WO2010071788A1 - Pancreatic cancer markers and uses thereof - Google Patents

Abstract

The present invention provides methods and reagents for assessing the probability of a subject having a pancreatic tumor, and for monitoring efficacy of treatments for pancreatic tumors.

Description

Pancreatic cancer markers and uses thereof Cross Reference

This application claims priority to U.S. Provisional Patent Application Serial Nos. 61/138,376 filed December 17, 2008 and 61/229118 filed My 28, 2009, incorporated by reference herein in their entirety.

Background

Pancreatic cancer, particularly ductal adenocarcinoma of the pancreas, is a malignant tumor of the pancreas with a generally poor prognosis, as less than 5% of those diagnosed are still alive five years after diagnosis. Since the symptoms of pancreatic cancer tend to occur after the disease has advanced beyond stage I disease, early detection is rare. Early detection of pancreatic cancer would permit treatment at an earlier stage, improving the patient prognosis. Currently available tests for pancreatic cancer are inadequate, and thus new methods are needed.

Summary of the Invention In one aspect, the present invention provides methods for assessing the probability of a pancreatic rumor in a subject, comprising analyzing a tissue sample of the subject for one or more peptides derived from one or both of QSOXl and SerpinF2, wherein an increased amount of the one or more peptides relative to a control correlates with a probability of a pancreatic tumor in the subject. In another aspect, the present invention provides methods for monitoring efficacy of treatment of a pancreatic tumor in a subject, comprising analyzing a tissue sample of a subject being treated for a pancreatic tumor for one or more peptides derived from one or both of QSOXl and SerpinF2, wherein the analyzing is carried out after one or more tumor treatments, and wherein a decrease in amount of the one or more peptides in the tissue sample after tumor treatment compared to an amount of the one or more peptides in a tissue sample from the subject after diagnosis of the pancreatic tumor but before tumor treatment began correlates with an effective tumor treatment.

In another aspect, the present invention provides an isolated peptide, selected from the group consisting of NEQEQPLGQWHLS (QSOXl) (SEQ ED NO: 1), NEQEQPLGQWH (QSOXl) (SEQ ID NO: 2), EQPLGQWHLS (QSOXl) (SEQ ID NO: 3), AAPGQEPPEHMAELQR (QSOXl) (SEQ ID NO: 4), AAPGQEPPEHMAELQ (QSOXl) (SEQ ID NO: 5), AAPGQEPPEHMAELQRNEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 6), NQEQVSPLTLLKLGN (SerpinF2) (SEQ ID NO: 7), NQEQVSPLTLLK (SerpinF2) (SEQ ID NO: 8), MEPLGRQLTSGP (SerpinF2) (SEQ ID NO: 9), NEQEQPL (SEQ ID NO: 10) (QSOXl), and GQWHLS (SEQ ID NO: 11) (QSOXl).

In a further aspect, the present invention provides isolated ligands, wherein the ligand selectively binds to a peptide selected from the group consisting of NEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 1), NEQEQPLGQWH (QSOXl) (SEQ ID NO: 2), EQPLGQWHLS (QSOXl) (SEQ ID NO: 3), AAPGQEPPEHMAELQR (QSOXl) (SEQ ID NO: 4), AAPGQEPPEHMAELQ (QSOXl) (SEQ ID NO: 5), AAPGQEPPEHMAELQRNEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 6), NQEQVSPLTLLKLGN (SerpinF2) (SEQ ID NO: 7), NQEQVSPLTLLK (SerpinF2) (SEQ ID NO: 8), MEPLGRQLTSGP (SerpinF2) (SEQ ID NO: 9), NEQEQPL (SEQ ID NO: 10) (QSOXl), and GQWHLS (SEQ ID NO: 11) (QSOXl). In a preferred embodiment, the ligand comprises an antibody.

In another aspect, the present invention provides methods for detecting the presence of a peptide selected from the group consisting of NEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 1), NEQEQPLGQWH (QSOXl) (SEQ ID NO: 2), EQPLGQWHLS (QSOXl) (SEQ ID NO: 3), AAPGQEPPEHMAELQR (QSOXl) (SEQ ID NO: 4), AAPGQEPPEHMAELQ (QSOXl) (SEQ ID NO: 5), AAPGQEPPEHMAELQRNEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 6), NQEQVSPLTLLKLGN (SerpinF2) (SEQ ID NO: 7), NQEQVSPLTLLK (SerpinP2) (SEQ ID NO: 8), MEPLGRQLTSGP (SerpinF2) (SEQ ID NO: 9), NEQEQPL (SEQ ID NO: 10) (QSOXl), and GQWHLS (SEQ ID NO: 11) (QSOXl), comprising

(a) contacting a sample to be tested with a ligand according to claim 16 or 17, under conditions in which the ligand selectively binds to its respective peptide to produce a binding complex; and

(b) detecting presence of the binding complex, wherein the presence of the binding complex indicates that the peptide is present in the test sample.

Brief Description of the Figures

Figure 1. Tandem mass spectra of NEQEQPLGQWHLS (SEQ ID NO: 1) (precursor m/z

783.36; +2 charge state) QSOXl-L peptide. Mass spectra from (A) peptide identified from <3 kDa plasma peptidome and (B) chemically re-synthesized peptide, y and b ions are labeled.

Figure 2. Anti-QSOXl IHC. Brown color (substrate is DAB) indicates reactivity of anti- QSOXl antibody with tumor cells expressing QSOXl . All staining of tumor was scored 3+. A. Tumor-specific staining from a patient with DAP. Left arrow indicates absence of staining in normal duct. Right arrow indicates tumor in ductal epithelium. Surrounding tissues including stroma and acinar cells do not express QSOXl . B. Peri-pancreatic lymph node from a patient with DAP. Arrow indicates staining of a micrometastasis in the lymph node. Of note is the general absence of staining of lymphocytes. C. High magnification of cytoplasmic QSOXl staining of DAP tissue section. Note that stroma is completely negative. D. Tissue section from patient with chronic pancreatitis. Light brown staining is likely due to fixation process or non-specific substrate development. E. Staining of dysplastic ductal cells. Left arrow shows normal cobblestone-like nuclei lining ducts. Right arrow shows dysplastic cells and concomitant expression of QSOXl .

Figure 3. Western blotting for QSOXl expression. Anti-QSOXl rabbit polyclonal antibody was incubated with PVDF membrane blotted with cell lysate made from 1 x 10⁷ cells from peripheral blood mononuclear cell (PBMC) from healthy donors and human pancreatic carcinoma cell lines: Panc-1, Capan-1, CFPac-1, and BxPC3. 20 μg of cell lysate protein was added to each lane followed by SDS-PAGE as outlined in methods. The protein ladder used was MagicMark XP Standard from Invitrogen. Bands at ~74 kDa and 65 kDa represent long and short isoforms of QSOXl .

Figure 4. Stability of QSOXl peptide in whole blood. A determination of QSOXl-L peptide (NEQEQPLGQWHLS (SEQ ID NO: I)) levels in plasma using LC-MRM (multiple reaction monitoring) was performed. Blood from a pancreas cancer patient was drawn pre-operatively and was immediately split into three aliquots and processed into plasma after O, 6, 24 hours of incubation at room temperature. Plasma was filtered through 3 kDa ultrafilter and 35 fmol of an internal peptide standard spiked in before analyzing using LC-MRM method. Peak area of QSOXl-L peptide was normalized to the signal of an internal standard. Error bars represent standard deviations of three technical replicates. Figure 5. Quantitation of QSOXl peptide in plasma. Box plot shows ranges of NEQEQPLGQWHLS (SEQ ID NO: 1) peptide in 14 normal plasmas, 14 DAP patient plasmas for which LC-MS/MS were run and 14 DAP patient plasmas that had not been run on LC-MS/MS. A one-way analysis of variance (ANOVA) calculation of the means indicates P value <0.05. Values for each sample in ng/ml were calculated based on a , standard curve for which the linear portion of the curve was used from 500 ng/ml to 2.4 ng/ml of NEQEQPLGQWHLS (SEQ ID NO: 1) peptide.

Figure 6. Amino acid sequence of (A) QSOXl-L with peptide sequence that was identified in plasma shown in blue and (B) QSOXl-S obtained from www.uniprot.org.

Detailed Description of the Invention hi a first aspect, the present invention provides methods for assessing the probability of a pancreatic tumor in a subject, comprising analyzing a tissue sample of the subject for one or more peptides derived from one or both of QSOXl and SerpinF2, wherein an increased amount of the one or more peptides relative to a control correlates with a probability of a pancreatic tumor in the subject.

The methods of the invention are useful, for example, in assessing the probability of a pancreatic tumor in a patient. The methods thus provide early testing for pancreatic cancer (both initial diagnosis and recurrence after remission), and thus provide a means to improve the prognosis of pancreatic cancer patients. As disclosed in detail below, the inventors have identified peptides from QSOXl and SerpinF2 in patients with pancreatic cancer. In addition, the inventors have identified peptides from QSOXl and SerpinF2 in patients with a potentially pre-malignant condition called intraductal papillary mucinous neoplasm (IPMN), indicating that tissue sources of QSOXl and/or SerpinF2 can be used as a predictor of which patients might progress to develop ductal adenocarcinoma of the pancreas (DAP) or to pancreatic islet cell tumors.

The pancreatic tumor may be of any type; in one embodiment, the pancreatic tumor is an adenocarcinoma, such as a ductal adenocarcinoma, or a pancreatic islet cell tumor. The pancreatic tumor may be localized, or may include metastases to other tissue sites.

