired by any of the methods currently used in the field of cytopathology. For example, cells can be collected from sputum samples (see Example 1), cervical swabs, bronchial washes, fine needle aspiration, and breast and thyroid core biopsies, bladder washes, urine, mouthwashes, enemas. and other biopsies known in the art. Other sources of cell samples include blood or fractions thereof, lymph, cerebrospinal fluid, bone, and bone marrow, to name a few. The method of the present invention is applicable to any sample of cells from any tissue or organ in the body.

IS 2 380 261 T3

Optionally, cells can be fixed by standard procedures prior to exposure to TCPP, including but not limited to solutions containing formaldehyde, methanol, ethanol, or isopropanol. In one embodiment, the cells are fixed in 95% ethanol.

The assay can be performed in solution by measuring total fluorescence by cell density, or by adhesion of cells to a surface. The cells do not need to be treated with a fixative, but fixation of the cells is preferred in some embodiments, particularly those in which the cells adhere to a solid support. In one embodiment, the cells adhere as a monolayer to a slide. In other embodiments, the MonoPrep2 or MonoPrepG liquid-based slide preparation system (MonoGen, Inc., Herndon, Virginia) or the ThinPrep Processor (Cytyc Corporation, Marlborough, MA) is used.

The method of the present invention can also be used to detect precancerous and cancerous cells in situ, as well as an aid in resection surgery. For example, the method can be used to detect dysplastic cells in the lung in situ by injection of TCPP in a suitable medium followed by fluorescence bronchoscopy. A similar method can also be used to detect abnormal cells for excision during surgery. In situ applications can be found for any of the organs of the body, including without limitation, breast, prostate, lung, cervix, throat, bladder, oropharynx, skin, and gastrointestinal tract through the use of a similar endoscopic device. The preferred amount of TCPP for use in this embodiment is determined by the mode of administration and the site of application. For example, if TCPP is injected into the bloodstream, the effective concentration of TCPP will depend on its maximum solubility in saline or blood (eg, approximately 100 pg / ml). For injection directly into the affected tissue, an effective amount of TCPP will depend on the target tissue and the proximity of the injection to the tissue (eg, about 1-20 mg). In the lung, an aerosol delivery, for example 5-10 ml, at a concentration of 20-50 pg / ml should be adequate. Methods for determining such amounts of TCPP to be administered as a diagnostic agent are well known to chemists and other skilled in the art.

The TCPP concentration in the working solution and the length of time the cells are exposed to the TCPP solution are two variables that can be altered in a coordinated way. The TCPP concentration is preferably 4-100 pg / ml, more preferably 4-40 pg / ml, and most preferably 20-40 pg / ml. The exposure time can range from about 0.2 minutes to about 2 hours in one embodiment, and from 10 to 60 minutes in a more preferred embodiment. When low concentrations of TCPP solution are used, a longer exposure time is appropriate, and when high concentrations of TCPP are used, a shorter exposure time is suitable. For example, in preferred embodiments, the slides are exposed for 10 minutes in 40 pg / ml TCPP and alternatively for 60 minutes in 4 pg / ml TCPP. The method for optimizing TCPP concentration in working solution and exposure time is well known to those skilled in the art of cytology, with the goal of achieving maximum specific TCPP binding to cellular components while simultaneously Background and other non-specific absorptions and fluorescence are minimized.

The TCPP solution comprises TCPP in a buffered aqueous medium at approximately 36 ° C. In one embodiment, the buffering capacity is due to 100 mM MES, however a concentration range of 20 to 200 mM can be used in the method with equal efficiency. In one embodiment, the solution has a pH of about 6.1; however, a pH range of 5.8 to 6.7 can be used with sufficient efficiency. Other buffer compounds that are effective in the pH range 5.8-6.7 can also be used. While the exposure step is not particularly temperature sensitive, a temperature somewhat above room temperature is desirable for optimization. The suitable temperature range for the exposure step is about 23 ° C to about 42 ° C in a preferred embodiment and about 30 ° C to about 40 ° C in a more preferred embodiment.

Other compounds can be added to the working solution to reduce background fluorescence, increase stability, or reduce autofluorescence or quenching. For example, detergents can be used to decrease background fluorescence and reducers, antioxidants, and other inhibitors of the generation or diffusion of active oxygen species can be used to prevent oxidation of TCPP or reduce photobleaching. Compounds of interest include, but are not limited to, polyethylene glycols, tritons, dithiothreitol, dithioerythritol, 2-mercaptoethanol, or the Antifade kits supplied by Molecular Probes, Inc. (Eugene, OR, 7481-P, S-2828, S7461). In addition, hematoxylin can be included with TCPP, to act as a counterstain and facilitate white light microscopy.

The wash solution is generally similar to the aqueous solution used for the TCPP working solution, but without TCPP. If microscope slides are used, the slides should be washed at least once, more preferably three times, and preferably with shaking the excess wash solution. If the assay is performed in solution, unbound TCPP can be removed by centrifugation of the cells, decanted the supernatant, and the cells resuspended in fresh buffer. This stage can be repeated if necessary. Alternative means of separating cells from the staining solution can also be used, such as filtration with capture of the cells on a membrane or rapid dialysis (including rotary dialysis). Suitable washing conditions can be determined by monitoring the fluorescence of the cells. Washing should be sufficient to remove background and other non-specific junctions, but not so excessive as to remove TCPP

ES 2 380 261 T3 specifically bound to cellular components. Optimization of the wash step is well known to those skilled in the art of cytology. Compositions that can be added to the wash solution to improve efficacy or stability include, but are not limited to, low molar alcohols, detergents, or salt solutions.

An important step in the detection method is to allow the time between when the slide has been exposed to the TCPP working solution and when the slide is read to be more than 1 hour, but less than 24 hours. When the slide is read after 24 hours, there may be too much deterioration of the TCPP to obtain an accurate result. When cells are read within 1 hour, the background fluorescence level may be excessively high.

