WO2012100023A1 - Detection of pancreatic ductal adenocarcinoma - Google Patents

Abstract

The invention provides methods and compositions for diagnosing pancreatic ductal adenocarcinoma in a subject based on the detection or quantification of alternatively spliced COL6A3 transcripts, or the encoded polypeptides, in patient samples. The alternatively spliced COL6A3 transcripts comprise one or more of COL6A3 exons 3, 4 or 6.

Description

DETECTION OF PANCREATIC DUCTAL ADENOCARCINOMA

Reference to Government Grant

This invention was made with government support under Grant

RO1AR053251 awarded by the U.S. National Institutes of Health. The government has certain rights in the invention.

Cross-Reference to Related Application

The benefit of the filing date of U.S. Provisional Patent Application No. 61/434,132, filed January 19, 201 1, is hereby claimed. The entire disclosure of the aforesaid application is incorporated herein by reference.

Field of the Invention

The invention relates to the diagnosis of pancreatic ductal adenocarcinoma

(PDA).

Background of the Invention

The collagens are a superfamily of proteins that play a role in maintaining the integrity of various tissues. Collagens are extracellular matrix proteins and have a triple-helical domain as their common structural element. Collagen VI is a major structural component of microfibrils. The basic structural unit of collagen VI is a heterotrimer of the alphal(VI), alpha2(VI), and alpha3(VI) collagen chains. The alphal(VI) and alpha2(VI) chains are encoded by the COL6A1 and COL6A2 genes, respectively. The protein encoded by the COL6A3 gene is the alpha 3 subunit of type VI collagen (alpha3(VI) collagen chain) (Bertini et al., 2002 Eur. J. Paediatr. Neurol 6:193-8).

Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease in which a prominent desmoplastic reaction is a defining characteristic. Fibrillar collagens, such as collagen I and to a lesser extent, collagen III, and V make up the majority of this stromal fibrosis. Type VI collagen (COL6) is a nonfibrillar collagen that forms a microfibrillar network associated with type I collagen fibrils. The alpha 3 chain of COL6 (COL6A3) has been linked to inflammation and survival (Pasarica el al ,

2009 J Clin Endocrinol Metab. 94:5155-62).

Alternative splicing is a mechanism for increasing protein diversity by excluding or including specific exons during post-transcriptional processing.

Alternatively spliced proteins (the resulting spliced variants) may contribute to the etiology or progression of cancer and may provide selective drug targets or serve as markers for cancer diagnosis. Using exon array analysis studies, alternative splicing of exons 3, 4 and 6 of the COL6A3 transcript, encoding the collagen VI alpha 3 chain, was identified to be tumor-specific in colon cancer (Gadina et al. , 2006 BMC Genomics 7:325) and alternative splicing of exon 6 of the COL6A3 transcript was tumor-specific in colon, bladder, and prostate cancer (Thorsen et al , 2008 Mol Cell

Proteomics 7: 1214-1224).

PDA is one of the deadliest forms of cancer. It is the 4th leading cause of cancer death in the USA and world - wide with the number of diagnosed cases virtually equaling the number of deaths. Due to the silent nature in its early stages, most PDA patients present at the time of diagnosis with advanced unresectable disease.

There is currently no effective chemo or radiation therapy for PDA, and drugs like gemcitabine, capecitabine, and 5-fluorouracil have minor to modest activity. The delay in PDA diagnosis is mainly due to the absence of effective imaging or blood tests that can identify early disease. Benign lesions such as intraductal papillary mucinous neoplasms (IPMN) or pancreatic cystadenomas, chronic pancreatitis and PDA may present with similar symptoms and are not mutually exclusive, which can lead to incorrect diagnosis.

A lack of reliable molecular markers for early detection makes it difficult to predict when high risk patients (smokers, chronic inflammation) are likely to progress to malignant disease. While the incidence of pancreatic cancer continues to rise, this cancer remains notoriously difficult to diagnose in its early stages. At the time of detection, about 50% of all patients have distant disease and 25% have regional spread. The relative 1-year survival is only 24% and the overall 5-year survival is less than 5%.

Currently, there is no diagnostic marker for PDA. C A 19-9 is a serum marker that is considered the standard of care for postsurgical follow up tests. It has been the most thoroughly evaluated serum marker used for the diagnosis of pancreatic cancer (Goonetilleke et al. , European Journal of Surgical Oncology (EJSO) 33(3):266-270 (2007)). CA 19-9 has thus far been considered the most useful for the staging and monitoring of patients with pancreatic carcinomas

(Szymendera, Tumour Biol. 7:333-342 (1986)). However, it is not recommended for initial diagnosis as it is nonspecific and can give false positive findings. CA 19- 9 has failed to provide usefulness in screening for pancreatic cancer, as it may be consistently elevated in many normal patients. One study on the feasibility of CA 19-9 for screening showed that of 27 patients who had high levels of CA 19-9, only 1 had PDA (Zubarik et al , Gastrointestinal Endoscopy, 74(l):87-95 (201 1)).

Therefore, there is an urgent unmet need for an early diagnostic test for PDA. There is an urgent unmet need for tests that detect the presence and predict the invasive potential of early stage pancreatic cancers. Such diagnostics can improve the life expectancy for many affected patients and can facilitate treatment decisions. Identification of informative biomarkers for the early detection of pancreatic cancer, and for assessing prognosis and efficacy of treatment, is urgently need. The present invention meets this need.

Summary of the Invention

The invention is based on the discovery that certain alternative splice variants of COL6A3, which include one or more of exons 3, 4 and 6, are associated with pancreatic ductal adenocarcinoma (PDA).

In one embodiment, the invention provides a marker for diagnosing PDA, wherein the marker is a COL6A3 splice variant containing exon 3, exon 4 and/or exon 6. In one embodiment, the invention provides a method for diagnosing PDA in a subject comprising

a) determining the level of a COL6A3 transcript in a test sample from a subject, wherein said COL6A3 transcript is selected from the group consisting of:

(i) a COL6A3 transcript comprising the nucleotide sequence of

COL6A3 exon 3;

(ii) a COL6A3 transcript comprising the nucleotide sequence of COL6A3 exon 4;

(iii) a COL6A3 transcript comprising the nucleotide sequence of COL6A3 exon 6; and

(iv) any combination thereof;

b) comparing the level of said COL6A3 transcript determined in said test sample to the level of the COL6A3 transcript determined in a control sample, wherein an elevated level of the COL6A3 transcript in said test sample relative to the level of the COL6A3 transcript in said control sample is diagnostic of PDA in said subject.

In another embodiment, the invention provides a method for diagnosing PDA in a subject comprising

a) determining the level of a COL6A3 protein in a test sample from a subject, wherein said COL6A3 protein is selected from the group consisting of:

(i) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 3;

(ii) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 4;

(iii) the COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 6; and

(iv) any combination thereof,

b) comparing the level of said COL6A3 protein determined in said test sample to the level of the COL6A3 protein determined in a control sample, wherein an elevated level of the COL6A3 protein in said test sample relative to the level of the COL6A3 protein in said control sample is diagnostic of PDA in said subject. In another embodiment, the invention provides a method for diagnosing PDA in a subject comprising

a) determining the level of a COL6A3 protein fragment in a test sample from a subject, wherein said COL6A3 protein fragment is a fragment of a COL6A3 protein selected from the group of COL6A3 proteins consisting of:

(i) COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 3 ;

(ii) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 4;

(iii) the COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 6; and

(iv) any combination thereof,

b) comparing the level of said COL6A3 protein fragment determined in said test sample to the level of the COL6A3 protein fragment determined in a control sample, wherein an elevated level of the COL6A3 protein fragment in said test sample relative to the level of the COL6A3 protein fragment in said control sample is diagnostic of PDA in said subject.

In another embodiment, the invention provides a method of monitoring the progression of PDA in a subject, said method comprising:

a) obtaining a first sample from a subject at a first time point and a second sample from said subject at a second time point;

b) determining the level of a COL6A3 transcript in said first and second samples, wherein said COL6A3 transcript is selected from the group consisting of:

(i) a COL6A3 transcript comprising the nucleotide sequence of COL6A3 exon 3;

(ii) a COL6A3 transcript comprising the nucleotide sequence of COL6A3 exon 4;

(iii) a COL6A3 transcript comprising the nucleotide sequence of COL6A3 exon 6; and

(iv) any combination thereof;

b) comparing the level said COL6A3 transcript determined in said first sample to the level of the COL6A3 transcript in said second sample, wherein an elevated level of the COL6A3 transcript in said second sample relative to the level of said COL6A3 transcript in said first sample is an indication that the cancer has progressed in said subject.

In yet another embodiment, the invention provides a method of monitoring the progression of a PDA in a subject, said method comprising:

a) obtaining a first sample from a subject at a first time point and a second sample from said subject at a second time point;

b) determining the level of a COL6A3 protein in a test sample from a subject, wherein said COL6A3 protein is selected from the group of:

(i) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 3;

(ii) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 4;

(iii) the COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 6; and

(iv) any combination thereof,

c) comparing the level of said COL6A3 protein determined in said first sample to the level of the COL6A3 protein in said second sample, wherein an elevated level of the COL6A3 protein in said second sample relative to the level of said COL6A3 protein in said first sample is an indication that the cancer has progressed in said subject.

