JP2010537191A - A new molecular marker for detection of squamous cell carcinoma and adenocarcinoma and its high-grade precursor lesions - Google Patents

Abstract

The present invention provides new markers comprising the genes of the present invention for the detection of HPV-induced or pulmonary squamous cell carcinoma and / or adenocarcinoma and its high-grade precursor lesions. Detection can be accomplished by detection of overexpression or downregulation of the gene, either by nucleic acid assays or assays for proteins produced by the gene.

Description

  The present invention relates to the field of diagnosis, particularly cancer diagnosis, and more particularly to the diagnosis of squamous cell carcinoma and adenocarcinoma and their high-grade precursor lesions.

  The development of squamous cell and adenocarcinoma is characterized by a series of precancerous lesions that are staged as mild dysplasia, moderate dysplasia, and severe dysplasia / in situ carcinoma . Squamous cell and adenocarcinoma can develop from the mucosal lining of various organs such as the cervix, vagina, anus, head and neck, and lungs.

Over the past decade, it has been well established that some squamous cell carcinomas and adenocarcinoma can be initiated by infection with high-risk human papillomavirus (hrHPV). This causality is clear from epidemiological and functional studies on cervical cancer (
zur Hausen, Nat Rev Cancer 2002; 2: 342-350; Bosch et al., J Clin Pathol. 2002; 55: 244-265). Up to 99.7% of cervical squamous cell carcinoma (SCC) (Walboomers et al., J. Pathol. 1999: 189: 12-19) and at least 94% of cervical adenocarcinoma and adenosquamous carcinoma ( HrHPV DNA was detected in Zielinski et al., J Pathol 2003: 201: 535-543). Expression of the viral oncogenes E6 and E7, which disrupt the p53 and Rb tumor suppressor pathways, respectively, has been shown to be essential for both the onset of tumorigenesis and maintenance of the malignant phenotype. However, in line with the multi-step process of carcinogenesis, the progression of hrHPV infected cells to invasive cancer requires additional changes in the host cell genome.

In squamous cell carcinomas not related to HPV, these pathways are disrupted by other means such as genetic mutations and / or deletions that have been recognized as early events in the pathogenesis of these tumors. As in the case of hrHPV-induced lesions, additional changes in the cellular genome are also required for the progression of perturbed p53 and Rb pathway lesions to precancerous lesions and cancer. These additional changes would be equal in lesions caused by HPV and lesions that disrupt the p53 and Rb pathways by other means such as the result of exposure to carcinogens in tobacco smoke. There is an example of inactivation of the tumor suppressor gene TSLC1 / CADM1, which is part of hrHPV-induced cervical cancer (Steenbergen et al., J Natl Cancer).
Inst. 2004: 96: 294-305) and lung cancer (Kuramochi et al., Nature Genetics 2001: 27: 427-430), the latter usually developing independently of hrHPV.

  Only a few patients with epithelial cells showing evidence of disruption of the p53 and Rb pathways, including those infected with hrHPV, develop pre-cancerous lesions or cancer that meet the numerous events that underlie carcinogenesis It is an observation to do.

  Currently, there is a lack of markers that predict with high specificity high-grade precancerous lesions and squamous cell carcinomas and adenocarcinoma among those at risk, such as women with cervical hrHPV infection. Such markers are most important for cost-effective early detection programs. However, the heterogeneity of events that can drive malignant transformation of cells with disrupted p53 and Rb pathways indicates that a single marker lacks sufficient sensitivity to malignant disease. Therefore, the inventors conclude that a panel of various markers covering all cancers and their high-grade progenitor stages, where the p53 and / or Rb pathway can emerge from perturbed cells, is essential. did.

  Three data obtained by genome profiling and expression profiling of in vitro model systems of cervical squamous cell carcinoma, cervical adenocarcinoma, and HPV immortalized epithelial cells in combination with innovative statistical methods By using sets, we now have a high correlation of overexpression and / or down-regulation of numerous genes with human cervical squamous cell carcinoma and adenocarcinoma and its high-grade precursor lesions I discovered that. As shown in the examples, a marker panel of three genes was sufficient to detect all cervical squamous cell carcinomas and adenocarcinomas that have been analyzed so far.

  Thus, the present invention is based on evaluating cells of a clinical sample for overexpression of one or more genes listed in Table 1 and / or for downregulation of one or more genes of Table 2. It includes a method for detecting squamous cell carcinoma or adenocarcinoma or its high-grade precursor lesion. Preferably, the method comprises detection of cervical cancer or pre-cancerous cervical lesions, more preferably the squamous cell carcinoma or adenocarcinoma or its high-grade precursor lesion is hrHPV-infected invasive cancer or its high-grade malignant Is a progenitor lesion. In another embodiment, the method includes detection of lung squamous cell carcinoma or high grade precursor lesions thereof.