QSOXl is Quiescin Sulfhydryl Oxidase 1, also called QSCN6. The protein accession number for the long variant of QSOXl on the NCBI database is NP 002817 (SEQ ID NO:13), and the accession number for the short form is NP_001004128 (SEQ ID NO: 14). The full length sequence of QSOXl is provided in Figures 6A-6B below.

SerpinF2 is Serine protease inhibitor 2, also called 2 anti-plasmin (SEQ ID NO: 15). The protein accession number on the NCBI database is NP_000925. As used herein, "peptides derived from" the recited protein sequences means any contiguous region of the protein sequence less than the full sequence of the protein, such as protease digestion fragments of the protein. In one preferred embodiment, such "peptides derived from" the recited proteins are peptides of 3 kD or less. In a further preferred embodiment, the one or more peptides are selected from the group consisting of NEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 1), NEQEQPLGQWH (QSOXl) (SEQ ID NO: 2), EQPLGQWHLS (QSOXl) (SEQ ID NO: 3), AAPGQEPPEHMAELQR (QSOXl) (SEQ ID NO: 4), AAPGQEPPEHMAELQ (QSOXl) (SEQ ID NO: 5), AAPGQEPPEHMAELQRNEQEQPLGQWHLS (QSOXl) (SEQ DD NO: 6), NQEQVSPLTLLKLGN (SerpinF2) (SEQ ID NO: 7), NQEQVSPLTLLK (SerpinF2) (SEQ ID NO: 8), MEPLGRQLTSGP (SerpinF2) (SEQ ID NO: 9), NEQEQPL (SEQ ID NO: 10) (QSOXl), and GQWHLS (SEQ ID NO: 11) (QSOXl).

The tissue sample may be any suitable sample from which tumor-derived peptides may be obtained. In various preferred embodiments, the tissue sample is selected from the group consisting of plasma, serum, urine, saliva, cells from fine needle aspirate, and pancreatic juice.

In one preferred embodiment, the tissue sample comprises blood plasma. Methods for preparing blood plasma are well known in the art; such methods include those described below. In one embodiment, plasma is prepared by centrifuging a blood sample under conditions suitable for pelleting of the cellular component of the blood. Any suitable technique for analyzing a tissue sample of the subject for one or more peptides may be used, hi various non-limiting embodiments, techniques that can be used in the analysis include mass spectrometry (MS), two dimensional gel electrophoresis, Western blotting, immunofluorescence, ELISAs, antigen capture assays (including dipstick antigen capture assays) and mass spec immunoAssay (MSIA). hi one preferred embodiment, ligands for the one or more peptides are used to "capture" antigens out of the tissue sample. Such ligands may include, but are not limited to, antibodies, antibody fragments and aptamers. hi one embodiment, the ligand(s) are immobilized on a surface and the sample is passed over the surface under conditions suitable for binding of any peptides in the sample to the ligand(s) immobilized on the surface. Such antigen capture assays permit determining a concentration of the peptides in the tissue sample, as the concentration likely correlates with extent of disease. Exemplary MS techniques are described below. hi one preferred embodiment, the analysis is performed on a sample of peptides of 3 kD or less isolated from the tissue sample, hi this embodiment, use of an antigen capture assay preferably comprises use of ligands that selectively bind to a peptide selected from the group consisting of NEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 1), NEQEQPLGQWH (QSOXl) (SEQ ID NO: 2), EQPLGQWHLS (QSOXl) (SEQ ID NO: 3), AAPGQEPPEHMAELQR (QSOXl) (SEQ ID NO: 4), AAPGQEPPEHMAELQ (QSOXl) (SEQ ID NO: 5), AAPGQEPPEHMAELQRNEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 6), NQEQVSPLTLLKLGN (SerpinF2) (SEQ ID NO: 7), NQEQVSPLTLLK (SerpinF2) (SEQ ID NO: 8), MEPLGRQLTSGP (SerpinF2) (SEQ ID NO: 9), NEQEQPL (SEQ ID NO: 10) (QSOXl), and GQWHLS (SEQ ID NO: 11) (QSOXl). As used herein, "selectively bind" means that the ligand binds specifically under suitable assay conditions to its peptide binding partner in a complex solution, such as a sample of isolated peptides of 3 kD or less from a tissue sample, hi another preferred embodiment, use of antibodies to the ligand of interest can be carried out on the tissue sample, without fractionating the sample to obtain peptides of 3 kD or less.

In a preferred embodiment, an antibody that selectively binds one of the peptides is generated that is capable of selectively binding the peptide in the tissue sample and also capable of binding to the same peptide when coated on an ELISA plate, such as antibodies disclosed below, hi this embodiment, an ELISA can be carried out based on peptide present in the tissue sample, such as a plasma sample, blocking binding of the antibodies to the same peptide coated on an ELISA plate, hi a particularly preferred embodiment, the tissue sample (such as a plasma sample) is incubated under time and conditions suitable for selective binding of the peptide (if present in the tissue sample) to the peptide antibody. If the peptide is present in the tissue sample, it binds to the antibodies and inhibits them from binding to the peptide on an ELISA plate. The antibody-tissue sample mixture is then transferred to the ELISA plate pre-coated with peptide, and incubated under time and conditions suitable for binding of the coated peptide to the antibody in the absence of any peptide present in the tissue sample. AU unbound antibodies and peptides are subsequently washed from the ELISA plate, and labeled secondary antibodies (or any other suitable detection technique) is used to detect antibody that was not inhibited by peptide in the tissue sample from binding to the plate- bound peptide; in this way, an amount of peptide present in the tissue sample can be quantitated. In another embodiment, mass spec is used, in which a know concentration of a heavy isotope of the peptide of interest is added to the sample. This allows one to calculate the concentration of the natural peptide in plasma compared to the heavy peptide of known concentration.

Any suitable control can be used for comparison to an amount of peptide in the subject's tissue sample. In one embodiment, the control comprises an amount of one or more peptides of interest from a tissue sample from a normal subject or population (ie: known not to be suffering from a pancreatic tumor), or from a subject or population of subjects suffering from a pancreatic tumor, using the same detection assay. The control tissue sample will be of the same tissue sample type as that assessed from the test subject. In one preferred embodiment, a standard concentration curve of one or more peptides of interest in the control tissue sample is determined, and the amount of the one or more peptides of interest in the test subject's tissue sample is compared based on the standard curve, hi another preferred embodiment for use in monitoring progress of pancreatic cancer therapy, samples are obtained from patients over time, during or after their therapy, to monitor levels of one or more peptides in plasma as an indication about tumor burden in patients. The control may comprise a time course of concentration of the one or more peptides of interest in a given tissue type of the test subject, to monitor the effect of treatment on the concentration; this embodiment is preferred, for example, when assessing efficacy of pancreatic cancer treatments.

The subject can be any mammal, preferably a human, hi one preferred embodiment, the subject is identified as having one or more risk factors for pancreatic cancer prior to analysis of the blood sample, hi this embodiment, the methods are selectively carried out on subjects most likely to benefit from such a test. Non-limiting risk factors that may predispose a subject to pancreatic cancer include intraductal papillary mucinous neoplasm (EPMN), jaundice, pain in the upper abdomen, significant weight loss, being 50 years old or older, male gender, African ethnicity, smoker, obesity, diabetes, chronic pancreatitis, Heliobacter pylori infection, family history of pancreatic cancer, autosomal recessive ataxia-telangiectasia, autosomal dominantly inherited mutations in the BRCA2 gene, Peutz-Jeghers syndrome due to mutations on the STKl 1 tumor suppressor gene, hereditary non-polyposis colon cancer (Lynch syndrome), familial adenomatous polyposis, familial atypical multiple mole melanoma-pancreatic cancer syndrome (FAMMM-PC) due to mutations in the CDKN2A tumor suppressor gene, gingivitis or periodontal disease, and alcoholism.

As used herein, the "probability of a pancreatic tumor" can comprise determining a probability that the subject will develop a pancreatic tumor (prognostic), or may comprise determining a probability that the subject already has a pancreatic tumor (diagnostic). For example, a subject in whose tissue sample one or more of the recited peptides are identified can then undergo further tests, such as ultrasound, abdominal CT, endoscopic ultrasound or any other test suitable to visualize whether a pancreatic tumor is present. If such imaging tests do not show the presence of a tumor, an attending physician can determine whether early treatment interventions (chemotherapy, radiation therapy, etc.), are warranted, based on all circumstances. Alternatively, an attending physician can use the results from the methods of the invention, in light of other circumstances, to determine whether the subject should begin treatment for pancreatic cancer. Thus, the methods of the invention lead to earlier prognosis, diagnosis, and/or treatment of pancreatic cancer, which will lead to improved patient prognosis. In cases where imaging techniques and/or fine needle aspirate may be inconclusive with a pathological diagnosis of "atypical cells suspicious of malignancy", the presence of QSOXl and/or SerpinF2 in the tissue sample or in cells from fine needle aspirate (by IHC) combined with imaging may confirm diagnosis of DAP. The methods of the invention may be used in combination with other markers for pancreatic cancer, including but not limited to analysis of CAl 9-9 in the subject's serum or other suitable tissue sample. hi another aspect, the present invention provides methods for monitoring efficacy of treatment of a pancreatic tumor in a subject, comprising analyzing a tissue sample of a subject being treated for a pancreatic tumor for one or more peptides derived from one or both of QSOXl and SerpinF2, wherein the analyzing is carried out after one or more tumor treatments, and wherein a decrease in amount of the one or more peptides in the tissue sample after tumor treatment compared to an amount of the one or more peptides in a tissue sample from the subject after diagnosis of the pancreatic tumor but before tumor treatment began correlates with an effective tumor treatment. Thus, this aspect of the invention provides a valuable tool for monitoring effectiveness of treatments for pancreatic cancer. All embodiments of the first aspect of the invention are suitable in this aspect as well, unless the context dictates otherwise. The tissue sample may be any suitable sample from which tumor-derived peptides may be obtained, hi various preferred embodiments, the tissue sample is selected from the group consisting of plasma, serum, urine, saliva, cells from fine needle aspirate, and pancreatic juice. Treatment options to be assessed for efficacy in this aspect of the invention include, but are not limited to, surgical removal/resection of the tumor, chemotherapy (including, but not limited to gemcitabine. 5-fluorouracil, oxaliplatin, erlotinib, abraxane or any combination of chemotherapies, and/or radiation therapy. In one preferred embodiment, the method comprises analyzing the blood plasma sample of the subject for peptides of 3 kD or less; in a further preferred embodiment, the method comprises isolating peptides of 3 kD or less from the blood plasma sample, hi another preferred embodiment, the analyzing comprises a technique selected from the group consisting of mass spectrometry (MS), two dimensional gel electrophoresis, Western blotting, immunofluorescence, ELISAs, antigen capture assays (including dipstick antigen capture assays) and mass spec immunoAssay (MSIA) In a further preferred embodiment, the one or more peptides are selected from the group consisting of NEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 1), NEQEQPLGQWH (QSOXl) (SEQ ID NO: 2), EQPLGQWHLS (QSOXl) (SEQ ID NO: 3), AAPGQEPPEHMAELQR (QSOXl) (SEQ ID NO: 4), AAPGQEPPEHMAELQ (QSOXl) (SEQ ID NO: 5), AAPGQEPPEHMAELQRNEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 6), NQEQVSPLTLLKLGN (SerpinF2) (SEQ ID NO: 7), NQEQVSPLTLLK (SerpinF2) (SEQ ID NO: 8), MEPLGRQLTSGP (SerpinF2) (SEQ ID NO: 9), NEQEQPL (SEQ ID NO: 10) (QSOXl), and GQWHLS (SEQ ID NO: 11) (QSOXl).