The wavelengths used for the detection stage are within wide ranges in which specific peak ranges will be most efficient. The excitation peak of TCPP in an aqueous solution of pH 5.0-7.0 occurs at approximately 415 nm, while an emission peak occurs at approximately 645 nm and a secondary emission peak occurs at approximately 706 nm. . In general, TCPP can be detected by illuminating the sample with ultraviolet (UV) light and detecting the light emitted from the sample above about 500 nm. The wavelengths used to excite TCPP in the method of the present invention may preferably span part or all of the range from about 380 to about 450 nm, and most preferably a narrow band of wavelengths near about 415 nm. Also, the detected wavelengths may span part or all of the range from about 610 nm to about 740 nm and more preferably a narrow range near about 650 nm. Under certain circumstances apparent to those skilled in the art of fluorescence microscopy, it may be preferred to detect emissions in a narrow range close to about 706 nm. Wavelength selection can be achieved more easily by optical filters. Filter sets for popular fluorescent dyes for example are readily available from Molecular Probes (Eugene, OR). The selection of the appropriate wavelength to excite and detect TCPP in the method of the present invention can be obtained in the easiest way by using a set of filters designed for the detection of fluorescein isothiocyanate (hereinafter FITC), which it generally has an excitation filter of 400 to 490 nm and a barrier filter for emission above 500 nm. The selection of other filter systems is well known to those skilled in the art of microscopy.

Fluorescence can be detected visually or mechanically, manually or by automated means. Cells that have even moderate fluorescence compared to non-fluorescent cells are easily distinguishable by the human eye. Therefore, certain embodiments of the present invention comprise simply observing TCPP-treated cells under a fluorescence microscope and manually quantifying the percentage of fluorescent cells in the sample. However, preferred embodiments comprise automated methods well known in the art, and mechanical quantitation in which a cell is counted as fluorescent if it shows fluorescence above a predetermined threshold level programmed into the counting device, as is well known in the art. technique. For example, in embodiments comprising a monolayer of cells adhered to a microscope slide, an automated plate reader can be programmed to count as fluorescent any cell that has predetermined fluorescence that is statistically significant compared to a normal equivalent cell, as determined by standard statistical methods (such devices can also be programmed to count cells in a certain way, which can provide a second indicator of precancerous or cancerous abnormalities). Alternatively, for embodiments comprising a solution-based assay, TCPP-stained and washed cell samples can be analyzed to sort fluorescence-activated cells (FACS), where the FACS is programmed to separate cells that have a predetermined level of fluorescence, as can be statistically determined by comparison with normal cells.

The total number of cells present in a sample is determined in order to calculate a percentage of that total that are fluorescent by TCPP. This determination can be made in a variety of ways known in the art. In one embodiment, all cells are stained with hematoxylin and counted by white light microscopy. In another embodiment, cells are stained with a suitable fluorescent counter dye (eg, one that stains the outer or inner membranes of a cell) that fluoresces at a different wavelength than TCPP. In this latter embodiment, the ratio of TCPP fluorescence to cell marker fluorescence is quantified.

As mentioned above, the novelty of the methods of the present invention resides in the present inventors' appreciation that TCPP staining not only identifies cancer cells, as is already known, but also identifies precancerous dysplastic cells. Because of this, the method described above produces much more information than previously thought possible. Consequently, the results of the TCPP fluorescence quantification will be decisive in deciding whether and in what way further analytical steps are carried out.

For example, the method will identify a percentage of cells in a sample that are fluorescent by TCPP. If about 1-3%, more particularly about 2-3%, of the cells in a sample are

ES 2 380 261 T3 fluorescent, then the sample contains cells that are at least abnormal precancerous (dysplastic) or cancerous. Therefore, a simple analytical scheme is to determine whether a sample contains at least about 1% cells fluorescent by TCPP. If not, the sample is diagnosed as negative (normal). If it does, additional tests are recommended for the patient. It should be noted in connection with this embodiment that even if a sample contains less than 1% fluorescent cells, other factors (eg, predisposition of the patient to cancer, or a pre-existing cancer in other tissue) may suggest that further testing be performed. . An advantage of the present invention, which is described in greater detail below, is that an enriched population of fluorescent cells can be obtained from the patient via FACS.

Furthermore, the level of fluorescence of a given cell in a sample has been found to correlate with the cancer-associated state of that cell (see Example 1). Consequently, individual cells or groups of cells can be evaluated for their overall fluorescence intensity and a determination of whether further testing is required may be based in part on this evaluation.

The terms high, medium, and low fluorescence and related terms as used herein, will be understood by one of ordinary skill in the art to be comparative terms in which the fluorescence intensity of a single cell or group of cells in a sample The test sample is compared, at a minimum, with cells from an equivalent source (eg, sputum) that are known to be normal relative to cancer (negative control). This comparison can be done by visual estimation, or, in automated systems, it can be programmed using statistical parameters such as the variation of the median fluorescence of a sample population, as described in Example 2. In preferred embodiments, cells in a test sample are compared by fluorescence intensity to additional control cells whose cancer status has been predetermined and precorrelated to a fluorescence intensity of TCPP (eg, as described in Example 1). .

The terms fluorescent and non-fluorescent are also used herein. According to the aforementioned definitions of various levels of fluorescence intensity, the terms non-fluorescent and fluorescent are used as comparative terms, in which the fluorescence is compared to normal cells of an equivalent origin, and / or against the fluorescence of background in general resulting from the reagents or equipment used in the detection of fluorescence. Therefore, if a cell or sample of cells is determined to be fluorescent, then fluorescence is present at some intensity above the background fluorescence or fluorescence observed in known normal cells. If a cell or sample of cells is determined to be non-fluorescent, then the observed fluorescence is minimally or not at all in excess of the background fluorescence or the fluorescence observed in normal cells. This comparison is obvious to a person skilled in the art.

A cytomorphological evaluation combined with TCPP fluorescence is particularly useful with cell cultures that have a low level of fluorescence because a visual evaluation of the cells with standard evaluation techniques can easily differentiate the slightly fluorescent (non-cancerous) metaplastic cell from dysplastic (precancerous) cells. One embodiment of the method comprises an additional step of cytomorphological evaluation in addition to fluorescence quantification, especially using a standard cytological stain such as hematoxylin to help visualize the cell and nuclear contours. Another embodiment employs cytomorphological evaluation as a later stage, if certain threshold requirements are met, eg, the sample contains more than 1% fluorescent cells.