  Assessment of overexpression or downregulation is preferably performed by nucleic acid assays or by assessing the concentration of gene products, preferably by immunoassays.

  In another embodiment, the invention relates to at least one gene of Table 1 or Table 2, more preferably at least 2 genes, more preferably at least 3 genes, more preferably at least 4 genes, more preferably at least 5 It includes one or more nucleic acid assays that target one gene, more preferably at least 6 genes, most preferably at least 7 genes. Preferably, the assay targets 3-6 genes listed in Tables 1 and 2. A greater number of genes from Table 1 or Table 2 can also be evaluated in the assays of the invention. In addition, the assay additionally targets a control gene.

  The present invention also provides a kit for detecting squamous cell carcinoma or adenocarcinoma and its high-grade precursor lesions, the detection kit comprising means for collecting a sample from a subject, optionally storing the sample. Means, and means for detecting overexpression or downregulation of one or more genes listed in Tables 1 and 2.

  In yet another embodiment, the present invention relates to polypeptides of the genes listed in Table 1 that function as tumor-associated antigens that can be recognized by cytotoxic T lymphocytes (CTLs) and / or that can induce CTLs. A method of treating squamous cell carcinoma or adenocarcinoma and its high-grade precursor lesions by antigen-specific immunotherapy based on peptides or gene sequences. The (poly) peptide sequence may include an amino acid sequence in which one or more amino acids of a specific amino acid sequence are subjected to substitution, deletion, insertion or addition, and further have immunity-inducing activity.

It is a figure of the result of real-time RT-PCR. Control patients (both normal cervical vagina and cervix), as well as squamous cell carcinoma (SSC) and adenocarcinoma (AdCA), against the cell line RNA used for the standard curve by real-time RT-PCR Of ITGAV gene in samples taken from. The Y axis gives the expression level (2 log) relative to the standard curve, based on tumor cell line RNA. It is a figure of the result of real-time RT-PCR. Similarly for SYCP2. It is a figure of the result of real-time RT-PCR. Similarly for DTX3L. FIG. 5 is a real-time RT-PCR of AQP3 for HPV immortalized cells and controls. FIG. 2 is a real-time RT-PCR of AQP3 for normal cervical epithelium and cervical cancer. FIG. 6 shows the fold change (FC) as determined by microarray analysis compared to normal cervical epithelium for the 7 genes identified by the integration of microarray CGH and expression analysis. It is the arrangement | sequence of the gene of Table 1 and Table 2. FIG.

  The present invention is based on an innovative approach that combines a unique set of cervical tissue specimens and three data sets derived from HPV immortalized keratinocyte cell lines.

  The first data set is based on mRNA expression profiling of cervical squamous cell carcinoma, cervical adenocarcinoma and normal epithelial control, and is differentially expressed in cervical cancer compared to normal control including. The second data set is the integration of expression profiling and genomic profiling using the comparative genomic hybridization array (CGH array) of the same set of cervical squamous cell carcinoma and cervical adenocarcinoma used for expression profiling (Wilting et al., J Pathol 2006: 209: 220-230). The third data set is based on mRNA expression profiling of a well-defined in vitro model system of HPV immortalized keratinocytes (Steenbergen et al., J Natl Cancer Inst 2001: 93: 865-872). Since all cell lines in this model system have the same genetic background and non-genetic background, genes that are identified differently in this model system represent marker genes, and these markers Rather than being the result of a (non) genetic background difference, genes are specifically associated with immortalization, an important feature of cancer cells.

  Comparison of these data sets revealed that changes in DNA copy number do not necessarily reflect changes in the expression level of genes seated in the altered chromosomal region. This indicates that the expression marker cannot be inferred solely from the DNA profile. Conversely, changes in gene expression in a chromosomal region are not always reflected by changes in the DNA copy number in that region. These findings highlight the value of combining various data sets for selection of the most comprehensive panel of candidate marker genes.

  By combining the three data sets, 64 marker genes were identified, which are expressed differently in some cervical cancers and HPV immortalized cells compared to normal epithelial cells. Thirty of these genes listed in Table 1 revealed overexpression in cervical cancer / HPV immortalized cells compared to normal epithelial controls. The other 34 genes listed in Table 2 were found to be down-regulated in cervical cancer / HPV immortalized cells compared to normal epithelial controls.

  The genes listed in Tables 1 and 2 and their gene products provide a valuable source of molecular markers from which to diagnose invasive cancer and high-grade precursor lesions of this cancer A marker panel can be configured. Second, the (poly) peptide sequence of the genes listed in Table 1 provides a valuable source of tumor antigens for the treatment of cancer and high-grade precursor lesions of this cancer by immunotherapy.

  “Expression” refers to transcription of a gene into structural RNA (rRNA, tRNA) or messenger RNA (mRNA) and, if applicable, subsequent translation into protein.