Li another aspect, the present invention provides isolated peptides, selected from the group consisting of NEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 1), NEQEQPLGQWH (QSOXl) (SEQ ID NO: 2), EQPLGQWHLS (QSOXl) (SEQ ID NO: 3), AAPGQEPPEHMAELQR (QSOXl) (SEQ ID NO: 4), AAPGQEPPEHMAELQ (QSOXl) (SEQ ID NO: 5), AAPGQEPPEHMAELQRNEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 6), NQEQVSPLTLLKLGN (SerpinF2) (SEQ ID NO: 7), NQEQVSPLTLLK (SerpinP2) (SEQ ID NO: 8), MEPLGRQLTSGP (SerpinF2) (SEQ ID NO: 9), NEQEQPL (SEQ ID NO: 10) (QSOXl), and GQWHLS (SEQ ID NO: 11)

(QSOXl). These isolated peptides are useful as controls for the assays of the invention (ie: to verify identification of peptides in serum), as well as for generating ligands, including but not limited to antibodies and aptamers, that can be used in antigen capture assays of the invention, as well as in therapeutic aspects of the invention described below. The isolated peptides of the invention maybe recombinantly produced or chemically synthesized. Preferably, the polypeptides of the present invention are chemically synthesized. Synthetic polypeptides, prepared using the well known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can include natural and unnatural amino acids. Amino acids used for peptide synthesis may be standard Boc (Nα-amino protected Nα-t-butyloxycarbonyl) amino acid resin with the standard deprotecting, neutralization, coupling and wash protocols of the original solid phase procedure of Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154), or the base-labile Nα-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpino and Han (1972, J. Org. Chem. 37:3403- 3409). Both Fmoc and Boc Nα-amino protected amino acids can be obtained from Sigma, Cambridge Research Biochemical, or other chemical companies familiar to those skilled in the art. hi addition, the polypeptides can be synthesized with other Nα-protecting groups that are familiar to those skilled in this art. Solid phase peptide synthesis may be accomplished by techniques familiar to those in the art, or using automated synthesizers. The polypeptides of the invention may comprise L-amino acids, D-amino acids (which are resistant to L-amino acid-specific proteases in vivo), or a combination of D- and L- amino acids to convey special properties. In addition, the polypeptides can have peptidomimetic bonds, such as ester bonds, to prepare peptides with novel properties. For example, a peptide may be generated that incorporates a reduced peptide bond, i.e., R₁- CH₂-NH-R₂, where R₁ and R₂ are amino acid residues or sequences. A reduced peptide bond may be introduced as a dipeptide subunit. Such a polypeptide would be resistant to protease activity, and would possess an extended half-live in vivo. The isolated peptides may be further modified with other groups as desired to provide special properties, such as linkage to polyethylene glycol to increase plasma half-life for therapeutic use.

The polypeptides may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, adjuvants, etc., prior to being disposed on the heparin coating. The polypeptides may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers, or may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, dextran sulfate, heparin-containing gels, and/or polyvinyl alcohol prior to being disposed on the heparin coating.

In a further aspect, the present invention provides substantially purified nucleic acids encoding a peptide of the present invention. The substantially purified nucleic acid sequence may comprise RNA or DNA. Such nucleic acids are useful, for example, for preparing recombinant expression vectors to make large amounts of the peptides of the invention. As used herein, "substantially purified nucleic acids" are those that have been removed from their normal surrounding nucleic acid sequences in the genome or in cDNA sequences. Such substantially purified nucleic acid sequences may comprise additional sequences useful for promoting expression and/or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. In one preferred embodiment, the substantially purified nucleic acid coding region consists of a nucleic acid of encoding a peptide of the invention.

Li another aspect, the present invention provides recombinant expression vectors comprising the substantially purified nucleic acid of the invention operatively linked to a promoter. Such recombinant expression vectors are useful, for example, for generating host cells that produce large amounts of the peptides of the invention. "Recombinant expression vector" includes vectors that operatively link a nucleic acid coding region or gene to any promoter capable of effecting expression of the gene product. The promoter sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). The construction of expression vectors for use in transfecting prokaryotic cells is also well known in the art, and thus can be accomplished via standard techniques. (See, for example, Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. EJ. Murray, The Humana Press Inc., Clifton,

N. J.), and the Ambion 1998 Catalog (Ambion, Austin, TX). The expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA. hi a preferred embodiment, the expression vector comprises a plasmid. However, the invention is intended to include other expression vectors that serve equivalent functions, such as viral vectors. hi a further aspect, the present invention provides host cells that have been transfected with the recombinant expression vectors disclosed herein, wherein the host cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably transfected. Such transfection of expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art, including but not limited to standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection. (See, for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: A Manual of Basic Technique, 2^nd Ed. (R.I. Freshney. 1987. Liss, Inc. New York, NY).

In a further aspect, the present invention provides isolated ligands, wherein the ligand selectively binds to a peptide selected from the group consisting of

NEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 1), NEQEQPLGQWH (QSOXl) (SEQ ID NO: 2), EQPLGQWHLS (QSOXl) (SEQ ID NO: 3), AAPGQEPPEHMAELQR (QSOXl) (SEQ ID NO: 4), AAPGQEPPEHMAELQ (QSOXl) (SEQ ID NO: 5), AAPGQEPPEHMAELQRNEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 6), NQEQVSPLTLLKLGN (SerpinF2) (SEQ ID NO: 7), NQEQVSPLTLLK (SerpinF2) (SEQ ID NO: 8), MEPLGRQLTSGP (SerpinF2) (SEQ ID NO: 9), NEQEQPL (SEQ ID NO: 10) (QSOXl), and GQWHLS (SEQ ID NO: 11) (QSOXl). The ligands of the invention can be used, for example, in the antigen capture assays of the invention disclosed herein. In one preferred embodiment, the ligand is selected from the group consisting on antibodies, antibody fragments, and aptamers. It is well within the level of those of skill in the art to make antibodies and aptamers to the peptides disclosed herein. Such antibodies or aptamers are those that selectively bind to the peptide of interest, as defined above, hi a preferred embodiment, the ligand is an antibody. Suitable antibodies include, but are not limited to, polyclonal, monoclonal, and humanized monoclonal antibodies.

Antibodies can be made by well-known methods, such as described in Harlow and Lane, Antibodies; A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., (1988). hi one example, preimmune serum is collected prior to the first immunization. A substantially purified peptide of the invention, or a fragment thereof, together with an appropriate adjuvant, are injected into an animal in an amount and at intervals sufficient to elicit an immune response. Animals are bled at regular intervals, preferably weekly, to determine antibody titer. The animals may or may not receive booster injections following the initial immunization. At about 7 days after each booster immunization, or about weekly after a single immunization, the animals are bled, the serum collected, and aliquots are stored at about -20° C. Polyclonal antibodies against the proteins and peptides of the invention can then be purified directly by passing serum collected from the animal through a column to which non-antigen-related proteins prepared from the same expression system without GPBP-related proteins bound. Monoclonal antibodies can be produced by obtaining spleen cells from the animal.

(See Kohler and Milstein, Nature 256, 495-497 (1975)). hi one example, monoclonal antibodies (mAb) of interest are prepared by immunizing inbred mice with a peptide of the invention, or an antigenic fragment thereof. The mice are immunized by the IP or SC route in an amount and at intervals sufficient to elicit an immune response. The mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks.