In a particularly preferred embodiment of the present invention, the selected cytomorphological characteristics are combined with the fluorescence intensity to produce a classification system that is very useful for the efficient and reproducible diagnosis of the various stages of metaplasia, dysplasia and carcinoma that may be present. present in a sample of cells. Said classification system is described in detail in Example 1. In this embodiment, TCPP fluorescent cells are assigned to one or more numerical classes, based on fluorescence intensity and simple morphological characteristics including cell shape and size, number or size of nuclei, presence of clumps of cells and the degeneration of cells or groups of cells, the presence of irregular anisoid cells, the visibility of the cell membrane, and the presence and nature of nuclear debris. The technician or scientist who performs the cytomorphological evaluation of fluorescent cells by TCPP can use the classification as a checklist, that is, a cell being examined can be marked to indicate plus or minus with respect to each of the numerical classes. The number of numerical classes assigned to a particular cell and the pattern of specific classes assigned to a cell are informative of the cancerous or precancerous condition of that cell. By way of illustration, Example 1 shows a classification system comprising 14 numerical classes. As shown in Table 2 of that example, which presents the sputum sample assays, negative or metaplastic cells can generally be assigned to some of the classes, while severely carcinomic cells are assignable to several. As a further illustration, negative or metaplastic cells are frequently assigned to class 11, while moderately dysplastic cells are not carcinomic, and carcinomic cells are frequently assigned to class 6, whereas normal dysplastic or metaplastic cells are not. .

IS 2 380 261 T3

In another embodiment of the present invention, cells treated with TCPP in solution from a single patient determined to be carcinomic can be separated by flow cytometry based on their level of fluorescence. Cells showing a higher level of fluorescence are considered cancerous while cells with moderate to low level fluorescence are considered dysplastic and cells without fluorescence are considered normal. This type of separation allows a patient's dysplastic or cancer cells to be compared to the patient's own normal cells, thus providing an ideal internal control population.

In another embodiment, by separating cancer and normal cells from the same patient, they can be tested to test the efficacy of various chemotherapeutic agents. Separated cells are dispensed in aliquots. A selected therapeutic agent can be mixed at the same concentration with an aliquot of highly fluorescent cells and an aliquot of low-fluorescent cells. This step can be repeated with fresh aliquots and a different therapeutic agent. Cell death rates can be assessed using techniques known in the art. The most preferred therapeutic agent for treatment can then be determined by choosing the chemotherapeutic agent in which the highest number of cells determined to be cancerous died (i.e., highly fluorescent cells) and destroyed the fewest normal cells (i.e. i.e. cells with little or no fluorescence after TCPP treatment).

In conjunction with the screening or diagnostic detection method of the present invention, a method for dissolving TCPP has been developed for use in the method, as well as other applications. This method comprises dissolving TCPP in about 50% to about 90% alcohol with a pH greater than about pH 8.5 and less than about pH 12.5. Lower alcohols such as methanol, ethanol, isopropanol and n-propanol are preferred for use in the present invention. More preferably, the alcohol is isopropanol and its pH is adjusted with sodium bicarbonate or ammonium hydroxide to a pH greater than 8.5 and less than 10.0. The isopropanol concentration can be between 50% and 90% and the sodium bicarbonate can be between 20mM and 100mM in some embodiments. The TCPP concentration can be up to about 2 mg / ml. In one embodiment, TCPP is dissolved at 1 mg / ml in a 50 mM sodium bicarbonate solution in 50% isopropanol.

The present invention also comprises a composition useful for use in any method using TCPP comprising TCPP in alcohol with a pH greater than 7. This solution is preferably prepared by the method of dissolving TCPP detailed above. This composition should preferably be stored at about 4 ° C in the dark.

The present invention further encompasses pre-cancer and cancer cell detection kits comprising TCPP in a container, optionally with instructions. In one embodiment, the kit is designed to be used with the detection method of the present invention. In one embodiment, the kit comprises the composition of the present invention comprising alcohol-based solubilized TCPP in a container. This TCPP solution can be used as a stock solution that is diluted in a buffered aqueous solution in order to detect precancerous and cancerous cells. The kit may comprise cell sample collection components, such as the sputum collection container of Example 1, or alternatively may comprise items for the detection of precancerous cells in samples already acquired. The kit can be adapted for use with slide preparation systems, eg, MonoPrep2 or MonoPrepG (Monogen, Inc., Herndon, VA) or the ThinPrep Processor (Cytyc Corporation, Marlborough, MA), to name three. These kits can also be designed to be used with other different microscope slide formats, such as microtiter plates or flow cytometry devices. In any of the above embodiments, the kit may comprise positive or negative controls, or both, as would be used by one of ordinary skill in the art in performing the assays of the present invention.

The following examples are provided to describe the present invention in greater detail. They are intended to illustrate the present invention and do not constitute a limitation thereof.

EXAMPLE 1

Glass Slide Assay for Detection of Precancerous and Cancer Cells with TCPP

This example compares the diagnostic results obtained by standard cytomorphological analysis of PAP-stained sputum slides with slides treated with TCPP and analyzed by fluorescence microscopy. The results indicate that the TCPP detection technique of the present invention is equivalent to conventional sputum cytology in the detection of neoplastic cells (dysplasia and carcinoma in situ) and pure carcinomas. The results also indicate that one of skill in the art can use the method, in conjunction with simple classification rules, to estimate the degree of dysplasia present in a tissue sample.

IS 2 380 261 T3

Methods

Sputum processing procedures used in the preparation of monolayer slides. All monolayer slides selected for analysis in this study were prepared from sputum samples collected from patients who performed the early morning spontaneous cough technique. Specifically, patients were instructed to cough up any material by coughing over three consecutive mornings into a container filled with a fixative consisting of 2% Carbowax in 50% alcohol / 50% Saccomanno fluid with 0.03-0 .05 mg / ml rifampicin. Rifampin was added to the fixative solution to serve as a prophylactic against patients with M. tuberculosis or those patients who may be asymptomatic carriers of N. meningitis.

The 2% Carbowax solution was prepared by adding 2 ml of molten Carbowax (150) to 98 ml of 50% ethanol and mixing for 30 minutes. The glassware used to prepare the solution was kept warm to prevent hardening of the wax on the surface during preparation, which can cause inaccurate measurements. Carbowax was removed prior to exposure to the TCPP working solution by immersion in 95% alcohol for at least 15 minutes.

Rifampin solution (3 mg / ml) was prepared by dissolving 300 mg rifampin capsules in 100 ml ethyl alcohol and mixed in a Waring mixer at high speed. One ml of this solution was added to each 30 ml of Saccomano solution or 20 ml per liter of Saccomano solution and mixed vigorously. The preparation of the Saccomano solution was carried out by standard methods well known to those skilled in the art of cytology.