The term “invasive cancer” refers to a cancer that invades the surrounding tissue.
The term “HPV-induced invasive cancer” refers to a cancer induced by high-risk HPV that invades surrounding tissues. The term “invasive cervical cancer” refers to cervical cancer that invades the surrounding tissue.

  The term “invasive lung cancer” refers to lung cancer that invades the surrounding tissue. The terms “high-grade precancerous lesion” and “high-grade precursor lesion” represent aspects in multi-stage cell evolution to cancer that have significantly increased the chance of progressing to cancer. With conventional morphology, pathologists cannot predict whether precancerous lesions will progress or regress in individual patients. This patent application refers to a method by which progression to invasive cancer can be predicted.

The term “invasive potential” refers to the potential to infiltrate the surrounding tissue and eventually become a tumor.
The terms “high-grade precancerous cervical lesion” or “high-grade cervical progenitor lesion” refer to an aspect in multistage cell evolution to cervical cancer that has significantly increased the chance of progression to cervical cancer. To express. With conventional morphology, pathologists cannot predict whether cervical precancerous lesions will progress or regress in individual patients.

  The term “can specifically hybridize to” refers to a nucleic acid sequence that can specifically base pair with and bind to a complementary nucleic acid sequence to form a nucleic acid dimer.

  A “complement” or “complementary sequence” is a nucleotide sequence that forms a hydrogen bond dimer with another nucleotide sequence according to the Watson-Crick base-pairing rules. For example, the complementary base sequence of 5'-AAGGCT-3 'is 3'-TTCCGA-5'.

The term “stringent hybridization conditions” refers to hybridization conditions that affect the stability of the hybrid, such as temperature, salt concentration, pH, formamide concentration, and the like. These conditions are optimized empirically to maximize specific binding and minimize non-specific binding of the primer or probe to the target nucleic acid sequence. The term used includes a reference to a condition in which a probe or primer hybridizes to a target sequence and the extent of detection is greater than other sequences (eg, at least twice background). Stringent conditions are sequence-dependent and will be different in different circumstances. Larger sequences hybridize specifically at higher temperatures. In general, stringent conditions are selected to be about 5 ° C. lower than the melting temperature (T _m ) for the specific sequence at a defined ionic strength and pH. T _m is the temperature at which 50% of the complementary target sequence (at a defined ionic strength and pH) hybridizes to a perfectly matched probe or primer. Typically, stringent conditions are such that the salt concentration is less than about 1.0 M Na ion, usually about 0.01 to 1.0 M Na ion concentration (or other salt) at pH 7.0 to 8.3. Where the temperature is at least about 30 ° C. for short probes or primers (eg 10-50 nucleotides) and at least about 60 ° C. for long probes or primers (eg greater than 50 nucleotides). Stringent conditions may be achieved by the addition of destabilizing agents such as formamide. Typical low stringency conditions or “reduced stringency conditions” are hybridization with 30% formamide, 1M NaCl, 1% SDS buffer solution at 37 ° C. and 2 × SSC at 40 ° C. Includes cleaning. Typical high stringency conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37 ° C. and washing in 0.1 × SSC at 60 ° C. Hybridization procedures are well known in the art and are described, for example, in Ausubel et al, (Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994).

  The term “oligonucleotide” refers to a short sequence of nucleotide monomers (usually 6-100 nucleotides) joined by phosphate linkages (eg, phosphodiester, alkyl and allyl phosphates, phosphorothioates) or non-phosphorus linkages (eg, peptides, sulfamates, etc.). ). Oligonucleotides have modified bases (eg 5-methylcytosine) and modified sugars (eg 2′-O-methylribosyl, 2′-O-methoxyethylribosyl, 2′-fluororibosyl, 2′-aminoribosyl, etc.). Modified nucleotides may be included. Oligonucleotides can be circular, branched or linear, double- and single-stranded DNA and double- and single-stranded RNA, naturally occurring or synthetic molecules, optionally stable secondary Domains that can also form structures may be included (eg, stem and loop structures and loop-stem-loop structures).

  The term “primer” as used herein refers to the conditions under which synthesis of a primer extension product complementary to a nucleic acid strand is induced, ie, the presence of a nucleotide and a polymerizing agent such as a DNA polymerase, and an appropriate temperature and When under pH conditions, it represents an oligonucleotide that anneals to the amplification target and allows the DNA polymerase to attach and serve as a starting point for DNA synthesis. The (amplification) primer is preferably single stranded for maximum efficiency in amplification. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be long enough to facilitate synthesis of the extension product in the presence of the polymerizing agent. The exact length of the primer depends on many factors, including temperature and primer source. As used herein, “bidirectional primer pair” refers to one forward primer and one reverse primer commonly used in DNA amplification techniques such as PCR amplification.

  The term “probe” refers to a single stranded oligonucleotide sequence that recognizes a complementary sequence in a target nucleic acid sequence analyte or a cDNA derivative thereof and forms a hydrogen bond dimer therewith.