In yet another embodiment, the invention provides a method of monitoring the progression of a PDA in a subject, said method comprising:

a) obtaining a first sample from a subject at a first time point and a second sample from said subject at a second time point;

b) determining the level of a COL6A3 protein fragment in a test sample from a subject, wherein said COL6A3 protein fragment is a fragment of a COL6A3 protein selected from the group of COL6A3 proteins consisting of:

(i) a COL6 A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 3; (ii) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 4;

(iii) the COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 6; and

(iv) any combination thereof,

c) comparing the level of said COL6A3 protein fragment determined in said first sample to the level of the COL6A3 protein fragment in said second sample, wherein an elevated level of the COL6A3 protein fragment in said second sample relative to the level of said COL6A3 protein fragment in said first sample is an indication that the cancer has progressed in said subject.

Abbreviations

The following abbreviations are used herein:

AP: Alkaline Phosphatase

COL6: Type VI collagen

COL6A3: Alpha 3 chain of COL6

ELISA: Enzyme-Linked Immunosorbent Assay

GST: Glutathione S-transferase

HRP: Horseradish Peroxidase

LCR: Ligase Chain Reaction

PCR: Polymerase Chain Reaction

PDA: Pancreatic ductal adenocarcinoma

RIA: Radioimmunoassay

RT-PCR: Reverse Transcriptase PCR

SAGE: Serial Analysis of Gene Expression

Brief Description of the Figures

For the purpose of illustrating the invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings. Figure 1 is a schematic diagram of the three collagen VI chains (COL6A1, COL6A2 and COL6A3). N-globular subdomains are designated N1-N10. The N10, N9 and N7 subdomains are either present or absent in the final COL6A3 chain due to alternative splicing of exons 3, 4, and 6 (E3, E4, E6).

Figure 2 is an image depicting analysis of COL6A3 alternative splice transcripts containing exons 3, 4 and 6 mRNA in PDA tumor ("T_") and adjacent normal surrounding tissue control ("N_") samples from eight patients afflicted with PDA, amplified by RT-PCR. PCR products were electrophoresed on agarose gels.

Figure 3 is a graph showing the results of real-time RT-PCR of COL6A3 transcript containing exon 6 in eight PDA test samples vs. the level in normal surrounding tissue.

Figures 4A, 4B and 4C are graphs showing the results of real-time RT-PCR of COL6A3 transcript containing exons 3, 4 and 6, respectively in serum samples from patients afflicted with PDA, neuroendocrine tumors (NET), intraductal papillary mucinous neoplasm (IPMN), cystadenoma and chronic pancreatitis (Ch pancreatitis).

Definitions

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

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The term "about" will be understood by persons of ordinary skill in the art and will vary to some extent depending on the context in which it is used. As used herein, "about" is meant to encompass variations of ±20% or ±10%, more preferably ±5%), even more preferably ±1%, and still more preferably ±0.1%.

Unless otherwise specified herein, the terms "antibody" and "antibodies" broadly encompass naturally-occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site. Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.

As used herein, an alternative splice form which is "substantially absent from normal cells" means the alternative splice form is not present in the normal cells or is present in a negligible amount.

The term "complementary" refers to nucleic acid sequences that base-pair according to the standard Watson-Crick complementary rules, or that are capable of hybridizing to a particular nucleic acid segment under relatively stringent conditions. Nucleic acid polymers are optionally complementary across only portions of their entire sequences.

"Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.

Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

"Gene expression" or "expression" as used herein refers to the process by which information from a gene, such as the COL6A3 gene, is made into a functional gene product, such as RNA or protein. Thus, the "level of expression" of a gene product, such as a COL6A3 gene product, in a sample of interest, refers to the level of RNA, particularly the level of mRNA, or the level of the encoded protein, and is not intended to be limited to either.

The term "gene" refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA). The term "gene" encompasses both cDNA and genomic forms of a gene.

A genomic form of a gene contains the coding region or "exons" interrupted with non-coding sequences termed "introns" or "intervening regions" or

"intervening sequences." Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. In addition to containing introns, genomic forms of a gene can also include sequences located on both the 5' and 3' end of the sequences that are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript).

Both of transcription and translation are included in the "gene expression" of the present invention. Accordingly, both of detection at the transcription level (mRNA, cDNA) and detection at the translation level (protein) are included in the "detection of spliced COL6A3 expression".

The term "hybridization" refers to the process in which two single-stranded nucleic acids bind non-covalently to form a double-stranded nucleic acid; triple- stranded hybridization is also theoretically possible. Complementary sequences in the nucleic acids pair with each other to form a double helix. The resulting double- stranded nucleic acid is a "hybrid." Hybridization may be between, for example two complementary or partially complementary sequences. The hybrid may have double-stranded regions and single stranded regions. The hybrid may be, for example, DNA:DNA, RNA:DNA or RNA:RNA. Hybrids may also be formed between modified nucleic acids. One or both of the nucleic acids may be immobilized on a solid support. Hybridization techniques may be used to detect and isolate specific sequences, measure homology, or define other characteristics of one or both strands.

As used herein, an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression, which can be used to communicate the usefulness of the invention in the kit for determining the progression of a disease. The instructional material of the kit of the invention may, for example, be affixed to a container, which contains a reagent of the invention or be shipped together with a container, which contains a reagent. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the reagent be used cooperatively by the recipient.

A "marker" is a protein, or associated gene or other nucleic acid, whose altered level of expression (or abundance) in a tissue or cell from its expression level in normal or healthy tissue or cell is associated with a disease state, such as cancer.

"Measuring" or "measurement," or alternatively "detecting" or "detection," or alternatively "determine" or "determining" means assessing the presence, absence, quantity or amount of either a given substance within a clinical or subject- derived sample, including the derivation of qualitative or quantitative concentration levels of such substances.

"Primer" refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, i.e., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization such as DNA polymerase. Typical uses of primers include, but are not limited to, sequencing reactions and amplification reactions. A primer is typically single-stranded, but may be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally-occurring primers are useful for many applications. A primer is complementary to the template to which it is designed to hybridize to serve as a site for the initiation of synthesis, but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with, e.g., detectable moieties, such as chromogenic, radioactive or fluorescent moieties, or moieties for isolation, e.g., biotin.

"Probe" refers to a polynucleotide that is capable of specifically hybridizing to a designated sequence of another polynucleotide. "Probe" as used herein encompasses oligonucleotide probes. A probe may or may not provide a point of initiation for synthesis of a complementary polynucleotide. A probe specifically hybridizes to a target complementary polynucleotide, but need not reflect the exact complementary sequence of the template. In such a case, specific hybridization of the probe to the target depends on the stringency of the hybridization conditions.

As used herein, the term "a reagent that specifically detects expression levels" refers to one or more reagents used to detect the expression of one or more genes (e.g., a gene selected from the 24 marker genes provided herein). Examples of suitable reagents include, but are not limited to, nucleic acid probes capable of specifically hybridizing to the gene of interest, PCR primers capable of specifically amplifying the gene of interest, and antibodies capable of specifically binding to proteins expressed by the gene of interest. The term "amplify" is used in the broad sense to mean creating an amplification product. "Amplification", as used herein, generally refers to the process of producing multiple copies of a desired sequence, particularly those of a sample. A "copy" does not necessarily mean perfect sequence complementarity or identity to the template sequence.

"Sample" or "biological sample" as used herein means a biological material isolated from an individual. The biological sample may contain any biological material suitable for detecting the desired biomarkers, and may comprise cellular and/or non-cellular material obtained from the individual.

The term "solid support," "support," and "substrate" as used herein are used interchangeably and refer to a material or group of materials having a rigid or semirigid surface or surfaces. In one embodiment, at least one surface of the solid support will be substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions for different compounds with, for example, wells, raised regions, pins, etched trenches, or the like. According to other embodiments, the solid support(s) will take the form of beads, resins, gels, microspheres, or other geometric configurations. See US Pat No 5,744,305 for exemplary substrates.

"Specifically binds" as used herein in the contexts of an antibody refers to antibody binding to a predetermined antigen with a preference that enables the antibody to be used to distinguish the antigen from others to an extent that permits the diagnostic assays described herein. Specific binding to alternatively spliced COL6A3 means that the antibody preferentially binds alternatively spliced COL6A3 versus other proteins.

As used herein, a "splice variant" refers to naturally occurring nucleic acid sequences and proteins encoded therefrom which are products of alternative RNA splicing.

As used herein, the term "subject" or "patient" refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, suspected of having PDA or which is to be the subject of a particular diagnosis. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

As used herein, a "normal subject" or "control subject" refers to a subject not suffering from PDA.

As envisioned in the present invention with respect to the disclosed compositions of matter and methods, in one aspect the embodiments of the invention comprise the components and/or steps disclosed herein. In another aspect, the embodiments of the invention consist essentially of the components and/or steps disclosed herein. In yet another aspect, the embodiments of the invention consist of the components and/or steps disclosed herein.