Two "Thin-prep" microscope slides (Cytyc Corporation, Marlborough, MA) and a 50 ml plastic centrifuge tube were labeled with patient information. The sputum sample was poured into a 50 ml plastic centrifuge tube and additional 50% ethyl alcohol solution was added to bring the volume to 50 ml if necessary. The contents of the centrifuge tube were poured into a 250 ml Eberbach semi-micro-liquefier container and homogenized for 10 to 60 seconds, depending on visual examination of the sample and mucosa content. Samples with thick mucosa sometimes required longer liquefaction times. The sample was poured back into the centrifuge tube and centrifuged at 1850 rpm for 10 minutes. The supernatant was decanted, leaving 1 to 2 ml in the centrifuge tube to mix with the pellet (spin). The tube was shaken on a rotary mixer for approximately 10 seconds. One to three drops of the pellet were placed in a PreservCyt vial (Cytyc Corporation, Milford, MA). The sample was incubated for 5 minutes to inactivate all microbial and viral organisms.

The single cell layers of the samples were fixed on the slides using the Thinprep Processor (Cytyc Corporation, Milford, MA) according to the manufacturer's instructions. On the Thinprep Processor, cells were collected on a polycarbonate filter (0.5mm pore size) and transferred to a glass slide. The Thinprep Processor was then immediately placed on the slide in a fixative bath containing 95% ethanol.

TCPP stock solution. 400 mg of sodium bicarbonate was added to approximately 90 ml of 50% isopropanol (50 mM sodium bicarbonate) and mixed until completely dissolved to prepare 50% isopropanol base. One hundred milligrams of TCPP was slowly added to the 50% isopropanol base (50 mM sodium bicarbonate) and mixed for 3 to 5 minutes until dissolved. The TCPP solution was made up to 100 ml volumetrically with the 50% isopropanol base, mixed well, and stored in an amber reagent bottle covered with aluminum foil in a refrigerated area. The final concentration of TCPP in the stock solution was 1 mg / ml.

TCPP working solution. Fresh TCCP working solution was prepared each day. Approximately 10 ml of TCPP stock solution with a concentration of 1 mg / ml was brought to room temperature. Eight milliliters of TCPP stock solution (1 mg / ml) were placed in a 200 ml volumetric flask and approximately 100 ml of MES buffer was added slowly. The solution was mixed gently. Additional MES buffer was added to bring the solution to 200 ml volumetrically. The solution was mixed for 3 to 5 minutes and stored at 24 ° C in an amber bottle. The final concentration of TCPP in the working solution was 40 pg / ml.

TCPP exposure procedure. The slides were fixed in 95% alcohol for 30 minutes at room temperature. The slides were exposed to TCPP immediately after fixation or up to 3 days later. The slides were immersed in the TCPP solution at 40 pg / ml for 10 minutes at 36 ° C, then washed three times in 100 mM MES buffer, one minute each, at room temperature with shaking. The slides were observed after more than 1 hour but not more than 24 hours later.

Microscope information. The microscope used for viewing the TCPP-treated sputum cell slides was an Olympus model BH-1 microscope with a top illuminator and a top mercury lamp for reflected light fluorescence microscopy. The mercury lamp has primary emission lines at 365nm, 405nm, 436nm, and 545nm. The assembled fluorescence filter consisted of

ES 2 380 261 T3 two dicrotic cubes. The green cube (490 nm) contained a filter system with an excitation filter passing 400-490 nm and an emission barrier filter passing above 500 nm.

Sputum slide staining procedures using modified PAP staining technique. Procedure sequence (no.), Reagent, and time (min .: sec.) Were as follows: (1) 95% alcohol 15:00, (2) tap water 1:00, (3) Gil-i hemotox 2:30, (4) tap water 1:00; (5) bluish reagent: 30; (6) tap water 1:00; (7) 95% alcohol: 10; (8) og-6 1:30; (9) 95% alcohol: 10; (10) 95% alcohol: 10; (11) EA-50 1:15; (12) 95% alcohol: 20;

(13) 95% alcohol: 30; (14) 100% alcohol 1:00; (15) 100% alcohol 1:00; (16) 100% alcohol 1:30; (17) xylene 1:00; (18) xylene 1:00; and (19) xylene 1:00.

Methods of routine cytopathological analysis of slides with Papanicolaou staining. The PAP stained slides were subjected to semiquantitative cytomorphological evaluation. (1) dysplastic and neoplastic cells were identified using traditional morphological criteria, and (2) the expression levels of seven fundamental indicators of lung inflammation were quantified (alveolar macrophages, neutrophils, columnar cells, mucus, mucous spirals, macrophages pigmented, metaplastic cells). The methodology for the quantification of these indicators of inflammation has been previously discussed in the literature (Roby et al., 1989, Acta Cytol 34: 147-154; Roby et al., 1990, Acta Cytol 34: 140-146; Schumann et al., 1989, Am Rev Respr Dis 139: 601-603). The criteria used to determine cell morphology using PAP staining cytology are discussed below.

There are no significant anomalies. Cells were identified without significant abnormalities if the following requirements are met:

1. basophilic ciliated epithelial cells mixed with grade 1-2 pigmented macrophages along with inflammatory cells;

2. round nuclei of basal oriented epithelium;

3. uniformly dispersed chromatin;

Four. nuclear membranes barely visible;

5. nucleoli barely visible; Y

6. no metaplastic or dysplastic cells are present.

Squamous metaplasia (without dysplasia). Cells were identified as squamous metaplastic without dysplasia if the following requirements are met:

1. groups of basophilic cells without cilia;

2. uniform cell and nuclear size;

3. low nucleus / cytoplasm ratio (N / C);

Four. finely granular nuclear chromatin; Y

5. small rounded nucleoli may be present (usually alone).

Mild dysplasia (squamous atypia). Cells were identified as mild dysplasia if the following requirements are met:

1. smaller than metaplastic cells;

2. they are seen in cohesive groups, or singularly;

3. cells appear flat (leaves) both nuclei and cytoplasm in focus;

Four. cells vary slightly in size;

5. the cytoplasm can be eosinophilic or basophilic;

6. thin cytoplasmic borders;

7. nuclei vary slightly in size, generally round to oval, if divided into 2 nuclei halves they are mirror images, the N / C ratio may vary slightly;

IS 2 380 261 T3

8. smooth nuclear membrane;

9. finely granular nuclear chromatin (slightly increased), occasional chromocenter; Y

10. fiber-like cells, elongated cells with elongated cytoplasm and nucleus different nuclear membrane fine to granular reticular cytoplasm usually bright orange-yellow-simple keratinizing, can form eddies around the central keratin nucleus to make epithelial beads.