  “Expression” refers to transcription of a gene into structural RNA (rRNA, tRNA) or messenger RNA (mRNA) and, if applicable, subsequent translation into protein.

  Polynucleotides are “heterologous” to each other if they do not occur simultaneously in the same organism. A polynucleotide is heterologous to an organism if it is not naturally occurring in that particular form and configuration in the organism.

A polynucleotide has a “homologous” sequence if the sequence of nucleotides in the two sequences is the same when aligned with maximal match as described herein. Sequence comparison between two or more polynucleotides is usually performed by comparing portions of the two sequences over a comparison window to identify and compare local region sequence similarity. The comparison window generally ranges from about 20 to 200 contiguous nucleotides. A “percent sequence homology” of a polynucleotide, such as 50, 60, 70, 80, 90, 95, 98, 99 or 100 percent sequence homology, compares two sequences that are optimally aligned over the comparison window. The portion of the polynucleotide sequence in the comparison window may contain additions or deletions (ie gaps) when compared to the reference sequence (not including additions or deletions) of the optimal alignment of the two sequences. Percentages are: (a) determine the number of positions where the same nucleobase occurs in both sequences to determine the number of matching positions; (b) determine the number of matching positions by the total number of positions in the comparison window. And (c) multiply the result by 100 to determine the percentage of sequence homology. Optimal alignment of sequences for comparison may be done by computer implementation of known algorithms or by inspection. Easily available sequence comparison and multi-sequence alignment algorithms are the Basic Local Alignment Search Tool (BLAST) (Altschul, SF et al. 1990. J. Mol. Biol. 215: 403; Altschul, SF et al. 1997. Nucleic Acid Res. 25: 3389-3402) and the ClustalW program, both available on the Internet. Other suitable programs are the GAP from the Wisconsin Genetics Software Package (Genetics Computer Group (GCG), Madison, Wis., USA)
Includes BESTFIT and FASTA.

  As used herein, “substantially complementary” is a sequence in which two nucleic acid sequences are at least about 65%, preferably about 70%, more preferably 80%, even more preferably 90%, and most preferably about 98%. It means that they have complementarity with each other. This means that the primer and probe must exhibit sufficient complementarity to hybridize to the template and target nucleic acid, respectively, under stringent conditions. Accordingly, the primer sequences disclosed herein need not reflect the exact sequence of the binding region of the template, and degenerate primers can be used. A substantially complementary primer sequence is a sequence that has sufficient sequence complementarity to effect primer binding and second strand synthesis to the amplification template.

  The term “hybrid” refers to a double-stranded nucleic acid molecule or dimer formed by hydrogen bonding between complementary nucleotides. The term “hybridize” or “anneal” refers to the process by which single strands of nucleic acid sequences form double-helix segments through hydrogen bonding between complementary nucleotides.

  “Overexpression” or “upregulation” of a gene in a particular cell or sample means that the mRNA is transcribed more from the gene of the particular cell or sample than in the control cell or control sample. Alternatively, this increase in expression can be measured from the concentration of the gene product (protein) in the cell. The increase in expression should be at least 1.5 times, preferably at least 2 times, more preferably at least 3 times greater than the expression in the control. This increase in expression can be measured relative to the expression of the gene in the control sample or relative to the expression of the control gene within the same sample.

  “Underexpression” or “downregulation” of a gene in a particular cell or sample means that the mRNA is transcribed less from the gene of the particular cell or sample than the control cell or control sample. Alternatively, this decrease in expression can be measured from the concentration of the gene product (protein) in the cell. The reduction in expression should be at least 0.5 times, preferably at least 0.25 times, more preferably at least 0.1 times or less of the expression in the control. This reduction in expression can be measured relative to the expression of the gene in the control sample or relative to the expression of the control gene within the same sample.

  Expression of the genes in Table 1 and Table 2 may be detected by measuring gene transcripts. For this reason, the coding region of the protein in these genes provides a marker sequence for detection of the transcript of the gene. In yet another alternative, gene expression may be detected directly by measuring the protein produced by the gene.

  Thus, the cellular component used in the assay can be RNA, preferably a nucleic acid such as mRNA, or a protein. When the cellular component is a protein, the reagent is generally an antibody against the protein produced by the gene. Where the component is a nucleic acid, the reagent is generally a nucleic acid (DNA or RNA) probe or (PCR) primer. By using such probes or primers, gene expression products such as mRNA may be detected, for example.

  Test cell components may be detected directly in situ or may be isolated from other cell components by common methods well known to those skilled in the art prior to contacting with the reagents (eg, “ Current Protocols in Molecular Biology, Ausubel et al., 1995. Fourth edition, John Wiley and Sons; “A Laboratory Guide to RNA: Isolation, analysis, and synthesis”, Krieg (ed.), 1996, Wiley-Liss; "Molecular Cloning: A laboratory manual", J. Sambrook, EF Fritsch. 1989. Vol. 3, 2nd edition, Cold Spring Harbor Laboratory Press).