Immunized mice are given one or more booster immunizations of by the intravenous (IV) route. Lymphocytes, from antibody positive mice are obtained by removing spleens from immunized mice by standard procedures known in the art. Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner under conditions which will allow the formation of stable hybridomas. The antibody producing cells and fusion partner cells are fused in polyethylene glycol at concentrations from about 30% to about 50%. Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art. Supernatant fluids are collected from growth positive wells and are screened for antibody production by an immunoassay such as solid phase immunoradioassay. Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press, 1973. "Humanized antibody" refers to antibodies derived from a non-human antibody, such as a mouse monoclonal antibody. Alternatively, humanized antibodies can be derived from chimeric antibodies that retains or substantially retains the antigen-binding properties of the parental, non-human, antibody but which exhibits diminished immunogenicity as compared to the parental antibody when administered to humans. For example, chimeric antibodies can comprise human and murine antibody fragments, generally human constant and mouse variable regions. Humanized antibodies can be prepared using a variety of methods known in the art, including but not limited to (1) grafting complementarity determining regions from a non-human monoclonal antibody onto a human framework and constant region ("humanizing"), and (2) transplanting the non-human monoclonal antibody variable domains, but "cloaking" them with a humanlike surface by replacement of surface residues ("veneering"). These methods are disclosed, for example, in, e.g., Jones et al., Nature 321:522-525 (1986); Morrison et al., Proc. Natl. Acad. ScL, U.S.A., 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyer et al., Science 239:1534-1536 (1988); Padlan, Molec. Irnmun. 28:489-498 (1991); Padlan, Molec. Immunol. 31(3):169-217 (1994); and Kettleborough, C. A. et al., Protein Eng. 4(7):773-83 (1991).

To generate an antibody response, the peptides of the present invention are typically formulated with a pharmaceutically acceptable carrier for parenteral administration. Such acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum-precipitate, water in oil emulsion containing Corynebacterium parvum and tRNA. The formulation of such compositions, including the concentration of the polypeptide and the selection of the vehicle and other components, is within the skill of the art.

The term antibody as used herein is intended to include antibody fragments thereof which are selectively reactive with the peptides of the invention. Antibodies can be fragmented using conventional techniques, and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab')₂ fragments can be generated by treating antibody with pepsin. The resulting F(ab')₂ fragment can be treated to reduce disulfide bridges to produce Fab' fragments.

In another aspect, the present invention provides methods for detecting the presence of a peptide selected from the group consisting of NEQEQPLGQ WHLS (QSOXl) (SEQ ID NO: 1), NEQEQPLGQWH (QSOXl) (SEQ ID NO: 2),

EQPLGQWHLS (QSOXl) (SEQ ID NO: 3), AAPGQEPPEHMAELQR (QSOXl) (SEQ ID NO: 4), AAPGQEPPEHMAELQ (QSOXl) (SEQ ID NO: 5), AAPGQEPPEHMAELQRNEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 6), NQEQVSPLTLLKXGN (SerpinF2) (SEQ ID NO: 7), NQEQVSPLTLLK (SerpinF2) (SEQ ID NO: 8), MEPLGRQLTSGP (SerpinF2) (SEQ ID NO: 9), NEQEQPL (SEQ ID NO: 10) (QSOXl), and GQWHLS (SEQ DD NO: 11) (QSOXl), comprising

(a) contacting a sample to be tested with a ligand of the invention, under conditions in which the ligand selectively binds to its respective peptide to produce a binding complex; and

(b) detecting presence of the binding complex, wherein the presence of the binding complex indicates that the peptide is present in the test sample.

The contacting occurs under conditions suitable to promote specific binding of the ligand (such as an antibody) to the peptide while minimizing non-specific binding. As used herein "specific binding" means that the ligands recognize one or more epitope within the recited protein under the selected conditions, but possess little or no detectable reactivity with other proteins under the same conditions.

The methods of this aspect of the invention can be used, for example, in the prognosis and diagnosis of pancreatic cancer, as well as to monitor protease degradation of the QSOXl and/or SerpinF2 protein in a sample of interest. All embodiments of each of the other aspects disclosed herein can be used in combination with the embodiments of this aspect, unless the context dictates otherwise. In a preferred embodiment, the detecting comprises quantitating an amount of the peptide in the tissue sample. The tissue sample may be any suitable sample from which tumor-derived peptides may be obtained. In various preferred embodiments, the tissue sample is selected from the group consisting of plasma, serum, urine, saliva, cells from fine needle aspirate, and pancreatic juice. In another embodiment, the method comprises analyzing the sample for peptides of 3 kD or less; in a further preferred embodiment, the method comprises isolating peptides of 3 kD or less from the sample. In another preferred embodiment, the analyzing comprises a technique selected from the group consisting of mass spectrometry (MS), two dimensional gel electrophoresis, Western blotting, immunofluorescence, ELISAs, antigen capture assays (including dipstick antigen capture assays) and mass spec immunoAssay (MSIA).

Examples Abbreviations:

CA-125: Cancer Antigen 125, a protein often found in patients with ovarian cancer CA- 19-9: Carbohydrate Antigen 19-9, a biomarker associated with pancreas cancer DAP: Ductal Adenocarcinoma of the Pancreas

EDTA: Ethylenediaminetetraacetic acid

HAP: Highly Abundant Proteins

HMW: High Molecular Weight HPLC: High Performance Liquid Chromatography

IHC: Immunohistochemistry

EPMN: Intraductal Papillary Mucinous Neoplasm kDa: kilodaltons

LC-MS/MS: Liquid Chromatography followed by tandem mass spectrometry LMW: Low Molecular Weight

MWCO: Molecular Weight Cutoff

PSA: Prostate Specific Antigen

PVDF: Polyvinylidene fluoride

QSOXl: Quiescin Sulfhydryl Oxidase 1, also called QSCN6 SerpinF2: Serine protease inhibitor 2, also called α2 anti-plasmin

Abstract

Blood circulates through nearly every organ including tumors. Therefore, plasma is a logical source to search for tumor-derived proteins and peptides. The challenge with plasma is that it is a complex bodily fluid composed of high concentrations of normal host proteins that obscure identification of tumor-derived molecules. To simplify plasma, we examined a low molecular weight (LMW) fraction (plasma peptidome) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. In the plasma peptidome of patients with ductal adenocarcinoma of the pancreas (DAP) a prominent peptide was identified from the QSOXl parent protein. This peptide is stable in whole blood over 24 hours and was present in 16 of 23 DAP patients and 4 of 5 patients with intraductal papillary mucinous neoplasm (IPMN). QSOXl peptides were never identified in the plasma peptidome from 42 normal healthy donors using the same methods. Similarly, a prominent and stable peptide from the SerpinF2 parent peptide was identified in the plasma peptidome of DAP patients. Immunohistochemical staining of DAP tissue sections with anti-QSOXl antibody, shows overexpression of QSOXl in tumor but not in adjacent stroma or normal ducts. Three of four pancreas tumor cell lines also express QSOXl protein by Western blot analysis. This is the first report of QSOXl and SerpinF2 peptides in plasma from DAP patients and makes the rare connection between a peptide in plasma from cancer patients and over-expression of the parent protein in tumors.

INTRODUCTION

Because blood circulates through almost every organ it samples proteins from a multitude of tissues, including tumors. As a result plasma is a complex collection of proteins, sugars, carbohydrates and lipids that span concentrations of almost 10 orders of magnitude '. Even though diagnoses as well as response to therapeutic treatment are correlated with quantitative and physiological changes of plasma proteins ' , analysis of plasma usually requires overcoming highly abundant proteins (HAP). Plasma protein concentration is estimated to be 60-80 mg/ml, of which twenty-two "classical" HAP constitute approximately ninety-nine percent of the total mass ^{1( "} . The remaining one percent of the plasma proteome offers insights into normal versus pathological states.

For screening purposes, interest has focused on proteins that are secreted or shed from tissues ^{1> 7}. For example, increased plasma levels of CA 19-9 ⁸, prostate-specific antigen (PSA) ⁹ CAl 25 ¹⁰, and cardiac myoglobin ^π have been used as markers for the detection of pancreatic cancer, prostatic cancer, ovarian cancer, and myocardial infarction, respectively. The effort of searching for disease markers within the past few decades has achieved limited success partly due to HAP and the large dynamic range of plasma protein concentration. Many investigators have tried to separate HAP away from less abundant, but more biologically informative molecules by using two-dimensional electrophoresis (2-DE), liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and immunoaffmity approaches ^{lj 7> 12"21}. However, instrument sensitivity and capacity still fall short of reliably and reproducibly detecting the lower range of plasma proteins.

Because a diagnosis of cancer late in the disease process may lead to poor prognoses for patients and significant healthcare expense, it is important to identify plasma-based biomarkers to detect cancer early or to monitor recurrence after surgery. As normal cells transition to tumor cells, they go through many genetic transformations that result in production of protein variants or over-production of proteins that can be distinguished from wild type proteins produced by normal cells due to their increased abundance in tissues and plasma ^{22> 23}.

If a protein is over-expressed by a tumor, it may be shed into circulation and subject to proteolytic digestion, resulting in LMW peptides from the parent protein. Several plasma-based proteolytically derived peptides have been reported ⁶' ^{19> 24"28} ₅ however, their use for disease diagnosis has been under-explored. For example, fibrinogen A-derived peptides have been reported to correlate with hepatocellular carcinoma ²⁹, ovarian carcinoma ³⁰, colorectal carcinoma ' , and severe acute respiratory syndrome (SARS) ³³. Using mass spectrometry alone to identify potential biomarkers in plasma has been criticized because of lack of confirmatory non-mass spectrometry based evidence to support plasma-based results ^{26> 34"38}. Here we report the use of mass spectrometry methods followed by confirmatory immunohistochemistry (IHC) and Western blotting. Mass spectrometry analyses led us to identify peptide fragments from QSOXl in pancreatic cancer patient plasma, and IHC with anti-QSOXl antibodies indicated that the QSOXl peptides identified in plasma are likely derived from pancreas tumors.