Moderate dysplasia (squamous atypia). Cells were identified as moderately dysplastic if the following requirements were met:

1. variation in size, usually larger, but may be less than in mild dysplasia;

2. more variation in shape and N / C ratio than in mild dysplasia;

3. dense cytoplasm, acidophilic predominates; increased number of atypical cells;

Four. the nucleus may have two unequal halves (they are not mirror images);

5. nuclear lobulations, cracks, and nodules are present; Y

6. nuclear material may show hyperchromasia with more stipple-like chromatin pattern.

Severe dysplasia (marked squamous atypia). Cells were identified as severe dysplasia if the following requirements are met:

1. cells vary greatly in size and shape;

2. usually slightly larger cell size than in moderate dysplasia;

3. the N / C ratio is high, but variable (with extremes);

Four. individual cells predominate, the nucleus is more central than in CIS;

5. the nucleus can follow the shape of the cytoplasm; the core shows less distortion than in CIS;

6. nuclear pleomorphism increases with the presence of thick chromatin and condensation along the nuclear envelope;

7. parachromatin, large nucleus, focally thickened multi-grouped nuclear membrane; Y

8. cells show predominant acidophilic cytoplasm.

Squamous cell carcinoma in situ (CIS, non-invasive). Cells were identified as squamous cell carcinoma in situ if the following criteria are met:

1. individual cells or in aggregates (groups);

2. variable cell size - may be smaller or larger than marked dysplasia cells usually smaller than invasive squamous cell carcinoma;

3. cells are large, rounded with symmetrically located nuclei;

Four. cell degeneration may be present;

5. scant cytoplasm, uniformly distributed, may be keratinized or non-keratinized concentrically around the nucleus, (orangiophilic or basophilic);

6. variable N / C ratio - higher or lower than normal;

7. Coarse dense nuclear chromatin granules may be interrupted by clear areas;

8. chromatin rim uniformly thickened with ripple of nuclear membrane;

9. lobulations of the nuclei can be seen;

IS 2 380 261 T3

10. cannibalism can be observed, but is unusual;

eleven. multinucleated cells may be present;

12. without nucleoli in the nucleus, a mitotic cell may be present; Y

13. light background.

Squamous cell carcinoma (well differentiated keratinizing invasive type). Cells were identified as squamous cell carcinoma if the following criteria are met:

1. cells usually individual, orangephilic, but can be in groups and degenerate;

2. large or small, angular cells with well-preserved nuclei and well-defined cell borders;

3. cells generally larger than in situ, and can be pleomorphic, wide range of size and shape;

Four. pearl formation (cancer pearls) can be observed;

5. moderate amount of cytoplasm with abnormal tail (consistent with invasion); foreign cells in the form of a tadpole, star, axis, an angular nucleate chromatin, with unpredictable clustering with hyperchromasia and clearly defined parachromatin and chromatin compensation, parachromatin interface;

6. the chromatin is lumpy, especially along the nuclear membrane;

7. nucleoli are large and acidophilic, if present;

8. the nuclear membrane itself may be thickened and irregular; edge thickness irregularity of nuclear chromatin;

9. the N / C ratio is very high;

10. marked nucleolar irregularity in shape, size, number (daughter nucleoli); abnormal mitoses, multinucleation;

eleven. cannibalism and multinucleation are common; Y

12. necrotic background material is common.

Results

In a blind study in which 60 samples were examined, the results indicate that abnormal cells (mild, moderate or severe dysplasia or cancerous) can be detected accurately with the TCPP detection procedure compared to the PAP staining procedure (Table 1). If 2-3% of the cells exposed to TCPP were fluorescent, then the sample reliably correlated, at a minimum, with the diagnosis of mild dysplastic. Fifty of the fifty sputum samples determined by the standard cytomorphological PAP staining procedure that were mildly dysplastic to cancerous were also identified as abnormal by TCPP detection. Among the ten samples characterized as normal or metaplastic based on the PAP staining procedure, four samples demonstrated the same morphology by the TCPP method. Samples diagnosed as normal showed minimal or no TCPP uptake.

TCPP uptake in cells determined to be negative or metaplastic by cytomorphology had characteristic fluorescence intensity and patterns that were recognizable and diagnostic. Table 2 presents a comparison between cell morphology and fluorescence as determined by PAP staining cytomorphology and TCPP techniques, respectively. Based on fluorescence intensity and pattern in TCPP-treated cell samples, cells were sorted with one of 14 possible numbered ranks relative to a morphological description. If the cells were class 11 using TCPP determination and less than one

2-3% of the cells on the slide were fluorescent above background levels, then that sample was determined to be metaplastic and not dysplastic. Metaplastic cells are easily differentiated from normal cells by their moderate fluorescence with a barely visible cell membrane. Of the ten cell samples that were determined to be negative or metaplastic by PAP staining, 6 were designated with a class 11 cell description based on TCPP fluorescence. Of the six TCPP samples with class 11 designation, three also indicated nuclear waste fluorescence (ie, either a class 13 or 14), and two also showed a class 10 designation (core fluorescence only).

IS 2 380 261 T3

Another pattern shown in Table 2 refers to the numerical classification 6 - irregular anisoid cells, low to medium fluorescence. It is notable that relatively few dysplastic cells were assigned to this classification, while the majority of carcinomic cells received classification 6. Therefore, this classification is expected to be of particular importance in distinguishing carcinomas from dysplasias using the methods herein. invention.

Another important observation revealed in Table 2 is that, as cell morphology progressed from normal to severe carcinomic, the total number of numerical classifications that were assignable to each cell examined also increased. By way of illustration, cells having a negative or metaplastic morphology were assigned an average of 2 numerical classifications, while cells showing adenocarcinoma, squamous cell carcinoma and small cell carcinoma were assigned an average of 5 numerical classifications. Since the numerical classifications contain descriptions of the different types of cellular abnormalities, a positive correlation between the degree of dysplasia or carcinoma and the number of different abnormalities observed in the cells is logical. However, this correlation has so far not been systematized and used to diagnose precancerous and cancerous conditions in a sample of cells.

Table 1. Correlation between TCPP results and cytomorphological results.

<td>Description of diagnosis</td><td>N = 60 Slides with morphology using TCPP / Slides with morphology using cytomorphology</td>

<td>Negative or metaplastic</td><td> 4/10</td>

<td>Mild dysplasia</td><td> 12/12</td>

<td>Moderate dysplasia</td><td> 9/9</td>

<td>Severe dysplasia</td><td> 8/8</td>

<td>Carcinoma in situ</td><td> 11/11</td>

<td>Adenocarcinoma, squamous cell carcinoma</td><td> 10/10</td>

<td>and small cells</td><td></td>

Table 2. Cell descriptions: TCPP fluorescence and cytomorphological characteristics.