  Detection methods include Northern blot analysis, RNase protection, immunoassay, in situ hybridization, PCR (Mullis 1987, US 4,683,195, US 4,683,202, and US 4,800,159), LCR (Barany 1991, Proc. Natl. Acad. Sci. USA 88: 189-193; EP320, 308), 3SR (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1874-1878), SDA (US 5,270,184 and US 5,455,166), TAS (Kwoh et al., Proc. Natl. Acad. Sci. USA 86: 1173-1177), Q-Beta replicase (Lizardi et al., 1988, Bio / Technology 6: 1197), rolling circle amplification (RCA) or other cDNA / DNA amplification methods Including. In an alternative method, RNA can be obtained from NASBA (L. Malek et al., 1994, Meth. Molec. Biol. 28, 36, edited by Isaac PG, Humana Press, Inc., Totowa, NJ) or TMA. It may be detected by a method. These include PCR analysis on a microfluidic array platform that allows simultaneous detection of multiple targets in one sample using a limited amount of input material. Nucleic acid probes, primers and antibodies can be detectably labeled with, for example, radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelators, enzymes, or biologically relevant binding structures such as biotin or digoxigenin . One skilled in the art knows other labels suitable for binding to the reagent or can determine this through routine experimentation.

Other detection methods include analyzes that can be performed with nucleic acid arrays (especially Chee).
et al., 1996, Science 274 (5287): 610-614). Such arrays include oligonucleotides having sequences that can hybridize to nucleic acid cell components under stringent conditions, and the level of nucleic acid cell components can be detected in the methods of the present invention.

  The present invention provides a nucleic acid assay or immunoassay for detection of squamous cell carcinoma and adenocarcinoma, wherein at least one gene of Table 1 or Table 2, more preferably 2 genes, more preferably 3 genes, More preferably 4 genes are targeted, more preferably 5 genes, more preferably 6 genes, most preferably at least 7 genes.

Another embodiment of the invention is a method for predicting the presence or risk of expression of squamous cell carcinoma and adenocarcinoma, or the development or risk of high-grade precancerous lesions:
a. Taking a tissue sample from the patient, eg a cervical specimen or a lung specimen;
b. Isolating nucleic acid and / or protein from the sample;
c1. Analyzing the gene expression profile of this nucleic acid by assaying with a nucleic acid assay according to the invention; or c2. Analyzing the gene expression of this protein lysate by assaying with an immunoassay according to the present invention;
d. Identifying the expression of one or more genes listed in Tables 1 and 2;
e. Assessing risk based on the expression found in step d. Preferably, the sample of the above method is a freshly collected sample.

Gene expression analysis is preferably done using nucleic acid assays or immunoassays.
In order to investigate gene expression, the assay may be performed on a polynucleotide molecule or protein derived from a clinically relevant source, in this case, for example, a patient suspected of having squamous cell or adenocarcinoma or its high-grade precursor lesions. The derived sample should be targeted. Therefore, preferably a freshly collected sample (within 2 days from sampling) or a sample collected in a preservation solution that ensures the preservation of RNA and / or protein needs to be available. The target polynucleotide molecule must be expressed RNA or nucleic acid derived therefrom (eg, cDNA or amplified RNA derived from a cDNA that incorporates an RNA polymerase promoter). Where the target molecule consists of RNA, this can be total cellular RNA, poly (A) + messenger RNA (mRNA) or a portion thereof, cytoplasmic mRNA, or RNA transcribed from cDNA (cRNA). Methods for preparing total RNA and poly (A) + messenger RNA are well known in the art, for example, Sambrook et al. (1989. Molecular Cloning-A Laboratory Manual (2nd edition) 1-3, Cold Spring Harbor. , New York). In one embodiment, RNA is extracted from cells using CsCl centrifugation after guanidinium thiocyanate lysis (Chrigwin et al., (1979) Biochem. 18: 5294-5299). In another embodiment, total RNA is extracted using a silica gel-based column, and commercially available examples of this column include RNeasy (Qiagen, Valencia, CA, USA) and StrataPrep (Stratagene, La Jolla, CA, USA). In another embodiment, total RNA is extracted using commercially available RNA isolation reagents, examples of reagents are Trizol (Life Technologies, Breda, The Netherlands) and RNAbee (Tel-Test Inc., Friendswood, Texas, USA). )including. In another embodiment, total RNA is extracted using an automated nucleic acid extraction platform, an example of which is Nuclisens EasyMAG (BioMerieux, Durham, NC, USA). Poly (A) + messenger RNA can be selected, for example, by selection with oligo-dT cellulose, or alternatively by reverse transcription of total cellular RNA, primed with oligo-dT or hexamer primers. . In another embodiment, the polynucleotide molecule analyzed by the present invention comprises cDNA, or amplified RNA or a PCR product of cDNA.