EXPERIMENTAL SECTION

Materials. LC-MS grade water, acetonitrile, and formic acid were obtained from Fisher Scientific (Fair Lawn, NJ). Blood collection and processing for mass spectrometric analysis. Blood samples from 42 normal subjects were collected under an IRB-approved protocol at Arizona State University. Participants included 22 male and 20 female normal subjects whose ages were between 18 and 63 years old. The age and sex of normal donors partially overlaps with the age bracket of pancreas cancer patients. Blood samples from normal subjects were drawn directly into sterile blood collection tubes containing EDTA (Fisher, Fair Lawn, NJ), as an anti-coagulant. Blood samples were separated into plasma, lymphocytes and red cells by performing standard Ficoll blood separation. Approximately 30% of blood samples collected after 4pm were processed the next morning after sitting at room temperature overnight (~ 16 to 18 hours later). Approximately 3 to 5 ml of plasma were collected and placed into cryovials (Wheaton, Millville, NJ) and stored at -8O⁰C for analysis. Prior to LC-MS/MS analysis, frozen plasma was thawed on ice, filtered through 0.45 μm polyvinylidene fluoride (PVDF) membrane ultrafilter (Millipore, Bedford, MA) and the filtrate was further passed through a 3 IcDa filter (Millipore, Bedford, MA). Using NanoDrop spectrophotometer (Thermo Scientific), the concentrations of the 0.45 μm and 3 kDa filtrates were measured to be 64.5 ± 0.8 mg/ml and 6.4 ± 0.6 mg/ml, respectively.

Blood samples from patients undergoing surgery for pancreas neoplasms were obtained from the Tissue Acquisition core of Dr. Daniel Von Hoff s pancreas cancer POl under the supervision of Dr. Demeure. Whole blood was collected from patients undergoing surgical resection for DAP at the Virginia Piper Cancer Institute in

Minneapolis, MN, and Banner Good Samaritan Medical Center in Phoenix, AZ under approved IRB protocols. Patients included 12 males and 16 females whose ages were between 34 and 84 years old. Blood samples were collected into tubes containing EDTA anti-coagulant and shipped to us by overnight express at ambient temperature (except in summer when cold packs were used). Blood samples from patients shipped to us were processed between 18 and 24 hours post phlebotomy. Blood samples collected locally from pancreas cancer patients were processed from 1 to 4 hours post phlebotomy. All blood samples were processed using the same methods as healthy donor plasma.

Immunohistochemistry (IHC). Formalin-fixed, paraffin-embedded tissue blocks from patients who underwent surgical resection for DAP were sectioned at 5μm thickness using water flotation for tissue section transfer and dried overnight at room temperature. The slides were dewaxed, rehydrated and antigen retrieved on-line on the Bond™ autostainer (Leica Microsystems, Inc. Bannockburn, IL). All slides were cut at 5μm and baked at 6O⁰C for 60 minutes. Slides were subjected to heat induced epitope retrieval using a proprietary citrate based retrieval solution for 20 minutes. Endogenous peroxidase was blocked. For QSOXl, the tissue sections were incubated for 30 minutes with anti-QSOXl polyclonal antibody at 1 :75 from Proteintech Group, Inc. (Chicago, IL). The sections were visualized using the Bond™ Polymer Refine Detection kit (Leica) using diaminobenzidine chromogen as substrate. IHC optimization and staining parameters were evaluated by Dr. Hostetter, a board-certified pathologist, with standard scoring based on stain intensity (0 to 3) with score of 0 representing no staining and score 3 intense staining. Criteria for IHC stain localization in tumor cells were nuclear, cytoplasmic or membranous.

SDS-PA GE- Western blotting. Cell lysates from Pane- 1 , CFPac- 1 , Bx-PC3 , and healthy donor PBMC were generated using an established protocol ³⁹. Twenty micrograms of cell lysate protein was loaded into each lane of a 4-12% gradient SDS-PAGE gel and electrophoresed under reducing conditions. The gel was transferred to PVDF membrane, blocked with 5% non-fat powdered milk and probed with anti-QSOXl rabbit polyclonal antibody (same as was used in IHC) at a concentration of 0.34ug/ml (1:1000 dilution) for 3 hours. The blot was washed free of anti-QSOXl primary antibody followed by addition of goat anti-rabbit IgG coupled to horse radish peroxidase (HRP) (Jackson Immunolabs, West Grove, PA) at a 1 :5000 dilution and incubated with the blot for 1 hour followed by washing. BCIP/NBT substrate (Pierce Chemical, Rockford, IL) was added and the blot was developed for 1 hour at room temperature.

Separation and analysis of peptides. Plasma samples from pancreatic cancer patients and normal subjects were filtered through centricon centrifugal filter devices with MWCO of 3 kDa at 10,000xg at 4⁰C. The filtrates were either analyzed immediately or transferred into siliconized tubes (VWR Scientific) and stored at -2O⁰C until analysis. The filtrates were resolved on an Agilent 1100 HPLC-Chip Cube Interface (Agilent Technologies). The Agilent HPLC-Chip integrates enrichment column (ZORBAX

300SB-C18, 40 nl, 5 μm), analytical column (ZORBAX 300SB-C18, 75 μm x 43 mm, 5 μm), and a nanospray emitter on a single and reusable microfluidic chip. Four microliters of each plasma sample was injected on the enrichment column using mobile phase containing 97% water, 3% acetonitrile, and 0.1% formic acid at a flow rate of 4 μl/min. The enrichment column was washed with 12 μl of mobile phase to remove salts in the plasma sample that got trapped along side peptides. Peptides that were trapped on the enrichment column were then eluted onto the analytical column for separation with a gradient from the nano-HPLC pump system. The gradient ran from 5 to 50% in 14 minutes and 50 to 100% in 4 minutes with mobile phase B (90% acetonitrile, 0.1% formic acid in LC-MS grade water) versus mobile phase A (3% acetonitrile, 0.1% formic acid in LC-MS water) at a flow rate of 0.3 μl/min.

The eluted gradient was analyzed on-line with nano-electrospray ionization (nano- ESI) ion trap mass spectrometer (LC/MSD Trap XCT Ultra, Agilent Technologies) in the positive ion mode. The high voltage capillary was set at 1880 V, while the dry gas flow and dry temperature were set at 5 1/min and 32O⁰C, respectively. Mass spectrometer (MS) full scans were acquired from 350 to 2000 m/z in data dependent mode. Six most abundant ion peaks above the background in each mass spectrum were selected as precursor ions for tandem mass spectrometry (MS/MS) using collision-activated dissociation (CAD). MS/MS scan of the same ion was not allowed in more than two MS spectra that were obtained within a period of 1 minute.

MS/MS Data interpretation. MS/MS data were searched against NCBI refseq protein database (May 2008) which contained approximately 30,000 protein entries using

Spectrum Mill software Rev A.03.03.078 (Agilent Technologies). Software parameters were adopted per manufacturer's recommendations except changing the manufacturer's recommended precursor mass tolerance of ±2.5 Da to ±1.5 Da to minimize false positive rate. Fragment ion mass tolerance of ± 0.7 Da was used. MS/MS spectra with at least four detected peaks and sequence tag length greater than three were considered for sequence interpretation. A peptide which had a charge of +2 with a score threshold of at least 10, or a charge of +1 or +3 with a score threshold of at least 13 was considered a good hit if it also has a scored peak intensity (SPI) of at least 70, and both forward-reverse score threshold and rank 1-2 score threshold of at least 2. Because we analyzed a low molecular weight fraction of the plasma proteome (peptidome), trypsin digestion was not employed. However, all proteins that were identified by only one peptide as well as all peptides that were ambiguously assigned to more than one protein were rejected from our list. A false positive rate of less than 7% was obtained by running our MS/MS spectra against a decoy database that was generated by shuffling target sequences in the NCBI refseq protein database ⁴⁰. All peptide sequences reported in this study were again searched using blastp (http://www.ncbi.nlm.nih.gov/blast/) to make sure each sequence was not found in more than one protein.

Synthetic peptides. Because of the relatively large precursor mass tolerance (± 2.5 Da) used in the database search, 30 peptides reported in this study were chemically synthesized and run on the LC-MS/MS under the same conditions as described above to make sure their retention times and spectra matched those of the natural peptides. Peptides were synthesized at the proteomics core facility at Arizona State University on a Milligen 9050 peptide synthesizer (Millipore, Bedford, MA). After HPLC purification, peptide purity was estimated to be greater than 95%. Mass spectrometric analysis was used to confirm amino acid composition of peptides. Stability of QSOXl peptide. To determine the stability of the QSOXl peptide, blood was drawn from a pancreas cancer patient just prior to surgical resection of tumor into a 7ml EDTA tube and provided to us within 1 hour of phlebotomy. It was split into three aliquots and processed into plasma as described above after 0, 6, and 24 hours incubation at room temperature. Six microliters of plasma sample that was filtered through 3 kDa ultrafilter and spiked with 35 fmol of an internal standard peptide, FLWGLTPKV (SEQ ID NO: 12), was analyzed to determine the levels of QSOXl peptide

(NEQEQPLGQWHLS (SEQ ID NO: I)) using LC-MRM (multiple reaction monitoring) technique. The internal standard was chosen because the peptide was available in our laboratory and the amino acid sequence does not occur in human proteome and, therefore, should not exist in the plasma peptidome. Plasma samples were injected on a Finnigan Surveyor LC system (Thermo

Electron Corp., San Jose, CA) online with a linear ion trap (LTQ, Thermo Electron Corp., San Jose, CA). The Surveyor LC system was equipped with a standard autosampler. The peptides were desalted and concentrated on a Paradigm Platinum Peptide Nanotrap (Michrom Bioresources, Inc) precolumn (0.15 x 50 mm) and subsequently to fused silica microcapillary Cl 8 column (75 mm internal diameter, 10 cm in length, 5 μm particle size) (PFC7515-PP2-10, Michrom Bioresources, Inc), at a flow rate of 160 μl/min. The samples were subjected to a 35 min (3-90% ACN) gradient and directly eluted into the microcapillary column set to 2.32 kV. The LTQ was operated in the positive-ion mode with collision energy of 35%. Each plasma sample was analyzed in three technical replicates.