Detailed Description of the Invention

Figure 1 shows the three collagen VI chains (COL6A1 , COL6A2 and COL6A3). Each chain contains a triple helical domain (TH) of 335-336 amino acids, which is flanked by N- and C-globular domains consisting of vWF-A modules (-200 amino acids/module), designated N1-N10 and C1-C2. The N10, N9 and N7 subdomains are either present or absent in the final COL6A3 chain due to alternative splicing of exons 3, 4, and 6 (E3, E4, E6).

The invention is based on the discovery that exons 3, 4 and 6 of the COL6A3 gene are alternatively spliced in a tumor-specific manner in PDA. COL6A3 gene transcripts (e.g., mRNA or cDNA made therefrom) containing one or more of exons 3, 4 and 6 are increased in pancreatic ductal adenocarcinoma (PDA) compared to a corresponding normal tissue. COL6A3 transcripts containing one or more of exons 3, 4 and 6 and encoded polypeptides thereof are therefore PDA-associated markers, and the detection of such markers thus provides for the detection of PDA. The three isoforms are highly expressed in blood, particularly serum, and tissues of patients with PDA, but not in blood or tissue of patients with benign pancreatic lesions or chronic pancreatitis.

Detection of or determination of the level of the PDA-associated markers may be incorporated into the initial patient work-up for the differential diagnosis of patients presenting with symptoms suggestive of PDA, and in the screening and monitoring of patients at high risk. Such high risk patients include, for example, individuals with a familiar history of PDA, chronic pancreatitis sufferers, and those with Peutz-Jeghers syndrome.

The test according to the present invention may also be used as an aid in determining the necessity for more invasive diagnostic biopsies, to monitor the effectiveness of treatment for PDA, and to detect recurrence. The test may also be ordered at regular intervals for all high risk groups. The advantage of the diagnosis panel of the invention is that it is indicative of the presence or absence of malignancy. In particular, identification of one or more of the three isoforms in patient peripheral blood will be diagnostic of malignancy and will be eliminative of benign lesions or chronic pancreatitis. This will be informative to the physician of whether a person is at an increased risk of having PDA and help with the decision of whether or not to proceed with a fine needle biopsy. The test of the present invention will also be useful in directing the subsequent treatment efforts by surgeons (extent of resection, with or without lymph node excision, etc), and medical oncologists (prescribing post surgical chemotherapy or radiotherapy).

Oligonucleotides, polypeptides or other binding entities that bind to the PDA-associated markers (as well as any other specific means for detecting them, such as antibodies), can be used for the detection of PDA and its diagnosis.

In embodiements of the invention, the difference in the level of expression of a COL6A3 splice variant transcript or encoded polypeptide comprising one or more of exon 3, 4 and 6 between patient samples and normal controls is at least about 1.5- fold, more preferably at least about two-fold, more preferably at least about threefold, more preferably at least about four-fold, more preferably at least about six-fold, more preferably at least about eight -fold most preferably at least about ten-fold.

The splice varaint assayed according to the present invention may comprise one or more COL6A3 transcripts comprising exons 3, 4 or 6, or the corresponding encoded polypeptides. In some embodiements all three of the splice variants are assayed. In some embodiments two of the three splice variants are assayed, e.g. the exon 3 variant and the exon 4 variant, the exon 3 variant and the exon 6 variant, or the exon 4 variant and the exon 6 variant. In some embodiments of the invention, a single splice variant is assayed. In one embodiment, the splice variant is a splice variant comprising exon 3, In an other embodiment, the splice variant is a splice variant comprising exon 4. In an other embodiment, the splice variant is a splice variant comprising exon 6.

In one embodiment, the invention provides kits for the diagnosis of PDA in a subject. The kits comprise at least one agent that binds to the PDA-associated markers of the invention, and instructional material for use.

Methods for detecting PDA-associated markers of the invention comprise any methods that determine the quantity or the presence of the markers either at the nucleic acid or protein level.

Detection of Gene Transcript

According to one embodiment of the invention, the method comprises detecting the presence, in a biological sample from a subject, a COL6A3 splice variant transcript comprising one or more of exons 3, 4 and 6. In one embodiment, nucleotide sequences of COL6A3 exons 3, 4 and 6 are set forth in SEQ ID NO: 1, 2 and 3, respectively.

The nucleotide sequence of COLA6A3 exon 3 is:

atgtcaaaaatggtgcggctgctgatataatatttctagtggattcctcttggaccattggagaggaacatttccaacttgttc gagagtttctatatgatgttgtaaaatccttagctgtgggagaaaatgatttccattttgctctggtccagttcaacggaaacc cacataccgagttcctgttaaatacgtatcgtactaaacaagaagtcctttctcatatttccaacatgtcttatattgggggaac caatcagactggaaaaggattagaatacataatgcaaagccacctcaccaaggctgctggaagccgggccggtgacg gagtccctcaggttatcgtagtgttaactgatggacactcgaaggatggccttgctctgccctcagcggaacttaagtctgc tgatgttaacgtgtttgcaattggagttgaggatgcagatgaaggagcgttaaaagaaatagcaagtgaaccgctcaatat gcatatgttcaacctagagaattttacctcacttcatgacatagtaggaaacttagtgtcctgtgtgcattcatccgtgagtcc agaaagggctggggacacggaaacccttaaagacatcacag (SED ID NO: 1 ).

The nucleotide sequence of COLA6A3 exon 4 is:

cacaagactctgctgacattattttccttattgatggatcaaacaacaccggaagtgtcaatttcgcagtcattctcgacttcct tgtaaatctccttgagaaactcccaattggaactcagcagatccgagtgggggtggtccagtttagcgatgagcccagaa ccatgttctccttggacacctactccaccaaggcccaggttctgggtgcagtgaaagccctcgggtttgctggtggggag ttggccaatatcggcctcgcccttgatttcgtggtggagaaccacttcacccgggcagggggcagccgcgtggaggaa ggggttccccaggtgctggtcctcataagtgccgggccttctagtgacgagattcgctacggggtggtagcactgaagc aggctagcgtgttctcattcggccttggagcccaggccgcctccagggcagagcttcagcacatagctaccgatgacaa cttggtgtttactgtcccggaattccgtagctttggggacctccaggagaaattactgccgtacattgttggcgtggcccaa aggcacattgtcttgaaaccgccaaccattgtcacacaag (SED ID NO:2).

The nucleotide sequence of COLA6A3 exon 6 is:

ttcactcaaacaaaagggatatcatctttcttttggatggatcagccaacgttggaaaaaccaatttcccttatgtgcgcgact ttgtaatgaacctagttaacagccttgatattggaaatgacaatattcgtgttggtttagtgcaatttagtgacactcctgtaac ggagttctctttaaacacataccagaccaagtcagatatccttggtcatctgaggcagctgcagctccagggaggttcgg gcctgaacacaggctcagccctaagctatgtctatgccaaccacttcacggaagctggcggcagcaggatccgtgaac acgtgccgcagctcctgcttctgctcacagctgggcagtctgaggactcctatttgcaagctgccaacgccttgacacgc gcgggcatcctgactttttgtgtgggagctagccaggcgaataaggcagagcttgagcagattgcttttaacccaagcct ggtgtatctcatggatgatttcagctccctgccagctttgcctcagcagctgattcagcccctaaccacatatgttagtggag gtgtggaggaagtaccactcgctcagccag (SED ID NO:3).

As a means to diagnose PDA in a subject, differences in the level of

COL6A3 splice variant transcripts between individuals suspected of affliction with PDA and controls from non-PDA tissues, such as tissues of unaffected subjects, or tissue from the same patient outside a suspected PDA lesion, is determined.

Increased level of or presence of COL6A3 splice variant transcripts comprising one or more of exons 3, 4 and 6 is indicative of PDA. Alterations in the transcript level can be assayed by comparison to a standard or control level of COL6A3 splice variant transcript. The detection of normal or altered levels of COL6A3 splice variant transcript of the invention will direct the medical practitioner to set an appropriate course of treatment for the patient.

An exemplary method for detecting the presence of a nucleic acid molecule corresponding to a COL6A3 splice variant transcript in a biological sample comprises obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting the nucleic acid (e.g., mRNA or cDNA) corresponding to the COL6A3 splice variant transcript. The detection methods of the invention can thus be used to detect mRNA or cDNA in a biological sample in vitro as well as in vivo.

A general principle of such diagnostic assays involves preparing a sample or reaction mixture that contains a suspected target nucleotide sequence as a marker, and a probe, under appropriate conditions and for a time sufficient to allow the marker and probe to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways.

It is considered that the COL6A3 splice variant transcript containing exons 3, 4 or 6 specifically expressed in PDA patients is involved in the onset of PDA.

Accordingly, when the COL6A3 splice variant transcript specifically expressed in a PDA patient as the PDA-associated marker is used as a target, a subject is judged to have a possibility having PDA if the expression level of the PDA-associated marker in the subject is significantly increased in comparison with that of a control. The expression level of the PDA-associated marker in a healthy individual, or disease- free tissue of the same individual, may be used as the control for the detection of the expression of the PDA-associated marker in a test subject. In one embodiment, the control comprises presumptively normal pancreatic tissue adjacent a lesion suspected of containing PDA.