  If one wishes to predict or determine the presence of squamous cell carcinoma or adenocarcinoma in a subject, the practitioner should take a sample from the subject and, after RNA isolation, at least one of Table 1 or Table 2, preferably The expression of 3-6 genes should be determined. To normalize these expression data, a control gene or element (such as a housekeeping gene) that is not affected by the tumor status present in the nucleic acid assay used to determine the expression profile of the subject being evaluated It is possible to correct the data related to the mutation by using the expression data. Instead of one control gene, the average value of the control gene pool can also be taken. This correction can be made, for example, by dividing the expression level of each gene tested by the expression level of the control gene / element.

  For purposes of the present invention, antibodies specific for the gene product from Table 1 or Table 2 are used to detect and quantify the presence of the polypeptide produced by the gene in a biological fluid or tissue sample. May be.

  For the purposes of the present invention, a probe specific for a gene polynucleotide from Table 1 or Table 2 may be used to detect and quantify the gene polynucleotide in a biological fluid or tissue sample. .

  Any specimen containing a detectable amount of a polynucleotide or encoded polypeptide of the genes of Tables 1 and 2 can be used. Nucleic acids can also be analyzed by RNA in situ methods, such as in situ hybridization, known to those skilled in the art. Preferred samples for testing according to the methods of the present invention include specimens such as (cervical) specimens and / or (cervical) biopsy materials. Preferably, cell (cervical) scrapes, self-sampling cervical / vaginal specimens, sputum and / or cervical biopsy materials are used as test samples. The subject can be any mammal, but preferably the subject is a human.

The methods of the present invention can utilize antibodies that are immunoreactive with the polypeptides encoded by the genes listed in Tables 1 and 2, and the predicted amino acid sequences of the polypeptides are listed in these tables. They can be obtained from the listed GenBank line numbers, or immunoreactive fragments of antibodies can be utilized. It is possible to use antibody preparations consisting essentially of pooled monoclonal antibodies with different epitope specificities and unique monoclonal antibody preparations. Monoclonal antibodies are raised against antigens containing protein fragments by methods well known to those skilled in the art (Kohler, et al.
al., Nature, 256: 495, 1975).

  The term antibody used in the present invention includes intact molecules and fragments thereof such as Fab and F (ab ′) 2 that can bind to the epitope determinants of the genes listed in Table 1 (and Table 2). Is intended to be. As used herein, antibody also refers to other protein or non-protein molecules with antigen binding specificity, such as miniantibodies, peptidomimetics, anticalins.

  Monoclonal antibodies can be used in the diagnostic methods of the invention, for example, in immunoassays where the monoclonal antibodies are available in the liquid phase or can be bound to a solid phase carrier. In addition, the monoclonal antibodies in these immunoassays can be detectably labeled in a variety of ways. Examples of immunoassay types that can utilize the monoclonal antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay. Detection of antigens using the monoclonal antibodies of the present invention can be performed using immunoassays that run in either forward, reverse, or simultaneous modes, such as immunohistochemical assays or immunocytochemistry of physiological samples. Includes assays. One of ordinary skill in the art knows or can easily identify other immunoassay formats without undue experimentation.

  Monoclonal antibodies can bind to many different carriers used to detect the presence of gene products of the genes in Tables 1 and 2. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose and magnetite. The nature of the carrier may be soluble or insoluble for the purposes of the present invention. One of ordinary skill in the art knows other carriers suitable for binding monoclonal antibodies or can determine this through routine experimentation.

  When conducting an assay, it is desirable to include a “blocker” in the culture medium (usually added with a soluble labeled antibody). A “blocker” is a non-specific protein, protease, or anti-heterophilic immunoglobulin against an immunoglobulin present in an experimental sample, cross-linked to an antibody on a solid phase support, or a radiolabeled indicator antibody, or It is added to ensure that the antibody is not destroyed and does not give false positive or false negative results. Accordingly, the choice of blocker can substantially increase the specificity of the assay described in the present invention. Many unrelated (ie non-specific) antibodies (eg, IgG1, IgG2a, IgM, etc.) of the same class or subclass (isotype) used in the assay can be used as “blockers”. The concentration of “blocker” (usually 1-100 μg / μL) is important in order to maintain adequate sensitivity to further inhibit any undesired interference with cross-reactive proteins that occur with each other in the specimen.

  Other diagnostic methods for detection of gene product production or gene expression of the genes listed in Table 1 and Table 2 include methods in which a test sample containing a cell preparation derived from cervical or other tissues is provided. . Preferably such samples are given as (cervical) scrapings, self-sampled cervical / vaginal specimens, or sputum. To provide an efficient test plan, hrHPV positive specimens are used as cervical test samples.