The QSOXl peptide precursor-to-product represents transition for the doubly charged parent ion (NEQEQPLGQWHLS, 2H+) with mlz 783.51 to the single-charge product y ions with mlz 937.37 and 1194.42, whereas the precursor-to-product of the internal standard represents transition for the doubly charged parent ion (SEQ ID NO: 12; GLTPKV, 2H+) with m/z 530.75 to single-charge product y ions with m/z 443.24 and 799.42. Data analysis and integration of peak areas were accomplished using Xcalibur 2.0. Peak area of the QSOXl peptide was normalized to the signal of the internal standard.

Quantitatitve ELISA for QSOXl peptide

Rabbit antisera were raised against NEQEQPLGQWHLS (SEQ ID NO: 1) peptide by Epitomics Inc. (Burlingame, CA) and affinity-purified on an NEQEQPLGQWHLS (SEQ ID NO: 1) peptide column. 50ng/ml affinity purified anti-NEQEQPLGQWHLS (SEQ DD NO: 1) antibody was incubated for one hour with plasma such that the plasma was diluted 1 :1 in the antibody preparation. This mixture was then added to ELISA plates pre-coated with streptavidin-biotin-NEQEQPLGQWHLS (SEQ ID NO: 1) peptide and further incubated at room temperature. After one hour, the plate was washed free of unbound antibody-peptide followed by addition of a pre-determined dilution of goat anti- rabbit IgG-HRP (Pierce, Rockland, IL). After another hour of incubation, the ELISA plate was washed as before, and tetramethyl benzidine substrate was added followed by termination of the HRP enzyme activity with IN H₂SO₄. Plates were read at OD 450nm and the concentration of NEQEQPLGQWHLS (SEQ ID NO: l)peptide in plasma was calculated based on the linear portion of a standard curve of NEQEQPLGQWHLS (SEQ ID NO: 1) peptide from 500ng/ml to 1.2ng/ml.

RESULTS

A peptide from QSOXl protein is prominent in plasma from patients with DAP. hi our LC-MS/MS analysis, two peptides from the C-terminus of the long isoform of QSOXl (QSOXl-L) were detected. The first peptide, NEQEQPLGQWHLS (SEQ ID NO: 1) (amino acid 631-643) is from the C-terminus of QSOXl-L and occurred in 16/23 patients with DAP and 4 of 5 patients with IPMN (Table IA and IB; Table 2). The other peptide, AAPGQEPPEHMAELQR (residues 615-630) is also derived from the C terminus of the protein, but just upstream of NEQEQPLGQWHLS (SEQ ID NO: 1) peptide. It occurred much less frequently in DAP and IPMN plasmas and will not be further discussed in this report.

Table 1. Detection of QSOXl and SerpinF2 peptides in plasma peptidome. A. LC- MS/MS analysis of OkDa fraction of plasma from surgically resectable patients diagnosed with ductal adenocarcinoma. B. LC-MS/MS analysis of OkDa plasma fraction from patients with IPMN. A total of 5 technical replicates of LC-MS/MS runs was performed for each patient and normal sample. Peptides from QSOXl or SerpinF2 were never detected in plasma from 42 healthy donors by LC-MS/MS.

TABLE 1A

TABLE IB

Table 2. Sequence and frequency of peptides from QSOXl and SerpinF2 proteins. "Position in protein" numbering is according to the NCBI sequence database. Obs (observed)/total indicates the number of patient plasmas which contained a peptide over the total number of patient plasmas analyzed.

TABLE 2

No peptide from QSOXl was found in plasma from 42 normal donors using the same processing and detection methods. Using Fisher's exact T-test, this is highly significant with a p-value of less than 0.001. Of particular interest is detection of QSOXl- L peptide corresponding to QSOXl in patients with IPMN. There is controversy about whether IPMN is a pre-malignant condition ⁴¹'⁴² ₃ but if IPMN predisposes patients to DAP, QSOXl could be a useful predictor for which patients with IPMN might progress to DAP. We are planning a retrospective study to address this hypothesis. The QSOXl gene locus is on human chromosome Iq24 in proximity to the prostate cancer locus (HPCl) ⁴³. It has short and long isoforms that are produced as splice variants. The long isoform (QSOXl-L) is 747 amino acids and the short isoform

(QSOXl-S) is 604 amino acids (Figure 6).

QSOXl is a sulfhydryl oxidase thought to participate in redox reactions during intracellular protein folding ⁴³ and has FAD-binding domains homologous to ERVIp from yeast ^{44> 4S}. Although there are relatively few publications related to QSOXl , one cancer-related study reported that over-expression of QSOXl might protect cells against oxidative stress-induced apoptosis ⁴⁶.

Using LC/MS-MS methods, we analyzed the plasma peptidome and did not perform trypsin digestion on plasma proteins. Therefore, identification of multiple peptides from the same protein may not occur. To confirm identification of

NEQEQPLGQWHLS (SEQ ID NO: 1), the peptide was chemically re-synthesized, run on

LC-MS/MS and LC retention time and MS/MS spectrum compared to those of the natural peptide detected in plasma. The retention times of the synthetic and natural

NEQEQPLGQWHLS (SEQ ID NO: 1) overlap perfectly. The mass spectra from the synthetic and natural sequences also show the same ion peaks as shown in Figure 1, confirming that the peptide is not a false positive.

Immunohistochemical Detection of QSOXl. Because peptides corresponding to QSOXl were identified frequently in plasma from patients with DAP, but not in plasma from healthy donors, we hypothesized that QSOXl might be over-expressed in DAP. To address our hypothesis, immunohistochemistry was performed with polyclonal anti- QSOXl antibody to stain tissue sections from patients with DAP. As shown in figure 2A, anti-QSOXl antibodies stain DAP tumor cells, but not normal adjacent ducts or surrounding stroma, hi figure 2B, anti-QSOXl antibodies stained a micrometastasis from a peripancreatic lymph node. Upon closer examination of the type of staining (figure 2C), it appears that QSOXl expression in DAP is cytoplasmic. Figure 2D demonstrates lack of specific staining in pancreas tissue from a patient with chronic pancreatitis. Figure 2E shows a pancreatic duct with normal and dysplastic-looking cells. Only the dysplastic cells show concomitant QSOXl expression. Because of this fortuitous result, we stained an array of 40 normal tissues. Only small bowel and pediatric kidney stained with the polyclonal anti-QSOXl antibody; all other tissues were negative (data not shown). This result supports our hypothesis that peptides from QSOXl detected in plasma from patients with DAP are tumor-derived. Since the anti-QSOXl antibody does not distinguish between QSOXl-L and QSOXl-S isofoπns of QSOXl, it is possible that either or both variants could be over-expressed in tumor.

QSOXl Western blotting. To further substantiate over-expression of QSOXl, SDS- PAGE was performed on cell lysates followed by Western blotting with anti-QSOXl antibody As shown in figure 3, the long and short isoforms of QSOXl, at 74 kDa and 65 kDa respectively, are most prominent in Panc-1, followed by Bx-PC3 and CFPac-1. Capan-1 and PBMC do not appear to express QSOXl protein within the detection limits of Western blotting. This result confirms in established tumor cell lines, the over- expression of QSOXl observed in IHC.

Stability of QSOXl peptide in blood. Because proteins and peptides are subject to proteases in blood, we performed a time course experiment with blood from a patient with DAP. LC-MS/MS analysis of the processed plasma using MRM techniques showed constant levels of peptide at each time point (Figure 4). This time course experiment suggests that the QSOXl peptide is stable in plasma over time. We also performed a similar experiment in which synthetic QSOXl peptide was spiked into whole blood from a healthy donor and observed the same result (data not shown).

Quantitative ELISA for QSOXl peptide

Since NEQEQPLGQWHLS (SEQ ID NO: 1) peptide was secreted from tumor cells and appeared to be stable in whole blood, we generated rabbit polyclonal antisera against the peptide and affinity purified the antisera on a peptide affinity column. Using this affinity purified anti- peptide antibody preparation, a quantitative ELISA was established in which natural NEQEQPLGQWHLS (SEQ ID NO: 1) peptide in plasma inhibited the anti-NEQEQPLGQWHLS (SEQ ED NO: 1) antibody from binding to ELISA plates coated with streptavidin-biotin-NEQEQPLGQWHLS (SEQ ID NO: 1) peptide. This assay was performed using plasma samples from 14 patients on which we did not run LC-MS/MS, 14 patients whose plasma contained NEQEQPLGQWHLS (SEQ ID NO: l)by LC-MS/MS analysis and 14 plasma samples from normal donors. Patient plasma samples that demonstrated the presence of NEQEQPLGQWHLS (SEQ ID NO: 1) peptide by LC-MS/MS ranged from 5.5 to 50.7 ng/ml with a mean of 20.5 ng/ml. DAP patient plasma samples on which we did not perform LC-MS/MS, ranged from 7.1 to 156.6 ng/ml with a mean of 49.4 ng/ml while normal donor plasma samples ranged from 3.6 to 13.4 ng/ml with a mean of 7.1 ng/ml as shown in figure 5.