<td></td><td colspan="6">Number of samples with cells that have a numerical description by fluorescence microscopy with TCPP / Number of samples with a cell description by cytomorphology</td>

<td>Number of</td><td>Negative or</td><td>Dysplasia</td><td>Dysplasia</td><td>Dysplasia</td><td>Carcinoma</td><td>Adenocarcinoma,</td>

<td>classification =</td><td>metaplastic</td><td>mild</td><td>moderate</td><td>serious</td><td>in situ</td><td>carcinoma of</td>

<td>cell description</td><td>(n = 10)</td><td>(n = 12)</td><td>(n = 9)</td><td>(n = 8)</td><td>(n = 11)</td><td>squamous cells and small cells (n = 10)</td>

<td>1 = core or large nuclei, low to medium fluorescence</td><td> 3/10</td><td> 12/12</td><td> 9/9</td><td> 7/8</td><td> 10/11</td><td> 6/10</td>

<td>2 = symmetric binuclear cells, medium fluorescence</td><td> 0/10</td><td> 5/12</td><td> 5/9</td><td> 3/8</td><td> 3/11</td><td> 2/10</td>

<td>3 = small oval cells, medium to high fluorescence</td><td> 0/10</td><td> 4/12</td><td> 4/9</td><td> 2/8</td><td> 6/11</td><td> 6/10</td>

<td>4 = small round cells, low fluorescence</td><td> 0/10</td><td> 0/12</td><td> 0/9</td><td> 0/8</td><td> 0/11</td><td> 2/10</td>

<td>5 = multinucleated cells, medium fluorescence</td><td> 0/10</td><td> 0/12</td><td> 3/9</td><td> 6/8</td><td> 9/11</td><td> 7/10</td>

<td>6 = irregular anisoid cells, low to medium fluorescence</td><td> 0/10</td><td> 0/12</td><td> 1/9</td><td> 1/8</td><td> 10/11</td><td> 7/10</td>

IS 2 380 261 T3

<td colspan="7">(Continuation)</td>

<td></td><td colspan="6">Number of samples with cells that have a numerical description by fluorescence microscopy with TCPP / Number of samples with a cell description by cytomorphology</td>

<td>Number of</td><td>Negative or</td><td>Dysplasia</td><td>Dysplasia</td><td>Dysplasia</td><td>Carcinoma</td><td>Adenocarcinoma,</td>

<td>classification =</td><td>metaplastic</td><td>mild</td><td>moderate</td><td>serious</td><td>in situ</td><td>carcinoma of</td>

<td>cell description</td><td>(n = 10)</td><td>(n = 12)</td><td>(n = 9)</td><td>(n = 8)</td><td>(n = 11)</td><td>squamous cells and small cells (n = 10)</td>

<td>7 = cell clumps, medium to high fluorescence</td><td> 0/10</td><td> 0/12</td><td> 1/9</td><td> 0/8</td><td> 1/11</td><td> 3/10</td>

<td>8 = degenerate single cells, medium to high fluorescence</td><td> 0/10</td><td> 0/12</td><td> 0/9</td><td> 0/8</td><td> 4/11</td><td> 7/10</td>

<td>9 = degenerate cell clumps, medium to high fluorescence</td><td> 0/10</td><td> 0/12</td><td> 0/9</td><td> 0/8</td><td> 1/11</td><td> 4/10</td>

<td>10 = cells uniform in size with small rounded nucleus, medium fluorescence (nucleus only)</td><td> 2/10</td><td> 2/12</td><td> 2/9</td><td> 1/8</td><td> 1/11</td><td> 2/10</td>

<td>11 = almost invisible cell membrane, medium fluorescence</td><td> 6/10</td><td> 6/12</td><td> 2/9</td><td> 2/8</td><td> 2/11</td><td> 0/10</td>

<td>12 = clustered nuclear debris, no fluorescence</td><td> 0/10</td><td> 1/12</td><td> 0/9</td><td> 0/8</td><td> 0/11</td><td> 0/10</td>

<td>13 = nuclear debris background, no fluorescence</td><td> 6/10</td><td> 5/12</td><td> 8/9</td><td> 2/8</td><td> 5/11</td><td> 3/10</td>

<td>14 = nuclear debris background, medium fluorescence</td><td> 1/10</td><td> 2/12</td><td> 2/9</td><td> 1/8</td><td> 1/11</td><td> 0/10</td>

<td>Average number of numerical descriptions per cell examined</td><td> 1,80</td><td> 3,08</td><td> 4,11</td><td> 3,13</td><td> 4,82</td><td> 4,90</td>

<td colspan="3">Each sample of cells can be designated and cells treated with TCPP</td><td colspan="4">with more than one of the 14 numerical cell descriptions for</td>

EXAMPLE 2

Suspension assay for detection and separation of precancerous and cancerous cells using 5 TCPP

This example describes the use of TCPP staining in conjunction with fluorescence flow cytometry in combination with cytomorphological slide microscopy to determine the abnormality of cells found in sputum samples. By virtue of the specificity of TCPP staining, the combination of

ES 2 380 261 T3 flow cytometry followed by slide microscopy is particularly powerful, providing an internal control for cytomorphological slide comparisons.

Methods

Sputum processing procedures used in the preparation of monolayer slides and suspensions. All suspensions and monolayer slides were prepared from sputum samples collected from patients who performed the early morning spontaneous cough technique. Specifically, patients were instructed to cough up any material by coughing over three consecutive mornings into a container filled with a fixative consisting of 2% Carbowax in 50% alcohol / 50% Saccomanno fluid with 0.03-0 .05 mg / ml rifampicin. Rifampicin was added to the fixative solution to serve as a prophylactic against patients with M. tuberculosis or those patients who may be asymptomatic carriers of N. meningitis.

The 2% Carbowax solution was prepared by adding 2 ml molten Carbowax (150) to 98 ml 50% ethanol and mixing for 30 minutes. The glassware used to prepare the solution was kept warm to prevent hardening of the wax on the surface during preparation, which can cause inaccurate measurements. Carbowax was removed prior to exposure to the TCPP working solution by immersion in 95% alcohol for at least 15 minutes.