  A cell or tissue sample obtained from a human is contacted with a reagent that detects the gene product of one or more genes listed in Table 1 and Table 2 and the labeled cell component of the test cell contained in the sample. Appropriately pretreated so as to be able to detect an increase or decrease compared to possible normal cells. The sample may be placed on a support suitable to allow observation of individual cells. Examples of well known supports include glass, polystyrene, polypropylene, polyethylene, polycarbonate, polyurethane, optionally provided with a layer such as poly-L-lysine or silane to improve cell adhesion and immobilization of the sample. . A cervical specimen or biopsy material may be prepared, for example, for the Papanicolaou (Pap) test, or any suitable modification of this test as known by those skilled in the art, with appropriate access of the reagents to the target components. It may be fixed by a procedure that allows In certain embodiments of the invention, the cytological specimen is known to those skilled in the art of a conventional specimen sample, a thin layer preparation of cervical cells, a self-sampling cervical / vaginal specimen, or any other type. Given as a preparation. If preservation is required, the routine procedure is then paraffin embedded using fixed buffered formalin to provide a well-preserved tissue base. In order to allow immunohistochemical or immunocytochemical staining, the antigenicity of the sample material must be restored, ie unmasked. One method for restoring the antigenicity of formaldehyde cross-linked proteins involves treatment of the sample with proteolytic enzymes. This method results in (partial) digestion of the material, and only a fragment of the original protein can be accessed by the antibody.

  Another method of restoring the immunoreactivity of formaldehyde cross-linking antigens involves thermal processes using heat treatment or high energy treatment of the sample. Such a method is described, for example, in US Pat. No. 5,244,787. Yet another method of antigen retrieval from formaldehyde-fixed tissue is described, for example, by Norton (1994. J. Pathol. 173 (4): 371-9) and Taylor et al. (1996. Biotech Histochem 71 (5): 263. Use of a pressure cooker (eg 2100-Retriever), either in combination with microwaves or in the form of an autoclave, as described in -70).

  Various alternatives to formaldehyde may be used, such as ethanol, methanol, methacan or glyoxal, acetone citrate, or a combination of fixatives. Alternatively, the sample may be air dried before further processing.

  In order to allow detection with nucleic acid probes, the sample material must be recovered or unmasked in the case of formalin-fixed and paraffin-embedded materials. One method involves treatment with a proteolytic enzyme and post-fixation with paraformaldehyde. There may be a denaturation process in HCl prior to proteolytic digestion. This method results in (partial) digestion of the material allowing the probe to enter the target. In the case of non-formalin fixed materials such as frozen materials, no specific unmasking procedure is required. Prior to hybridization, the sample can be acetylated by treatment with triethanolamine buffer.

  The present invention also provides a kit of parts for use in a method of detecting squamous cell carcinoma and adenocarcinoma and its high-grade precursor lesions in a subject's test cells. Such kits collect samples, such as a brush or spatula for scraping suspected mucosal tissue, such as a neck scraping or lung brush, and a container filled with collection media for collecting test cells. Means and storage means may suitably be included. Instead, to collect cervical cells by cervical-vaginal lavage, a sampling device consisting of a lavage syringe, a disposable female urinary catheter, and a container with lavage fluid is included.

  The kit according to the invention may have primers and probes for the detection of mRNA expression of the genes listed in Table 1 and Table 2. In another embodiment, a kit according to the present invention may comprise antibodies and reagents for detection of protein expression of proteins expressed by the genes of Tables 1 and 2 in cervical scraping or other tissue specimens. .

The invention is illustrated by the following non-limiting examples.
Example 1. Genes that are differentially expressed in tissues and cell lines To confirm the differential expression of the genes listed in Tables 1 and 2, the following genes: ITGAV, SYCP2, DTX3L, SEMP1, DEK, ATP2C1, SLC25A36 and PIK3R4 mRNA expression was measured by real-time RT-PCR analysis. For this purpose, RNA was added to 22 cervical squamous cell carcinomas, 8 cervical adenocarcinomas, 15 high-grade cervical carcinoma in situ (CIN) lesions and 24 normal epithelial controls (normal cervix). Extracted from vaginal n = 13 and normal cervix n = 11). ITGAV, SYCP2, DTX3L SEMP1, DEK, ATP2C1, PIK3R4 and SLC25A36 showed significant overexpression in squamous cell carcinoma compared to normal cervical vaginal region (see example in FIG. 1). In addition, DTX3L showed significant overexpression in adenocarcinoma compared to normal cervix (FIG. 1). Analysis of RNA isolated from 15 high-grade CIN lesions enriched for atypical cells by laser capture microdissection revealed that 60% (9/15) of cases had ITGAV and 40% (6/15) of cases. ) Demonstrated increased expression of ATP2C1, DEK in 80% (12/15) of cases, SLC25A36 in 73% (11/15) and PIK3R4 in 67% (7/15) of cases did.