In some embodiments, expression of a COL6A3 splice variant transcript according to the invention is detected by determining the level of the corresponding mRNA. Methods that may be utilized for determining the level of COL6A3 mRNA expression in a sample are well known in the art and include, but are not limited to, polymerase chain reaction analyses, Northern analyses, and probe arrays. Many expression detection methods use isolated RNA. Any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from pancreatic tissue samples (see, e.g., Ausubel, ed., 1999, Current

Protocols in Molecular Biology (John Wiley & Sons, New York). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski, 1989, U.S. Pat. No. 4,843,155). In some embodiments, the RNA sample may be depleted of one or more RNAs, for example, an RNA sample depleted of rRNA. General methods for total RNA extraction are well known in the art and are disclosed in standard textbooks on molecular biology. The practice of the invention is not limited to any one method of mRNA detection or quantification recited herein, but rather encompasses all presently known or heretofore unknown methods.

The control sample in one embodiment is a biological material representative of healthy, cancer- free subjects, and/or cells or tissues. The level of COL6A3 splice variant transcript comprising one or more of exons 3, 4 and 6 in a control sample is desirably typical of the general population of normal, cancer-free subjects or of a particular individual at a particular time (e.g. before, during or after a treatment regimen), or in a particular tissue. The control sample can be removed from subject expressly for use in the methods described in this invention, or can be any biological material representative of normal, cancer- free subjects, including cancer- free biological material taken from the same subject outside the suspected cancerous lesion. The control sample can also refer to an established level of COL6A3 splice variant transcript comprising one or more of exons 3, 4 and 6, representative of the cancer-free population, that has been previously established based on measurements from normal, cancer-free subjects.

In another embodiment, COL6A3 splice variant transcripts comprising one or more of exons 3, 4 and 6 in test versus control samples are detected and/or quantified using a Northern blot analysis. In brief, mRNA mRNA is isolated from a given cell sample using, for example, an acid guanidinium-phenol-chloroform extraction method. The mRNA is then electrophoresed to separate the mRNA species and the mRNA is transferred from the gel to a nitrocellulose membrane. Labeled probes are used to identify and/or quantify the COL6A3 transcript mRNA.

In another embodiment, COL6A3 splice variant transcripts comprising one or more of exons 3, 4 and 6 in test versus control samples are detected using a Southern blot analysis. Briefly, the COL6A3 mRNA is isolated and reverse transcribed to produce cDNA. The cDNA is then optionally digested and run on a gels in buffer and transferred to membranes. Hybridization is then carried out using nucleic acid probes specific for the target COL6A3 transcript variant.

In another embodiment, COL6A3 splice variant transcripts comprising one or more of exons 3, 4 and 6 in test versus control samples are detected or quantified using an amplification (e.g., PCR) assay. The target transcript nucleotide sequence act as a template in an amplification reaction. In a quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate controls provides a measure of the transcript level.

A variation of the RT-PCR comprises "real time" quantitative PCR, which measures PCR product accumulation through a dual-labeled fluorigenic probe (i.e., TaqMan™ probe). Real time PCR is compatible both with quantitative competitive PCR, where an internal competitor for each target sequence is used for

normalization, and with quantitative comparative PCR using a normalization gene contained within the sample, or a housekeeping gene for RT-PCR.

In lieu of PCR, alternative methods, such as Ligase Chain Reaction (LCR) may be used to detect the COL6A3 splice variant transcripts. See, e.g. , Wu and Wallace (1989) Genomics 4: 560.

In another embodiment of the invention, COL6A3 splice variant transcripts comprising one or more of exons 3, 4 and 6 in test versus control samples are detected by Serial Analysis of Gene Expression (SAGE). See, e.g. , Velculescu et al , Science 270(5235):484 (1995); Saha et al, Nat Biotechnol 20(5):508 (2002); Gowda et al. , Plant Physiol 134 (3)890 (2004); Matsumura et al, Cell Microbiol 7(1): 1 1 (2005). The probe that binds the COL6A3 splice variant transcript includes complementary nucleic acids. For example, the nucleic acid reagents may include oligonucleotides (labeled or non-labeled) fixed to a substrate, labeled

oligonucleotides not bound with a substrate, pairs of PCR primers, molecular beacon probes, and the like.

The probe may also comprise fragments of nucleotide sequences

complementary to the COL6A3 splice variant. A fragment may be defined to be at least about 10 nucleotides (nt), preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length. Such fragments are useful as diagnostic probes and primers as discussed herein and can be incorporated into detection kits to detect the COL6A3 splice variants of the invention in biological samples. Of course larger DNA fragments are also useful according to the present invention.

The nucleotide sequence of the probe molecule may be selected to hybridize to the target COL6A3 splice variant under stringent conditions. The particular hybridization technique is not essential to the invention. Any technique commonly used in the art is within the scope of the present invention. Typical probe technology is described in U.S. Pat. No. 4,358,535 to Falkow et al , incorporated by reference herein. For example, hybridization can be carried out in a solution containing 6XSSC (10XSSC: 1.5 M sodium chloride, 0.15 M sodium citrate, pH 7.0), 5XDenhardt's (lXDenhardt's: 0.2% bovine serum albumin, 0.2%

polyvinylpyrrolidones, 0.02% Ficoll 400), 10 mM EDTA, 0.5% SDS and about 10⁷ cpm of nick-translated DNA for 16 hours at 65°C.

Additionally, if hybridization is to an immobilized nucleic acid, a washing step may be utilized wherein probe binding to polynucleotides of low homology, or nonspecific binding of the probe, may be removed. For example, a stringent wash step may involve a buffer of 0.2XSSC and 0.5% SDS at a temperature of 65°C.

Additional information related to hybridization technology and, more particularly, the stringency of hybridization and washing conditions may be found in Sambrook et al. , Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989). Polynucleotides of the invention which are complementary to the nucleotide sequences set forth in SEQ ID NO: l , SEQ ID NO:2, or SEQ ID NO:3 may be used as hybridization probes for the COL6A3 splice variant transcripts. Such

hybridization techniques are known to those of skill in the art. Typically, these nucleotide sequences are at least about 90% identical, preferably at least 90% identical, and more preferably at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99%) or 100%) identical, to the nucleotide sequences of any one or more of SEQ ID NO: 1 , 2 and 3. In another embodiment, the probe is an isolated nucleic acid molecule having a nucleotide sequence at least 90% identical, and more preferably at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99% or 100% identical to a nucleotide sequence complementary to the nucleotide sequences of any one or more of SEQ ID NO: 1 , 2 and 3. The probes generally will comprise at least 15 nucleotides. Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides. In some embodiments, the probes will range between 30 and 50 nucleotides. In some embodiments, the probes will be at least about 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more nucleotides in length.

Conventional means utilizing known computer programs may be utilized to determine if a particular nucleic acid molecule is at least 90%>, 91%o, 92%>, 93%, 94%, 95%, 96%, 97%, 98% or 99%o identical to any one of the nucleotide sequences shown in SEQ ID NO: l , SEQ ID NO:2 or SEQ ID NO:3, or complement of such sequences.

Polynucleotides which are complementary to the nucleotide sequences set forth in SEQ ID NO: l , SEQ ID NO:2, or SEQ ID NO:3 may be used as primers in PCR. This procedure is well known and commonly used by those skilled in this art (see Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al. (1985) Science 230: 1350-1354) and commercial suppliers of reagents for PCR, real time PCR and reverse transcriptase PCR. PCR is based on the enzymatic amplification of a DNA fragment of interest that is flanked by two oligonucleotide primers that hybridize to opposite strands of the target sequence. The primers are oriented with the 3' ends pointing towards each other. Repeated cycles of heat denaturation of the template, annealing of the primers to their complementary sequences, and extension of the annealed primers with a DNA polymerase result in the amplification of the segment defined by the 5' ends of the PCR primers. Since the extension product of each primer can serve as a template for the other primer, each cycle essentially doubles the amount of DNA template produced in the previous cycle. This results in the exponential accumulation of the specific target fragment, up to several million- fold in a few hours. By using a thermostable DNA polymerase such as the Taq polymerase, which is isolated from the thermophilic bacterium Thermus aquaticus, the amplification process can be completely automated. Other enzymes which can be used are known to those skilled in the art.

PCR-based detection schemes are useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. Amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

Expression levels of COL6A3 splice variant transcripts comprising one or more of exons 3, 4 and 6 are normalized by correcting the absolute expression level of a marker by comparing its expression to the expression of a gene that is not a marker, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene. This normalization allows the comparison of the expression level in one sample, e.g., a test sample to a control sample.