Rifampin solution (3 mg / ml) was prepared by dissolving 300 mg rifampin capsules in 100 ml ethyl alcohol and mixed in a Waring mixer at high speed. One ml of this solution was added to each 30 ml of Saccomano solution or 20 ml per liter of Saccomano solution and mixed vigorously. The preparation of the Saccomano solution was carried out by standard methods well known to those skilled in the art of cytology.

The sputum sample was poured into a 50 ml plastic centrifuge tube and additional 50% ethyl alcohol solution was added to bring the volume to 50 ml if necessary. The contents of the centrifuge tube were poured into a 250 ml Eberbach semi-micro-liquefier container and homogenized for 10 to 60 seconds, depending on visual examination of the sample and mucosa content. Samples with thick mucosa sometimes required longer liquefaction times. The sample was poured back into the centrifuge tube and centrifuged at slow speed for 10 minutes. The supernatant was decanted, leaving 1 to 2 ml in the centrifuge tube to mix with the cell pellet. The tube was shaken in a mixer for approximately 10 seconds. The mixture was resuspended in 100 ml of MES buffer, pH ~ 6.15. The cells were centrifuged and washed two more times with 100 ml MES buffer, the last time leaving ~ 1 ml in the centrifuge tube in which the cells were resuspended. The cells were then resuspended in 15 ml of 95% ethanol (5% 100 mM MES buffer) at room temperature (~ 20 ° C) for 30 minutes, with gentle shaking. The centrifuge tube was then centrifuged at slow speed for 10 minutes. All supernatant except 1-2 ml was removed.

TCPP stock solution. 400 mg of sodium bicarbonate was added to approximately 90 ml of 50% isopropanol (50 mM sodium bicarbonate) and mixed until completely dissolved to prepare 50% isopropanol base. One hundred milligrams of TCPP was slowly added to the 50% isopropanol base (50 mM sodium bicarbonate) and mixed for 3 to 5 minutes until dissolved. The TCPP solution was made up to 100 ml volumetrically with the 50% isopropanol base, mixed well, and stored in an amber reagent bottle covered with aluminum foil in a refrigerated area. The final concentration of TCPP in the stock solution was 1 mg / ml.

TCPP working solution. Fresh TCCP working solution was prepared each day. Approximately 10 ml of TCPP stock solution with a concentration of 1 mg / ml was brought to room temperature. Eight milliliters of TCPP stock solution (1 mg / ml) were placed in a 200 ml volumetric flask and approximately 100 ml of MES buffer was added slowly. The solution was mixed gently. Additional MES buffer was added to bring the solution to 200 ml volumetrically. The solution was mixed for 3 to 5 minutes and stored at 24 ° C in an amber bottle. The final concentration of TCPP in the working solution was 40 pg / ml.

TCPP exposure procedure. Cell suspensions previously exposed to 95% alcohol for 30 minutes at room temperature were exposed to TCPP immediately after fixation or up to 3 days later. The cells were resuspended in 10 ml of the TCPP solution at 40 pg / ml for 10 minutes at 36 ° C with gentle shaking, then they were washed three times with 20 ml of 100 mM MES buffer, using low speed centrifugation to pellet cells for 10 minutes. The washed cell pellet was resuspended in 15-10 ml of MES buffer. These suspensions, or aliquots thereof, are passed through a fluorescence flow cytometry apparatus.

Fluorescence flow cytometry. First Pass. A minimum of 10,000 cells are passed through a flow cytometer with cell sorting capabilities. The flow cytometer must be equipped with a light source that provides radiation at approximately 415 nm, with filters that allow light to pass between approximately 390 nm and 490 nm. Fluorescence emission should be controlled between approximately 630 nm

ES 2 380 261 T3 and 730 nm (emission maximum at 645 nm and 706 nm). A barrier filter that passes light above 500 nm is satisfactory. In the first pass, individual cells are counted and their specific fluorescence is measured. The mean fluorescence is calculated and the standard deviation of that mean is calculated. In addition, the median value (the specific fluorescence value that is less than half of the values ​​and greater than half of the values) is determined.

Fluorescence flow cytometry with cell sorting. Second pass. A minimum of 100,000 cells are passed through the cell sorting capable fluorescence flow cytometer, equipped in the same way as for the first pass. Cells with less fluorescence than the median fluorescence + 1.3 standard deviations from the mean (approximately 90% of cells) are functionally defined as having low fluorescence, and stored in a test tube, and cells with specific fluorescence greater than or equal to the median fluorescence + 1.3 standard deviations from the mean (approximately 10% of cells) are functionally defined as having high fluorescence and stored in another test tube. Alternatively, cells can be sorted into tubes according to their fluorescence relative to the specific median fluorescence obtained in the first pass. Cells with less than twice the median specific fluorescence would be normal or low fluorescence, and then cells can be combined with 2-4x the median fluorescence, 4-6x, and greater than 6x the median fluorescence. Each of the sets of fluorescence with higher intensity is expected to be more enriched for abnormal cells. If more than 2-3% of cells have more than 3 times the median fluorescence, there would be support to presume, at the very least, an advanced precancerous condition.

Preparation of monolayer slides. The low and high fluorescence cell samples were centrifuged for 10 minutes at low rpm to pellet the cells. The supernatant was removed, leaving 1-2 ml in the centrifuge tube to mix with the cell pellet. The tube was shaken on a rotary mixer for approximately 10 seconds. One to three drops of the pellet were placed in a PreservCyt vial (Cytyc Corporation, Marlborough, MA). The sample was incubated for 5 minutes to inactivate all microbial and viral organisms.

The single cell layers of the samples were fixed on slides using the Thinprep Processor (Cytyc Corporation, Malborough, MA) according to the manufacturer's instructions. In the ThinPrep Processor, cells are collected on a polycarbonate filter (0.5mm pore size) and transferred to a glass slide. The ThinPrep Processor then immediately deposits the slides in a fixative bath containing 95% ethanol (holds for 30 minutes).

Sputum slide staining procedures using modified PAP staining technique. Procedure sequence (no.), Reagent, and time (min .: sec.) Were as follows: (1) 95% alcohol 15:00, (2) tap water 1:00, (3) Gil-i hemotox 2:30, (4) tap water 1:00; (5) bluish reagent: 30; (6) tap water 1:00; (7) 95% alcohol: 10; (8) og-6 1:30; (9) 95% alcohol: 10; (10) 95% alcohol: 10; (11) EA-50 1:15; (12) 95% alcohol: 20; (13) 95% alcohol: 30; (14) 100% alcohol 1:00; (15) 100% alcohol 1:00; (16) 100% alcohol 1:30; (17) xylene 1:00; (18) xylene 1:00; and (19) xylene 1:00.