  For another gene, AQP3 in both HPV immortalized cells (n = 8) compared to control cells (n = 4) and cervical cancer (n = 8) compared to normal cervical epithelial cells (n = 1) The decrease in the expression of was confirmed by real-time RT-PCR (FIG. 2).

  Based on the overexpression detected by microarray analysis over 2 fold, 100% cervical squamous cell carcinoma and 100% by a marker panel of 3 genes (ie, a combination of DTX3L, FLJ21291 and CCDC14 or ITGAV) Cervical adenocarcinoma could be detected (Fig. 3).

In addition, real-time RT-PCR analysis for the expression of ITGAV, DTX3L, DEK, ATP2C1, SLC25A36 and PIK3R4 mRNA was performed on 20 lung squamous cell carcinomas and 20 normal lung samples (adjacent to cancer). ITGAV, DTX3L, ATP2C1, SLC25A36 and PIK3R4 mRNA expression was significantly increased in cancer compared to normal control samples.
The primers used for RT-PCR are summarized in Table 3.

Increased expression of both proteins was confirmed by immunohistochemical analysis of PIK3R4 and DTX3L in 25 high-grade CIN lesions and 8 cervical squamous cell carcinomas. 100% (PIK3R4) and 85% (DTX3L) of cervical squamous cell carcinoma and 65% of high-grade CIN lesions (
PIK3R4) and 41% (DTX3L) revealed protein overexpression.

  Two pilot trials of squamous cell carcinoma of the lung also revealed increased PIK3R4 protein expression in these tumors.

2. Marker gene analysis of neck scraping
Using the nested case control design of women participating in the POBASCAM trial, 50 hrHPV positive women diagnosed with ≧ CIN 2 (including 1 cancer) within 18 months of follow-up, 18 Cervical scraping was studied in 100 hrHPV positive control women with CIN 1 or better within a follow-up period of months. Baseline cervical scrapes of these women were collected in universal collection media and subjected to expression analysis of DTX3L and PIK3R4 mRNA by real-time PCR. For the latter, the pool of hrHPV negative women's neck scrapings that had no signs of disease during the 5 years of follow-up served as a standard reference. Samples have an average ratio of repeated experiments [mRNA copy of DTX3L or PIK3R4 gene in case sample: mRNA copy of housekeeping gene] / [mRNA copy of DTX3L or PIK3R4 gene in standard reference sample: mRNA copy of housekeeping gene] ≧ In the case of 1.5, the differential expression was recorded as positive. Increased expression ratios of DTX3L and PIK3R4 to housekeeping genes were seen in 21 and 28 cases, respectively, relative to the control that had nothing. For one or more of these genes, a total of 36 cases revealed increased rates.

Claims (10)

  Squamous cell carcinoma or adenocarcinoma by evaluating cells of a tissue sample for overexpression of one or more genes listed in Table 1 and / or for downregulation of one or more genes of Table 2 Or a method for detecting its high-grade precursor lesions.   The method according to claim 1, wherein the squamous cell carcinoma or adenocarcinoma or a high-grade precursor lesion thereof is a cervical cancer or a precancerous cervical lesion.   The method according to claim 1 or 2, wherein the squamous cell carcinoma or adenocarcinoma or a high-grade precursor lesion thereof is a non-cervical origin hrHPV-infected invasive cancer or a high-grade precursor lesion thereof. .   The method according to claim 1 or 2, wherein the squamous cell carcinoma or adenocarcinoma or a high-grade precursor lesion thereof is lung cancer or a high-grade precursor lesion thereof.   The method according to any one of claims 1 to 4, characterized in that the evaluation of overexpression and / or down regulation is carried out by means of a nucleic acid assay.   5. Method according to any one of claims 1 to 4, characterized in that the evaluation of overexpression and / or downregulation is carried out by assessing the concentration of the gene product, preferably by immunoassay.   Said overexpression and / or said downregulation is preferably at least 2 genes, preferably at least 3 genes, more preferably at least 4 genes, more preferably at least 5 genes listed in Table 1 or Table 2. 7. The method of any one of claims 1-6, wherein is assayed for at least 6 genes, most preferably at least 7 genes.   8. The tissue sample of claim 1, wherein the tissue sample is a cervical specimen, a cervical (or lung) brush, a self-sampling cervical / vaginal specimen, sputum, lavage and / or cervical biopsy material. 2. The method according to item 1.   A kit for detection of squamous cell carcinoma or adenocarcinoma or its high-grade precursor lesion, comprising means for collecting a sample from a subject, optionally, means for storing said sample, and listed in Tables 1 and 2 A kit comprising a means for detecting overexpression or downregulation of one or more genes.   Using a peptide or polypeptide of amino acid sequence (modified / codon optimized) selected from the genes listed in Table 1, squamous cell carcinoma or adenocarcinoma or its high-grade precursor lesions (immunotherapy ) A therapeutic method, characterized in that the peptide is recognized by and / or induces cytotoxic T lymphocytes.

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