Detection of Expressed Protein

Determination of the presence of PDA may also be carried out by detecting and/or quantifying the expressed polypeptide translated from a COL6A3 splice variant. In this case, the protein sample is first isolated and prepared from a biological sample, e.g. a pancreatic tissue sample, derived from the subject, and the expression of respective proteins is detected. Detection of altered protein levels of the COL6A3 splice variants of the invention provides a diagnostic tool that can add to or define a diagnosis of PDA which results from altered expression of the COL6A3 splice variants. The detection of normal or altered expression levels of the COL6A3 splice variants of the invention will direct the medical practitioner to set an appropriate course of treatment for the patient. Any methods available in the art for detecting and quantifying polypeptide encoded by the nucleotide sequence of exons 3, 4 and/or 6 are encompassed in the invention.

The polypeptide encoded by the nucleotide sequences of exons 3, 4 and 6 is set forth in SEQ ID NO: 4, 5 and 6, respectively.

The amino acid sequence encoded by COLA6A3 exon 3, frame 3, is:

(D)VKNGAAADIIFLVDSSWTIGEEHFQLVREFLYDVVKSLAVGENDFHFAL VQFNGNPHTEFLLNTYRTKQEVLSHISNMSYIGGTNQTGKGLEYIMQSHLTK AAGSRAGDGVPQVIVVLTDGHSKDGLALPS AELKSADVNVFAIGVEDADEG ALKEIASEPLNMHMFNLENFTSLHDIVGNLVSCVHSSVSPERAGDTETLKDIT

(SED ID NO:4).

The amino acid sequence encoded by COLA6A3 exon 4, frame 3, is:

(A)QDSADIIFLIDGSN TGSVNFAVILDFLVNLLEKLPIGTQQIRVGVVQFSDE PRTMFSLDTYST AQVLGAVKALGFAGGELANIGLALDFVVENHFTRAGGS RVEEGVPQVLVLISAGPSSDEIRYGVVALKQASVFSFGLGAQAASRAELQHI ATDDNLVFTVPEFRSFGDLQEKLLPYIVGVAQRHIVLKPPTIVTQ (SED ID NO:5).

The amino acid sequence encoded by COLA6A3 exon 6, frame 3, is:

(V)HSNKRDIIFLLDGSANVG TNFPYVRDFVMNLVNSLDIGNDNIRVGLVQF SDTPVTEFSLNTYQT SDILGHLRQLQLQGGSGLNTGSALSYVYANHFTEAG GSRIREHVPQLLLLLTAGQSEDSYLQAANALTRAGILTFCVGASQANKAELE QIAFNPSLVYLMDDFSSLPALPQQLIQPLTTYVSGGVEEVPLAQP (SED ID NO:6).

The codon encoding for the first amino acid residue (shown in parentheses) of each of the sequences shown in SEQ ID NOS: 4, 5 and 6 results from the splicing of two exons, e.g., an upstream exon spliced to a downstream exon. Specifically, the last nucleotide of the upstream exon and the first two nucleotides of the downstream exon together form the codon encoding the first amino acid residue. For instance, the first in-frame codon of SEQ ID NO: l is GTC, corresponding to nucleotides 3-5 of SEQ ID NO: 1. This codon encodes residue 2, valine, of SEQ ID NO:4. Thus, the first entire codon in each of exons 3, 4 and 6 begins with nucleotide 3 (i.e., frame 3).

In one embodiment, the presence of PDA is determined by determining the presence of or increase in the level of polypeptide corresponding to a COL6A3 splice variant polypeptide comprising one or more of the sequences set forth in SEQ ID NOS: 4, 5 and 6. The level is determined and compared to the level in a non- malignant control sample. For example, detection of the presence or an increase in expression of a COL6A3 splice variant comprising one or more of the sequences set forth in SEQ ID NOS: 4, 5 and 6 in the sample derived from the subject compared to a control is an indication of PDA.

Sources of control samples for the expressed protein assay may comprise the same control sample sources as described above in connection with the gene transcript detection assay.

COL6A3 slice variant polypeptide expression level may be determined by any suitable method. Such methods may rely on utilizes a substance comprising a binding moiety for the polypeptide. Assays based on spliced COL6A3 protein- specific biomolecule interaction include, but are not limited to, antibody-based assays, aptamer-based assays, receptor and ligand assays, enzyme activity assays, and allosteric regulator binding assays. The invention is not limited to any one method of protein detection or quantification recited herein, but rather encompasses all presently known or heretofore unknown methods, such as methods that are discovered in the art.

In one embodiment, the substance comprises an antibody (inclusive of antibody fragments) that specifically binds to the COL6A3 splice variant polypeptide. Antibodies can be used in various immunoassay-based protein determination methods such as Western blot analysis, immunoprecipitation, radioimmunoassay (RIA), immunofluorescent assay, chemiluminescent assay, flow cytometry, immunocytochemistry and enzyme-linked immunosorbent assay

(ELISA).

The antibody used to detect spliced COL6A3 splice variant protein expression in a sample in an immunnoassay can comprise a polyclonal or monoclonal antibody. The antibody can comprise an intact antibody, or antibody fragments capable of specifically binding a COL6A3 splice variant comprising one or more of the sequences set forth in SEQ ID NOS: 4, 5 and 6. Such fragments include, but are not limited to, Fab and F(ab')₂ fragments.

When the antibody used in the methods of the invention is a polyclonal antibody (IgG), the antibody is generated by inoculating a suitable animal with a COL6A3 splice variant protein, peptide or a fragment thereof. Antibodies produced in the inoculated animal which specifically bind the COL6 A3 splice variant protein of the invention are then isolated from fluid obtained from the animal. Antibodies may be generated in this manner in several non-human mammals such as, but not limited to goat, sheep, horse, rabbit, guinea pig, rat, mouse and donkey. Methods for generating polyclonal antibodies are well known in the art and are described, for example in Harlow, et al. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, NY). These methods are not repeated herein as they are commonly used in the art of antibody technology.

When the antibody used in the methods of the invention is a monoclonal antibody, the antibody is generated using any well known monoclonal antibody preparation procedures such as those described, for example, in Harlow et al.

{supra) and in Tuszynski et al. (1988), Blood, 72: 109-1 15. Given that these methods are well known in the art, they are not replicated herein. Generally, monoclonal antibodies directed against a desired antigen are generated from mice immunized with the antigen using standard procedures as referenced herein.

Monoclonal antibodies directed against full length or peptide fragments of COL6A3 splice variant polypeptides of the invention may be prepared using the techniques described in Harlow, et al. {supra). Techniques for detecting antibody binding are well-known in the art.

Antibody binding to a COL6A3 splice variant polypeptide may be detected through the use of chemical reagents that generate a detectable signal that corresponds to the level of antibody binding and, accordingly, to the level of COL6A3 splice variant polypeptide expression. Examples of such detectable substances include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or

acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include ¹²⁵I,¹³¹1, ³⁵S, or ³H.

Antibody binding may be detected through the use of a secondary antibody that is conjugated to a detectable label. Examples of detectable labels include but are not limited to polymer-enzyme conjugates. The enzymes in these complexes are typically used to catalyze the deposition of a chromogen at the antigen-antibody binding site, thereby resulting in cell staining that corresponds to expression level of the biomarker of interest. Preferred enzymes of particular interest include horseradish peroxidase (HRP) and alkaline phosphatase (AP).

In some aspects of the invention, slides are reviewed microscopically by a cytotechnologist and/or a pathologist to assess cell and extracellular staining COL6A3 splice variant over-expression and/or subcellular distribution as either predominantly extracellular, cytoplasmic or nuclear). Alternatively, samples may be reviewed via automated microscopy or by personnel with the assistance of computer software that facilitates the identification of positive staining cells.

COL6A3 splice variants of the invention can be detected by aptamer-based assays, which are very similar to antibody-based assays, but with the use of an aptamer instead of an antibody. An aptamer can be any polynucleotide, generally a RNA or a DNA, which has a useful biological activity in terms of biochemical activity or molecular recognition attributes. Usually, an aptamer has a molecular activity such as having an enzymatic activity or binding to a polypeptide at a specific region (i.e., similar to an epitope for an antibody) of the polypeptide.

It is generally known in the art that an aptamer can be made by in vitro selection methods. In vitro selection methods include a well known method called systematic evolution of ligands by exponential enrichment (a.k.a. SELEX). Briefly, in vitro selection involves screening a pool of random polynucleotides for a particular polynucleotide that binds to a biomolecule, such as a polypeptide, or has a particular activity that is selectable. Generally, the particular polynucleotide represents a very small fraction of the pool, therefore, a round of amplification, usually via polymerase chain reaction, is employed to increase the representation of potentially useful aptamers. Successive rounds of selection and amplification are employed to exponentially increase the abundance of a particular aptamer. In vitro selection is described in Famulok and Szostak, Angew. Chem. 1992, 104, 1001. (Angew. Chem. Int. Ed. Engl. 1992, 31, 979-988.); Famulok and Szostak, Nucleic Acids and Molecular Biology, Vol 7, F. Eckstein, D. M. J. Lilley, Eds., Springer Verlag, Berlin, 1993, pp. 271 ; Klug and Famulok, Mol. Biol. Reports 1994, 20, 97- 107; and Burgstaller and Famulok, Angew. Chem. 1995, 707, 1303-1306 (Angew. Chem. Int. Ed. Engl. 1995, 34, 1 189-1192), US Patent No. 6,287,765, US Patent No. 6,180,348, US Patent No. 6,001 ,570, US Patent No. 5,861,588, US Patent No.