Methods of routine cytopathological analysis of slides with Papanicolaou staining. Stained slides were subjected to semiquantitative cytomorphological evaluation. (1) dysplastic and neoplastic cells were identified using traditional morphological criteria, and (2) the expression levels of seven fundamental indicators of lung inflammation were quantified (alveolar macrophages, neutrophils, columnar cells, mucus, mucous spirals, macrophages pigmented, metaplastic cells). The methodology for the quantification of these indicators of inflammation has been previously discussed in the literature (Roby et al., 1989, Acta Cytol 34: 147-154; Roby et al., 1990, Acta Cytol 34: 140-146; Schumann et al., 1989, Am Rev Respr Dis 139: 601-603). The criteria used to determine cell morphology using PAP staining cytology are discussed below.

There are no significant anomalies. Cells were identified without significant alterations if the following requirements are met:

1. basophilic epithelial hair cells mixed with grade 1-2 pigmented macrophages along with inflammatory cells;

2. round nuclei of basally oriented epithelium;

3. uniformly dispersed chromatin;

Four. nuclear membranes barely visible;

5. nucleoli barely visible; and no metaplastic or dysplastic cells are present.

IS 2 380 261 T3

Squamous metaplasia (without dysplasia). Cells were identified as squamous metaplastic without dysplasia if the following requirements are met:

1. groups of basophilic cells without cilia;

2. uniform cell and nuclear size;

3. low nucleus / cytoplasm ratio (N / C);

Four. finely granular nuclear chromatin; Y

5. small rounded nucleoli may be present (usually alone).

Mild dysplasia (squamous atypia). Cells were identified as mild dysplasia if the following requirements are met:

1. smaller than metaplastic cells;

2. they are seen in cohesive groups, or singularly;

3. cells appear flat (leaves) both nuclei and cytoplasm in focus;

Four. cells vary slightly in size;

5. the cytoplasm can be eosinophilic or basophilic;

6. thin cytoplasmic borders;

7. nuclei vary slightly in size, generally round to oval, if divided into 2 nuclei halves they are mirror images, the N / C ratio may vary slightly;

8. smooth nuclear membrane;

9. finely granular nuclear chromatin (slightly increased), occasional chromocenter;

10. fiber-like cells, elongated cells with elongated cytoplasm and nucleus different nuclear membrane fine to granular reticular cytoplasm usually bright orange-yellow-simple keratinizing, can form eddies around the central keratin nucleus to make epithelial beads.

Moderate dysplasia (squamous atypia). Cells were identified as moderately dysplastic if the following requirements are met:

1. variation in size, usually larger, but may be less than in mild dysplasia;

2. more variation in shape and N / C ratio than in mild dysplasia;

3. dense cytoplasm, acidophilic predominates; increased number of atypical cells;

Four. the nucleus may have two unequal halves (they are not mirror images);

5. nuclear lobulations, cracks, and nodules are present, and

6. nuclear material may show hyperchromasia with more stipple-like chromatin pattern.

Severe dysplasia (marked squamous atypia). Cells were identified as severe dysplasia if the following criteria are met:

1. cells vary greatly in size and shape;

2. usually slightly larger cell size than in moderate dysplasia;

3. the N / C ratio is high, but variable (with extremes);

Four. individual cells predominate, the nucleus is more central than in CIS;

5. the nucleus can follow the shape of the cytoplasm; the core shows less distortion than in CIS;

IS 2 380 261 T3

6. nuclear pleomorphism increases with the presence of thick chromatin and condensation along the nuclear envelope;

7. parachromatin, large nucleus, focally thickened multi-clustered nuclear membrane, and

8. cells show predominant acidophilic cytoplasm.

Squamous cell carcinoma in situ (CIS, non-invasive). Cells were identified as squamous cell carcinoma in situ if the following criteria are met:

1. individual cells or in aggregates (groups);

2. variable cell size - may be smaller or larger than marked dysplasia cells usually smaller than invasive squamous cell carcinoma;

3. cells are large, rounded with symmetrically located nuclei;

Four. cell degeneration may be present;

5. scant cytoplasm, uniformly distributed, may be keratinized or non-keratinized concentrically around the nucleus, (orangiophilic or basophilic);

6. variable N / C ratio - higher or lower than normal;

7. Coarse dense nuclear chromatin granules may be interrupted by clear areas;

8. chromatin rim uniformly thickened with ripple of nuclear membrane;

9. lobulations of the nuclei can be seen;

10. cannibalism can be observed, but is unusual;

eleven. multinucleated cells may be present;

12. without nucleoli in the nucleus, a mitotic cell may be present, and

13. light background.

Squamous cell carcinoma (well differentiated keratinizing invasive type). Cells were identified as squamous cell carcinoma if the following criteria are met:

1. cells usually individual, orangephilic, but can be in groups and degenerate;

2. large or small, angular cells with well-preserved nuclei and well-defined cell borders;

3. cells generally larger than in situ, and can be pleomorphic, wide range of size and shape;

Four. pearl formation (cancer pearls) can be observed;

5. moderate amount of cytoplasm with abnormal tail (consistent with invasion); foreign cells in the form of a tadpole, star, axis, an angular nucleate chromatin, with unpredictable clustering with hyperchromasia and clearly defined parachromatin and chromatin compensation, parachromatin interface;

6. the chromatin is lumpy, especially along the nuclear membrane;

7. nucleoli are large and acidophilic, if present;

8. the nuclear membrane itself may be thickened and irregular; edge thickness irregularity of nuclear chromatin;

9. the N / C ratio is very high;

10. marked nucleolar irregularity in shape, size, number (daughter nucleoli); abnormal mitoses, multinucleation;

eleven. cannibalism and multinucleation are common; Y

IS 2 380 261 T3

12. necrotic background material is common.

Cytopathology analysis. Because sputum samples contain cells from many locations in the lung, 5 intermixed with each other, there is little context to judge the normality or abnormality of a particular cell (unlike the case of thin-section staining). The availability of a low-fluorescence cell harvest provides an internal control sample of normal or near-normal patient cells, to which TCPP-stained high-fluorescence cells are compared. Using standard PAP staining, a skilled cytopathologist can easily determine the degree of abnormality of high fluorescence TCPP cells, which are 10-fold enriched for abnormal cells compared to an unfractionated monolayer.

The present invention is not limited to the embodiments described and exemplified above, but variations and modifications are possible within the scope of the following claims.