SerpinF2 peptides are prominent in plasma from patients with pancreatic ductal adenocarcinoma. As with QSOXl, peptides corresponding to SerpmF2 proteins occurred at high frequency (16 of 21) in the LMW fraction of plasma from patients with DAP

(Table IA). No peptide from SerpinF2 was found in plasma from 42 normal donors using the same processing and detection methods. Also similar to QSOXl, SerpinF2 peptides were detected in patients with intraductal papillary mucinous neoplasm (EPMN) (Table IB). Thus, if IPMN predisposes patients to DAP, SerpinF2 may be a useful biomarker of early disease, or possibly predict which patients with IPMN might progress to DAP.

SerpinF2 is a 452 amino acid glycoprotein that is synthesized in the liver. It is reported to have a serum half-life of 2.6 days with an average serum concentration of 70 μg/ml. Despite reports of 70 μg/ml SerpinF2 concentration in plasma, we never detected SerpinF2 in the low molecular weight fraction of plasma from normal donors (47). Three peptides corresponding to SerpmF2 protein were identified and derived from the N terminal region of the protein. One of the peptides was contained within the NQEQVSPLTLLKLGN (amino acid 40-54) parent peptide. These two peptides were found infrequently in 3 of 21 plasmas from patients with DAP. The other peptide, MEPLGRQLTSGP (amino acids 28-39) occurred with high frequency of 18 of 21 plasmas from patients with DAP and 5 of 5 plasmas from patients with EPMN. SerpinF2 is also called α-2 anti-plasmin. It is the main blood-derived inhibitor of fibrin clot- dissolving plasmin. To ensure the sequence identities of the SerpinF2 peptide, MEPLGRQLTSGP, it was chemically re-synthesized, run on LC-MS/MS and compared to the spectra from the natural peptide detected in plasma. The mass spectra from the synthetic and natural sequences show the same ion peaks (data not shown). .

DISCUSSION:

We reported three significant findings in this study. The first finding was the frequency for which a peptide derived from the QSOXl-L protein was identified in the plasma peptidome of patients with DAP. 16 of 23 DAP patients demonstrated one or more peptides from the long isoform of QSOXl in plasma using LC-MS/MS analysis, while we did not detect any QSOXl peptides in plasma from 42 healthy donors using the same methodology. As stated in the results, Fisher's probability test indicated these initial results were highly significant, although the numbers in this study are small. QSOXl peptide was also detected in 4 of 5 patients with IPMN, a potential precursor to DAP ⁴¹'⁴². Because our initial LC-MS/MS methods on the quadrupole ion trap were not able to accurately quantify peptides in plasma, we developed a quantitative ELISA to quantify the levels of QSOXl -L peptides in plasma, as disclosed herein.

One explanation for finding only C-terminal peptides from QSOXl-L is that tumor cells cleave the C-terminal end of QSOXl-L protein, releasing it into circulation. Another possibility is that the whole protein is secreted, and proteases at the cell surface or in plasma may cleave QSOXl-L at an unknown location. Our method of examining the <3 kDa fraction of plasma does not allow detection of the remainder of the molecule, or larger fragments of the proteolyzed protein. We are actively investigating these possibilities to determine the whether QSOXl-L is cleaved at the tumor cell surface, liberating peptide, or if plasma-derived proteases cleave longer versions of QSOXl-L.

The second finding was the connection between QSOXl-L peptides circulating in plasma and over-expression of the parent protein in tumors as detected by IHC.

Immunohistochemical staining of tissue sections (figure 2) from patients with DAP showed remarkable tumor-specific expression of QSOXl . It appears that pancreas tumor cells over-express QSOXl in the cytoplasm, while normal adjacent pancreatic tissue including normal ducts and stroma do not express enough QSOXl to be detected by IHC methods. Since the antibody used in this IHC study was generated against an N-terminal fragment of QSOXl (amino acids 1-329), it does not differentiate QSOXl-L and QSOXl-S isoforms, so it is possible that either or both variants could be over-expressed in rumor.

To address the question of whether QSOXl-L and QSOXl-S isoforms are expressed by tumor cells, four different pancreas tumor cell lines were analyzed in western blotting experiments in figure 3. This experiment demonstrated that 3 of the 4 cell lines tested express QSOXl-L and QSOXl-S, although it is clear from the western blot that the short form is more highly expressed than the long isoform. This may suggest that QSOXl-S is primarily detected by IHC. None-the-less, peptide 631-643 from the long isoform was detected in patient plasma, strongly suggesting that both are expressed in tumors in vivo. Lack of expression of QSOXl in Capan-1, and expression in three other pancreas tumor cell lines parallels our mass spectrometry data in which we only detected QSOXl-L peptide in -70% of patients with DAP. It is unclear at this point in time if tumor cells secrete the entire protein or secrete a C-terminal fragment of the protein such that it can be detected in blood. Since the QSOXl-L also contains a transmembrane domain, it may be retained at the cell membrane or even in Golgi bodies. Further studies will elucidate how peptide 631-643 might be cleaved if the long form is retained in the cell.

Approximately 70% (16/23) of patients with DAP demonstrated circulating QSOXl-L peptides by LC-MS/MS. Although we never found any QSOXl peptide in plasma from healthy donors using the same methods." we will be performing the same analysis on age and sex-matched patients with non-malignant pancreaticobiliary conditions such as cholecystitis and cancer patients with other tumor types such as cholangiocarcinoma, pending IRB approval. This is an important population to assess because oncologists need to differentiate pancreas cancer from non-malignant disease.

The third finding was the stability of QSOXl 631-643 peptide in plasma. Because we were surprised to find the same length QSOXl peptide in multiple patients, we assessed the stability of QSOXl peptide in whole blood at times O, 6 and 24 hours post- collection. Using MRM techniques as described in methods, QSOXl peptide was detected at each time point during incubation of whole blood from a DAP patient at room temperature (Figure 4). Degradation fragments of QSOXl-L peptide (NEQEQPLGQWHLS (SEQ ID NO: I)) were not observed, further suggesting stability of this peptide in blood. One possibility for the lack of proteolysis of the QSOXl peptide post phlebotomy is that blood was collected into EDTA-containing tubes. EDTA may chelate divalent cations necessary for proteases to function. Alternatively, it seems likely that most of the proteolytic activity would occur in vivo before blood is drawn. Villanueva et al. and others have reported that cancer patient blood may contain tumor- derived proteases and/or other metabolic processes that could affect detection of peptides in blood samples from cancer patients ¹⁹. As stated in methods, locally-obtained whole blood from DAP patients was processed within 1-4 hours, and blood that was shipped to us was processed between 18 and 24 hours post phlebotomy. QSOXl peptides were detected in blood that was processed within 1-4 hours as well as in blood that was shipped overnight. While not being bound by any specific mechanism of action, since cancer patient and normal donor blood samples were treated in the same manner, we suggest that peptide 631-643 is cleaved from the parent protein in vivo, either by the tumor cells or in plasma. As a companion to the stability of the peptide, we generated antibodies against NEQEQPLGQWHLS (SEQ ID NO: 1) peptide and developed an ELISA that quantifies the levels of the peptide in plasma. The fact that antibodies used in this ELISA inhibit natural NEQEQPLGQWHLS peptide present in plasma from binding to the synthetic peptide coated onto an ELISA plate addresses concerns that the NEQEQPLGQWHLS (SEQ ID NO: l)peptide may not be the correct sequence. Analysis of the data in figure 5 suggests that the means and medians of each group are significantly different (p value <0.05). Levels of QSOXl peptide in normal donors show little variation, but levels of QSOXl peptide in plasma from DAP patients demonstrated a broad concentration range. In one embodiment, a normal range of the peptide is from 2 ng/ml to 11 ng/ml; preferably 3.5 ng/ml to 11 ng/ml, with DAP suspected for a concentration above 11 ng/ml; preferably with DAP suspected for a concentration above 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more ng/ml.

This is the first report of detection of peptides from the long isoform of QSOXl in plasma from DAP patients. Our finding is novel because the QSOXl peptides map back to the parent protein which was over-expressed in malignant ductal epithelia in tumor tissue. We found that peptide 631-643 was stable and did not get degraded even in whole blood after 24 hours at room temperature. Finally, QSOXl is understudied in cancer. By investigating QSOXl expression in tumor cells in future studies, we hope to find the function of QSOXl or if it is simply a by-product that has potential as a biomarker.

Example 2 Monoclonal antibodies Supernatants from anti- NEQEQPLGQWHLS (SEQ ID NO: 1 ) hybridomas were tested to identify antibodies that i) bind only to the N-terrninal 7 amino acids (NEQEQPL) (SEQ ID N0:9), but not the C-terminal 6 amino acids, and ii) bind only to the C-terminal 6 amino acids (GQWHLS) (SEQ BD NO: 10), but not to the N-terminal 7 amino acids. The rationale for this is that the peptide is very small compared to the large binding site of the antibody protein. In the antigen capture, we want the best chance for both antibodies to be able to bind to the NEQEQPLGQWHLS (SEQ ID NO:1) peptide such that the binding of one antibody does not inhibit the binding of the other antibody. Reactivities of representative antibodies are listed in Table 5:

Example 3 Detection of QSOXl peptide NEQEQPLGQWHLS in Subtypes of Pancreatic cancer Using the inhibition ELISA methods disclosed above in Example 1, we analyzed samples from patients with pancreatic islet cell tumors. Of the 6 islet cell cancer plasmas, QSOXl peptide is elevated in 3 of them (50%).

References

1. Anderson, N. L., and Anderson, N. G., The human plasma proteome: history, character, and diagnostic prospects. MoI. Cell. Proteomics 2002, 1, 845-867.

2. Hutter G, S. P., Proteomics for studying cancer cells and the development of chemoresistance. Proteomics 2001, 1, 1233-48.