5,567,588, US Patent No. 5,475,096, and US Patent No. 5,270,163, which are incorporated herein by reference.

Substantially pure COL6A3 splice variant polypeptides of the invention, which can be used as an immunogen for raising polyclonal or monoclonal antibodies, or as a substrate for selecting aptamers, can be prepared, for example, by recombinant DNA methods. For example, the cDNA of the COL6A3 splice variant protein can be cloned into an expression vector by techniques within the skill in the art. An expression vector comprising sequences encoding the COL6A3 splice variant protein can then be transfected into an appropriate, for example bacterial or eukaryotic host, whereupon the protein is expressed. The expressed protein can then be isolated by any suitable technique.

For example, a COL6A3 splice variant polypeptide of the invention can be prepared in the form of a bacterially expressed glutathione S-transferase (GST) fusion protein. Such fusion proteins can be prepared using commercially available expression systems, following standard expression protocols, e.g., "Expression and Purification of Glutathione-S-Transferase Fusion Proteins", Supplement 10, unit 16.7, in Current Protocols in Molecular Biology (1990); Smith and Johnson, Gene 67: 34-40 (1988); and Frangioni and Neel, Anal. Biochem. 210: 179-187 (1993), the entire disclosures of which are herein incorporated by reference.

Briefly, DNA encoding for the COL6A3 splice variant of the invention is subcloned into a pGEX2T vector in the correct reading frame and introduced into E. coli cells. Transfectants are selected on LB/ampicillin plates after incubation for 12 to 15 hours at 37°C. The selected transfectants are then grown in liquid cultures in growth media containing isopropyl- -D-thiogalactoside, to induce expression of the alternatively spliced COL6A3 fusion protein. The cells are harvested from the liquid cultures by centrifugation, the bacterial pellet is resuspended and sonicated to lyse the cells.

To isolate the GST- COL6A3 fusion protein, the lysate is then contacted with glutathione-agarose beads. The beads, which bind the GST- COL6A3 fusion protein, are collected by centrifugation and the GDT- COL6A3 fusion protein is eluted. The GST agarose beads are removed by treatment of the fusion protein with thrombin cleavage buffer. The released COL6A3 splice variant protein is recovered and used to raise antibodies or aptamers, as described above.

Alternatively, the coding sequences of E3, E4 and E6 can be cloned into the pCEP-Pu episomal expression vector with the BM40 signal sequence and His tag. The expression constructs are transfected into the human embryonic kidney cells (293-EBNA; Invitrogen) and transfectants are selected by puromycin. Recombinant E3, E4 and E6 polypeptides secreted by the transfectants are isolated from the culture medium using nickel-affinity column purification. (Kohfeldt et al. , FEBS Lett. 1997;414:557-61 ; Mascarenhas et al. , EMBO J. 2003; 22: 529-536.). In some embodiments, the presence of PD A is determined by detecting the presence of or change in the level of a fragment of a COL6A3 splice variant polypeptide described herein. In particular, a fragment may be a degradation fragment, generated by proteolytic degradation of a COL6A3 splice variant polypeptide. In particular, such fragments may be detected in the blood. According the samples, reagents and techniques described herein for detection of COL6A3 splice variant polypeptides may be utilized for detection of COL6A3 splice variant polypeptide fragments, including degradation fragments. The reagents described herein for detection of COL6A3 splice variant polypeptides, e.g., antibodies and aptamers, may appropriately designed for detection of COL6 A3 splice variant polypeptide fragments by specifically binding thereto.

Samples

The sample source providing the polynucleotides or polypeptides for analysis according to the present invention may comprise, for example, pancreatic tissue. Pancreatic tissue may be harvested during surgical resection of the tumors. During this procedure, an adjacent tissue from the area surrounding the tumor will be collected for comparison. Alternatively, the sample source may comprise small pancreatic biopsies. Pancreatic biopsies may be collected by fine needle aspirate (FNA) of pancreatic tissue, a safe minor surgical procedure that is routinely done by an endoscopist and used for initial diagnosis of pancreatic lesions through collecting cells and small biopsies. This will allow for earlier detection of the lesions, before the surgical resection.

In another embodiment, the sample comprises a peripheral blood-derived sample, such as whole blood, or any of its components. Preferably, the blood- derived sample comprises plasma or serum. Blood is collected in e.g., heparin tubes (e.g., BD Vacutainer) and processed within 2 hours of collection by centrifugation at 1,300 x g at 4°C for 10 minutes. Plasma is transferred to a fresh tube and stored at 80°C for analysis. Peripheral blood-derived samples, especially serum, are appealing because they can be used as non-invasive screening methods in asymptomatic patients. In certain embodiments, a blood-derived sample, e.g., whole blood, plasma or serum, is a sample source for a COL6A3 splice variant polypeptide fragment.

As demonstrated hereinafter, analysis of COL6A3 E3, E4, and E6 isoforms by quantitative PCR in show the exclusive presence of E3, E4 and E6 mRNA isoforms in PDA patient sera with very low expression levels in non-malignant sera. Serum levels of CA19-9 in these patients are largely variable and inconclusive of malignancy.

Moreover, identification of the isoform mRNA in the serum is indicative of the presence of peptides that encode those isoform, which could be detected by antibodies. Additionally, since tumor tissue expresses high levels of matrix metalloproteinases, matrix degradation products released by the tumor stroma into serum would be expected to contain the expressed COL6A3 protein isoforms.

Kits

The invention also relates to diagnostic kits for assessing whether a patient is afflicted with PDA, or for monitoring disease progression. A kit comprises a set of reagents that specifically detects the expression level of COL6A3 splice variant of the invention, and instructions for using the kit for diagnosis of PDA, or monitoring the progress of disease.

In other embodiments, the set of reagents of the kit detects polypeptides encoded by one or more marker genes. In some embodiments, the set of reagents comprises antibodies or aptamers that specifically bind to the polypeptides encoded by one or more marker genes. For example, the kit can comprise an antibody, an antibody derivative, or an antibody fragment that binds specifically with a COL6A3 splice variant protein of the invention. Such kits may also comprise a plurality of antibodies, antibody derivatives, or antibody fragments wherein the plurality of such antibody agents binds specifically with a marker protein, or a fragment of the protein.

For antibody-based kits, the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) that binds to a marker protein; and, optionally, (2) a second, different antibody that binds to either the protein or the first antibody and is conjugated to a detectable label.

The kit can further comprise components necessary for detecting the detectable label (e.g., an enzyme or a substrate).

Antibody-based kits may further include instructions for use. Such instructions may comprise instructions to: (a) determine the level of a COL6A3 splice variant protein in a test sample from a subject, wherein said COL6A3 protein is selected from the group consisting of (i) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 3; (ii) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 4; and/or (iii) the COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 6; and (b) comparing the level of said COL6A3 protein determined in said test sample to the level of the COL6A3 protein determined in a control sample, wherein an elevated level of the COL6A3 protein in said test sample relative to the level of the COL6A3 protein in said control sample is a diagnosis of PDA in said subject. Alternatively, such instructions may comprise instructions to (a) determine the level of a COL6A3 splice variant protein fragment in a test sample from a subject, wherein said

COL6A3 splice variant protein fragment is a fragment of a COL6A3 protein selected from the group of COL6A3 proteins consisting of (i) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of

COL6A3 exon 3; (ii) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 4; and/or (iii) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 6; and (b) compare the level of said COL6A3 splice variant protein fragment determined in said test sample to a control.

In another embodiment, the invention provides a kit for assessing the presence of a COL6A3 splice variant transcript comprising one or more of exons 3, 4 and 6. The set of reagents in the kit detects the splice variant transcript. The reagents may comprise a nucleic acid molecules as a probes that binds specifically with a marker nucleic acid of the invention. Suitable reagents for binding with a marker nucleic acid include complementary nucleic acids. For example, the nucleic acid reagents may include oligonucleotides (labeled or non-labeled) fixed to a substrate, labeled oligonucleotides not bound with a substrate, pairs of PCR primers, molecular beacon probes, and the like. In some embodiments, the nucleic acid probes complementary to the COL6A3 splice variant transcript are immobilized on a substrate surface.

For oligonucleotide-based kits, the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence of a COL6A3 splice variant transcript comprising one or more of exons 3, 4 and 6 or (2) a pair of primers useful for amplifying a marker nucleic acid molecule of the invention.

Oligonucleotide-based kits may further include instructions for use. Such instructions may comprise instructions to: (a) determine the level of a COL6A3 splice variant transcript in a test sample from a subject, wherein said COL6A3 variant transcript is selected from the group consisting of (i) a COL6A3 transcript comprising the nucleotide sequence of COL6A3 exon 3, (ii) a COL6A3 transcript comprising the nucleotide sequence of COL6A3 exon 4; and/or (iii) a COL6A3 transcript comprising the nucleotide sequence of COL6A3 exon 6; and (b) comparing the level of said COL6A3 transcript determined in said test sample to the level of the COL6A3 transcript determined in a control sample, wherein an elevated level of the COL6A3 transcript in said test sample relative to the level of the COL6A3 transcript in said control sample is a diagnostic of PDA in said subject.

Kits for practice of the invention may also comprise, e.g., buffering agents, preservatives, or protein stabilizing agents . The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit. Disease Progression Monitoring

In another embodiment, the invention provides a method of monitoring the progression of PDA in a subject. Samples are obtained from the subject at different time points and analyzed as described above. An elevated level of the COL6A3 splice variant transcript or encoded polypeptide or polypeptide fragment in a later sample relative to an earlier sample is an indication that the PDA has progressed in said subject.

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Examples

The following experiments show the exclusive presence of COL6A3 isoforms containing exons 3, 4 and 6 in PDA and their absence from premalignant lesions.

Example 1; Expression of Alternatively Spliced COL6A3 in PDA Determined by RT-PCR

Paired pancreatic ductal adenocarcinoma PDA and paired surrounding normal samples from eight PDA patients were analyzed as follows by

semiquantitative PC using primers that amplify COL6A3 splice varinats containing exons 3, 4 or 6.

Total RNA was isolated from tumor and adjacent healthy tissue using Trizol reagent (Life Technologies, Gaithersburg, MD). RNAs were quantified after DNase digestion, and cDNAs were prepared using ImProm-II™ Reverse Transcription System (Promega), then subjected to semiquantitative PCR using master mix (Promega). The following primer sequences were used:

Primer sequences for exon 3 :

Forward: 5 '- AGC AGC AAGC AG ATGTC AA A -3' (SEQ ID NO:9); Reverse: 5 '-TTTCTCCC AC AGCTAAGGATTT-3 ' (SEQ ID

NO: 10).

Primer sequences for exon 4:

Forward: 5 '-ATTGTCTTGAAACCGCC AAC-3 ' (SEQ ID NO: 1 1); Reverse: 5'-AGCAATGAAGTCTCGGATGG-3' (SEQ ID NO: 12).

Primer sequences for exon 6:

Forward: 5'-TCTCAGGACCCTCTCTGGAAC-3' (SEQ ID

NO: 13);

Reverse: 5 '-TA AGGG AAATTGGTTTTTCC A A-3 ' (SEQ ID NO: 14).

PCR conditions were 120 seconds at 95 °C, 30 seconds at 55°C and 60 seconds at 72°C with a 7-minute final extension at 72°C after 40 cycles. Upstream and downstream primers that could anneal with the 3 '-untranslated region of human GAPDH were included in the PCR reaction as an internal standards:

Forward: 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' (SEQ ID

NO:7);

Reverse: 5 '-CATGTGGGCCATGAGGTCC ACC AC-3 ' (SEQ ID

NO: 8).

The linear range of amplification for each set of primers was determined to ensure that a number of cycles in the linear range were used. PCR products were electrophoresed on 2% agarose gels and band intensities were quantified using Kodak Electrophoresis Documentation and Analysis System 290 (EDAS 290).

The results are shown in Figure 2 where Tl, T2, T3, etc. represent tumor samples, and Nl, N2 and N3, etc. represents normal surrounding pancreatic tissue from the corresponding patient. Exons 3 and 4 were differentially expressed in PDA tissue compared to normal tissue.

Utilizing a similar protocol, COL6A3 isoforms containing exons 3, 4 and 6 were absent from the benign (premalignant) lesions intraductal papillary mucinous neoplasm (IPMN) (n=5) and cystadenoma (n=5; 3 serous and 2 mucinous) (results not shown). Example 2: Expression of COL6A3 E6 in PDA Determined by Real-Time RT-

PCR

Because the semi-quantitative PCR results for exon 6 in Example 2 did not show a consistent difference between samples of PDA and their matched adjacent tissue, the tissue was further analyzed by real-time PCR as follows, using primers that amplify exon 6.

Paired PDA and surrounding normal samples from eight PDA patients were analyzed by real-time PCR. For the PCR, total RNA was isolated from tumor and adjacent tissue using Trizol reagent (Life Technologies, Gaithersburg, MD). RNAs were quantified and input amounts were optimized for each amplicon. E6 and GAPDH primers were as in Example 1. The primers were diluted, and subjected to real-time PCR using SYBR-Green based detection technology (7500 Sequence Detector, Applied Biosystems). The relative mRNA levels are presented in Figure 3 as unit values of 2[CT(COL6A3-E6) ^~ CV(GAPDH)], where C τ is the threshold cycle value defined as the fractional cycle number at which the target fluorescent signal passes a fixed threshold above baseline. As shown in Figure 3, the COL6A3 splice variant transcript containing exon 6 was differentially expressed in tumor tissue of all eight PDA patients compared to adjacent normal tissue. There was consistent

upregulation of the exon 6 spice variant in PDA when compared with adjacent tissue.

Example 3: Serum Expression of COL6A3 E3, E4 and E6 in PDA Determined by Real-Time RT-PCR

RNA was isolated from 104 serum samples from patients afflicted with

PDA, neuroendocrine tumors (NET), IPMN, cystadenoma or chronic pancreatitis and subjected to quantitative real time PCR analysis using COL6A3 isoform- specific primers. The following primer sequences were used:

Primer sequences for exon 3 :

Forward: 5'-AAACATCGGCACTTGCCCTTAGTG-3' (SEQ ID

NO: 15 ); Reverse: 5 '-TCCTCTCCAATGGTCCAAGAGGAA-3 ' (SEQ ID

NO: 16).

Primer sequences for exon 4:

Forward: 5 '-AAGGCACATTGTCTTGAAACCGCC-3 ' (SEQ ID NO: 17);

Reverse: 5 '-TCC AGCCTCTGGATGACTTTAGC A-3 ' (SEQ ID

NO: 18).

Primers for exon 6 were provided by Applied Biosystems, Inc. (TaqMan Primers). GAPDH primers were included in the PCR reaction as an internal standards (Forward: SEQ ID NO:7; Reverse: SEQ ID NO. 8).

RNAs were quantified and input amounts were optimized for each amplicon. The primers were diluted, and subjected to real-time PCR using SYBR-Green based detection technology (7500 Sequence Detector, Applied Biosystems). The relative mRNA levels are presented in Figures 4A-4C as unit values of 2[CT(COL6A3-E₆) ^~ CT(GAPDH)] , where C χ is the threshold cycle value defined as the fractional cycle number at which the target fluorescent signal passes a fixed threshold above baseline.

As seen in Fig 4A, levels of the E3 isoform were significantly upregulated in PDA when compared to the individual benign lesions (P=0.0006 vs. IPMN; P= 0.002 vs. cystadenoma; P= 0.005 vs. chronic pancreatitis). Compared to

neuroendocrine tumors, E3 was significantly higher in PDA (P= 0.002), indicating that the appearance of the E3 isoform is PDA-specific. Similar results could be seen with isoform E4 (Fig 4B) and E6 (Fig 4C). In the figures, the symbols *, **, and # signify the relative level of the COL6A3 isoform in PDA versus the other lesions in the panel: * = isoform E3, 10-20-fold higher; ** = isoform E4, 12-20-fold higher; and # = isoform E6, 5-40-fold higher These data indicate that serum E3, E4 and E6 have a very high capability of differentiating PDA from any other pancreatic lesions, including another pancreatic malignancy, NET, benign lesions (cystadenoma) and chronic pancreatitis. The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed:

1. A method of diagnosing pancreatic ductal adenocarcinoma (PDA) in a subject, the method comprising:

a) determining the level of a COL6A3 transcript in a test sample from a subject, wherein said COL6A3 transcript is selected from the group consisting of:

(i) a COL6A3 transcript comprising the nucleotide sequence of COL6A3 exon 3;

(ii) a COL6 A3 transcript comprising the nucleotide sequence of

COL6A3 exon 4;

(iii) a COL6A3 transcript comprising the nucleotide sequence of COL6A3 exon 6; and

(iv) any combination thereof;

b) comparing the level of said COL6A3 transcript determined in said test sample to the level of the COL6A3 transcript determined in a control sample, wherein an elevated level of the COL6A3 transcript in said test sample relative to the level of the COL6A3 transcript in said control sample is diagnostic of PDA in said subject.

2. The method of claim 1, wherein said control sample is obtained from a healthy normal subject.

3. The method of claim 1, wherein said test sample comprises pancreatic tissue or fine needle aspirate of pancreatic tissue.

4. The method of claim 1 , wherein said test sample comprises a peripheral blood-derived sample.

5. The method of claim 4, wherein said test sample comprises serum.

6. The method of claim 1 , wherein said nucleotide sequence of COL6A3 exon 3 is set forth in SEQ ID NO: 1, said nucleotide sequence of COL6A3 exon 4 is set forth in SEQ ID NO: 2, and said nucleotide sequence of COL6A3 exon 6 is set forth in SEQ ID NO: 3.

7. The method of claim 1, wherein determining the level of said COL6A3 transcript is by Northern Blot analysis, in situ hybridization, polymerase chain reaction, reverse transcriptase- polymerase chain reaction, and any combination thereof.

8. The method of claim 1, wherein the level of COL6A3 transcript in said test sample is elevated at least 2-fold relative to the level of said COL6A3 transcript in said control sample.

9. The method of claim 1, wherein the COL6A3 transcript comprises the nucleotide sequence of COL6A3 exon 4.

10. A method of diagnosing pancreatic ductal adenocarcinoma (PDA) in a subject, the method comprising:

a) determining the level of a COL6A3 protein in a test sample from a subject, wherein said COL6A3 protein is selected from the group consisting of:

(i) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 3;

(ii) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 4;

(iii) the COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 6; and

(iv) any combination thereof,

b) comparing the level of said COL6A3 protein determined in said test sample to the level of the COL6A3 protein determined in a control sample, wherein an elevated level of the COL6A3 protein in said test sample relative to the level of the COL6A3 protein in said control sample is diagnostic of PDA in said subject.

11. The method of claim 10, wherein said control sample is obtained from a healthy normal subject.

12. The method of claim 10, wherein said test sample comprise pancreatic tissue or fine needle aspirate of pancreatic tissue.

13. The method of claim 10, wherein said test sample comprises a peripheral blood-derived sample.

14. The method of claim 13, wherein said test sample comprises serum.

15. The method of claim 10, wherein the level of COL6A3 protein in said test sample is elevated at least 2-fold relative to the level of said COL6A3 protein in said control sample.

16. The method of claim 10, wherein the COL6A3 protein comprises the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 4.

17. The method of claim 10, wherein determining the level of COL6A3 protein includes an assay selected from the group consisting of Western blot analysis, immunoprecipitation, radioimmunoassay, immunofluorescent assay, chemiluminescent assay, flow cytometry, immunocytochemistry, enzyme-linked immunosorbent assay, and any combination thereof.

18. A method of monitoring the progression of a pancreatic ductal adenocarcinoma (PDA) in a subject, said method comprising:

a) obtaining a first sample from a subject at a first time point and a second sample from said subject at a second time point; b) determining the level of a COL6A3 transcript in said first and second samples, wherein said COL6A3 transcript is selected from the group consisting of:

(i) a COL6A3 transcript comprising the nucleotide sequence of COL6A3 exon 3;

(ii) a COL6A3 transcript comprising the nucleotide sequence of COL6A3 exon 4;

(iii) a COL6A3 transcript comprising the nucleotide sequence of COL6A3 exon 6; and

(iv) any combination thereof;

b) comparing the level said COL6A3 transcript determined in said first sample to the level of the COL6A3 transcript in said second sample, wherein an elevated level of the COL6A3 transcript in said second sample relative to the level of said COL6 A3 transcript in said first sample is an indication that the cancer has progressed in said subject.

19. The method of claim 18, wherein said first and second samples comprise pancreatic tissue or fine needle aspirate of pancreatic tissue.

20. The method of claim 18, wherein said test sample comprises a peripheral blood-derived sample.

21. The method of claim 20, wherein said test sample comprises serum.

22. The method of claim 18, wherein the COL6A3 transcript comprises the nucleotide sequence of COL6A3 exon 4.

23. The method of claim 18, wherein the level of spliced COL6A3 transcript in said test sample is elevated at least 2-fold relative to the level of said spliced COL6A3 transcript in said control sample.

24. The method of claim 18, wherein said nucleotide sequence of COL6A3 exon 3 is set forth in SEQ ID NO: 1, said nucleotide sequence of COL6A3 exon 4 is set forth in SEQ ID NO: 2, and said nucleotide sequence of COL6A3 exon 6 is set forth in SEQ ID NO: 3.

25. The method of claim 18 wherein determining the level of said COL6A3 transcript is by Northern Blot analysis, in situ hybridization, polymerase chain reaction, reverse transcriptase- polymerase chain reaction, and any combination thereof.

26. A method of monitoring the progression of a pancreatic ductal adenocarcinoma (PDA) in a subject, said method comprising:

a) obtaining a first sample from a subject at a first time point and a second sample from said subject at a second time point;

b) determining the level of a COL6A3 protein in a test sample from a subject, wherein said COL6A3 protein is selected from the group consisting of:

(i) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 3;

(ii) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 4;

(iii) the COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 6; and

(iv) any combination thereof,

c) comparing the level of said COL6A3 protein determined in said first sample to the level of the COL6A3 protein in said second sample, wherein an elevated level of the COL6A3 protein in said second sample relative to the level of said COL6A3 protein in said first sample is an indication that the cancer has progressed in said subject.

27. The method of claim 26, wherein said first and second samples comprise pancreatic tissue or fine needle aspirate of pancreatic tissue.

28. The method of claim 26, wherein said test sample comprises a peripheral blood-derived sample.

29. The method of claim 28, wherein said test sample comprises serum.

30. The method of claim 26, wherein the COL6A3 protein comprises the acid sequence encoded by the nucleotide sequence of COL6A3 exon 4.

31. The method of claim 26, wherein the level of COL6A3 protein in said test sample is elevated at least 2-fold relative to the level of said COL6A3 protein in said control sample.

32. The method of claim 26, wherein determining the level of COL6A3 protein includes an assay selected from the group consisting of Western blot analysis, immunoprecipitation, radioimmunoassay, immuno fluorescent assay, chemiluminescent assay, flow cytometry, immunocytochemistry, enzyme-linked immunosorbent assay, and any combination thereof.

33. A method of diagnosing pancreatic ductal adenocarcinoma (PDA) in a subject, the method comprising:

a) determining the level of a COL6A3 protein fragment in a test sample from a subject, wherein said COL6A3 protein fragment is a fragment of a COL6A3 protein selected from the group of COL6A3 proteins consisting of:

(i) COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 3;

(ii) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 4;

(iii) the COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 6; and

(iv) any combination thereof, b) comparing the level of said COL6A3 protein fragment determined in said test sample to the level of the COL6A3 protein fragment determined in a control sample, wherein an elevated level of the COL6A3 protein fragment in said test sample relative to the level of the COL6A3 protein fragment in said control sample is diagnostic of PDA in said subject.

34. A method of monitoring the progression of a pancreatic ductal adenocarcinoma (PDA) in a subject, said method comprising:

a) obtaining a first sample from a subject at a first time point and a second sample from said subject at a second time point;

b) determining the level of a COL6 A3 protein fragment in a test sample from a subject, wherein said COL6A3 protein fragment is a fragment of a COL6A3 protein selected from the group of COL6A3 proteins consisting of:

(i) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 3;

(ii) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 4;

(iii) the COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 6; and

(iv) any combination thereof,

c) comparing the level of said COL6A3 protein fragment determined in said first sample to the level of the COL6A3 protein fragment in said second sample, wherein an elevated level of the COL6A3 protein fragment in said second sample relative to the level of said COL6 A3 protein fragment in said first sample is an indication that the cancer has progressed in said subject.

35. The method of claim 33 or 34, wherein said test sample comprises a peripheral blood-derived sample.

36. The method of claim 35, wherein said test sample comprises serum.

37. A kit for detecting the presence of pancreatic ductal adenocarcinoma in an individual, or for monitoring disease progression in an individual afflicted with pancreatic ductal adenocarcinoma, comprising:

(a) a set of reagents that specifically detects the expression level of COL6A3 splice variant comprising at least one of exon 3, exon 4 and exon 6; and

(b) instructions for using said kit for detecting the presence of pancreatic ductal adenocarcinoma, or for monitoring disease progression in an individual afflicted with pancreatic ductal adenocarcinoma.

38. A kit according to claim 37, wherein the instructions comprise instructions to:

(a) determine the level of a COL6A3 splice variant transcript in a test sample from a subject, wherein said COL6A3 splice variant transcript is selected from the group consisting of:

(i) a COL6A3 transcript comprising the nucleotide sequence of COL6A3 exon 3,

(ii) a COL6A3 transcript comprising the nucleotide sequence of COL6A3 exon 4; and/or

(iii) a COL6A3 transcript comprising the nucleotide sequence of COL6A3 exon 6; and

(b) compare the level of said COL6A3 transcript determined in said test sample to a control.

39. A kit according to claim 37, wherein the instructions comprise instructions to:

(a) determine the level of a COL6A3 splice variant protein in a test sample from a subject, wherein said COL6A3 splice variant protein is selected from the group consisting of:

(i) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 3; (ii) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 4; and/or

(iii) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 6; and

(b) compare the level of said COL6A3 protein determined in said test sample to a control.

40. A kit according to claim 37, wherein the instructions comprise instructions to:

(a) determine the level of a COL6A3 splice variant protein fragment in a test sample from a subject, wherein said COL6A3 splice variant protein fragment is a fragment of a COL6A3 protein selected from the group of COL6A3 proteins consisting of

(i) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 3;

(ii) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 4; and/or

(iii) a COL6A3 protein comprising the amino acid sequence encoded by the nucleotide sequence of COL6A3 exon 6; and

(b) compare the level of said COL6A3 splice variant protein fragment determined in said test sample to a control.