3. Tissot, J. D., Vu D. H., Aubert, V., Schneider, P., Vuadens, F., Crettaz, D., Duchosal, M. A., The immunoglobulinopathies: from physiopathology to diagnosis. Proteomics 2002, 2, 813-24.

4. Sasaki, K., Sato, K., Akiyama, Y., Yanagihara, K., Oka, M., Yamaguchi,; K., Peptidomics-based approach reveals the secretion of the

29-residue COOH-terminal fragment of the putative rumor suppressor protein DMBTl from pancreatic adenocarcinoma cell lines. Cancer Res. 2002, 62, 4894— 4898.

5. Kennedy, S., The role of proteomics in toxicology: identification of biomarkers of toxicity by protein expression analysis. Biomarkers 2002, 7, 269-290.

6. Tirumalai, R. S., Chan, K. C, Prieto, D. A., Issaq, H. J., Conrads, T. P., Veenstra, T. D., Characterization of the low molecular weight human serum proteome. MoI Cell Proteomics 2003, 2, 1096-103.

7. Adkins, J. N., Varnum, S. M., Auberry, K. J., Moore, R. J., Angell, N. H., Smith, R. D., Springer, D. L., and Pounds, J. G., Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry. MoI. Cell. Proteomics 2002, 1, 947-955.

8. Koopmann J, R. C, Zhang Z, Canto MI, Brown DA, Hunter M, Yeo C, Chan DW, Breit SN, Goggins M., Serum markers in patients with resectable pancreatic adenocarcinoma: macrophage inhibitory cytokine 1 versus CA 19-9. Clin Cancer Res. 2006, 12, 442-446.

9. Grossklaus, D. J., Smith, J. A., Shappell, S. B., Coffey, C. S., Chang, S. S.,; and Cookson, M. S., The free/total prostate-specific antigen ratio (%fPSA) is the best predictor of tumor involvement in the radical prostatectomy specimen among men with an elevated PSA. Urol. Oncol. 2002, 7, 195-198.

10. Whitehouse, C, and Solomon, E., Current status of the molecular characterization of the ovarian cancer antigen CA125 and implications for its use in clinical screening. Gynecol. Oncol. 2003, 88, S152-S157. 11. Cloonan, M. J., Bishop, G. A., Wilton-Smith, P. D., Carter, I. W., Allan,; R. M., a. W., D. E., An enzyme-immunoassay for myoglobin in human serum and urine. Method development, normal values and application to acute myocardial infarction. Pathology 1979, 11, 689-699. 12. Wu, S. -L., Choudhary, G., Ramstrom, M., Bergquist, J., and Hancock,; S., W., Evaluation of shotgun sequencing for proteomic analysis of human plasma using HPLC coupled with either ion trap or Fourier transform mass spectrometry. J. Proteome Res. 2003, 2, 383-393. 13. Pieper, R., Gatlin, C. L., Makusky, A. J., Russo, P. S., Schatz, C. R., Miller, S. S., Su, Q. McGrath, A. M., Estock, M. A., Parmar, P. P., Zhao, M., Huang, S.-T., Zhou, J., Wang, F., Esquer-Blasco, R., Anderson, N. L., Taylor, J., Steiner, S., The human serum proteome: Display of nearly 3700 chromatographically separated protein spots on two- dimensional electrophoresis gels and identification of 325 distinct proteins. Proteomics 2003, 3, 1345-1364. 14. Shen, Y., Jacobs, J. M, Camp, D. G., II, Fang, R., Moore, R. J., and Smith, R. D., Anal. Chem. 2004, 76, 1134-1144.

15. Hinerfeld, D., Innamorati, D., Pirro, J., and Tarn, S. W., Serum/plasma depletion with chicken immunoglobulin Y antibodies for proteomic analysis from multiple mammalian species. Journal of Biomolecular Techniques 2004, 15, 184—190. 16. Cho, S. Y., Lee, E.-Y., Lee, J. S., Kim, H.-Y., Park, J. M., Kwon, M.-S., Park, Y.- K., Lee, H.-J., Rang, M.-J., Kim, J. Y., Yoo, J. S., Park, S. J., Cho, J. W., Kim, H.-S. and Paik, Y.-K., Efficient prefractionation of low-abundance proteins in human plasma and construction of a two-dimensional map. Proteomics 2005, 5, 3386-3396.

17. Qian, W.-J., Jacobs, J. M., Camp, D. G., II, Monroe, M. E., Moore, R. J., Gritsenko, M. A., Calvano, S. E., Lowry, S. F., Xiao, W., Moldawer, L. L., Davis, R. W.,

Tompkins, R. G. and Smith, R. D., Comparative proteome analyses of human plasma following in vivo lipopolysacchari.de administration using multidimensional separations coupled with tandem mass spectrometry. Proteomics 2005, 5, 572-584.

18. Sato, A. K., Sexton, D. J., Morganelli, L. A., Cohen, E. H., Wu, Q. L., Conley, G. P., Streltsova, Z., Lee, S. W., Devlin, M., DeOliveira, D. B., Enright, J., Kent, R. B.,

Wescott, C. R., Ransohoff, T. C, Ley, A. C, and Ladner, R. C, Development of mammalian serum albumin affinity purification media by peptide phage display. Biotechnol. Prog. 2002, 18, 182-192. 19. Villanueva, J., Shaffer, D. R., Philip, J., Chaparro, C. A., Bromage, H. E., Olshen, A. B., Fleisher, M., Lilja, H., Brogi, E., Boyd, J., Sanchez-Carbayo, M., Holland, E. C, Cordon-Cardo, C, Scher, H. L, and Tempst, P., Differential exoprotease activities confer tumor-specific serum peptidome patterns. J. Clin. Invest. 2006, 116, 271-284. 20. States, D. J., Omenn, G. S., Blackwell, T. W., Fermin, D., Eng, J., Speicher, D. W., and Hanash, S. M., Challenges in deriving high-confidence protein identifications from data gathered by a HUPO plasma proteome collaborative study. Nat. Biotechnol. 2006, 24, 333-338.

21. Qian, W. J., Kaleta, D. T., Petritis, B. O., Jiang, H., Liu, T., Zhang, X., Mottaz, H. M., Varnurn, S. M., Camp, D. G. 2nd, Huang, L., Fang, X., Zhang, W. W., Smith, R. D.,

Enhanced detection of low abundance human plasma proteins using a tandem IgY 12 SuperMix immunoaffϊnity separation strategy. MoI. Cell. Proteomics. 2008, 7, 1963-73.

22. Hugosson, J., Aus, G., Lilja, H., Lodding, P., PiM, C. G., Pileblad, E., Prostate specific antigen based biennial screening is sufficient to detect almost all prostate cancers while still curable. J. Urol. 2003, 169, 1720-1723.

23. Ghosh, A., Wang, X., Klein, E., and Heston, W.D., Novel role of prostate-specific membrane antigen in suppressing prostate cancer invasiveness. Cancer Res. 2005, 65, 727-731.

24. Richter, R., Schulz-Knappe, P., Schrader, M., Standker, L., Jϋrgens, M., Tammen, H., and Forssmann, W. G., Composition of the peptide fraction in human blood plasma: database of circulating human peptides. J. Chromatogr. B Biomed. ScL Appl. 1999, 726, 25-35.

25. Koomen, J. M., Li, D., Xiao, L. C, Liu, T. C, Coombes, K. R., Abbruzzese, J., Kobayashi, R., Direct tandem mass spectrometry reveals limitations in protein profiling experiments for plasma biomarker discovery. J. Proteome Res. 2005, 4, 972-981.

26. Villanueva, J., Philip, J., Chaparro, C. A., Li, Y., Toledo-Crow, R., DeNoyer, L., Fleisher, M., Robbins, R. J., Tempst, P., Correcting common errors in identifying cancer- specific serum peptide signatures. J. Proteome Res. 2005, 4, 1060-1072.

27. Davis, M. T., Auger, P., Spahr, C. and Patterson, S. D., Cancer biomarker discovery via low molecular weight serum proteome profiling — Where is the tumor?

Proteomics Clin. Appl. 2007, 1, 1545-1558.

28. Villanueva, J., Philip, J., Entenberg, D., Chaparro, C. A., Tanwar, M. K., Holland, E. C, Tempst. P., Serum peptide profiling by magnetic particle-assisted, automated sample processing and MALDI-TOF mass spectrometry. Anal. Chem. 2004, 76, 1560- 1570.

29. Orvisky, E., Drake, S. K., Martin, B. M., Abdel-Hamid, M., Ressom, H. W., Varghese, R. S., An, Y., Saha, D., Hortin, G. L., Loffredo, C. A., and Goldman, R., Enrichment of low molecular weight fraction of serum for MS analysis of peptides associated with hepatocellular carcinoma. Proteomics 2006, 6, 2895-902.

30. Bergen, H. R., Ill, Vasmatzis, G., Cliby,W. A., Johnson, K. L., Oberg, A. L., and Muddiman, D. C, Discovery of ovarian cancer biomarkers in serum using NanoLC electrospray ionization TOF and FT-ICR mass spectrometry. Dis. Markers 2003-2004, 19, 239-249.

31. Yu, J. K., Chen, Y. D., and Zheng, S., An integrated approach to the detection of colorectal cancer utilizing proteomics and bioinformatics. World J. Gastroenterol. 2004, 10, 3127-3131.

32. Engwegen, J. Y., Helgason, H. H., Cats, A., Harris, N., Bonfrer, J. M., Schellens, J. H., Beijnen, J. H., Identification of serum proteins discriminating colorectal cancer patients and healthy controls using surfaceenhanced laser desorption ionisation-time of flight mass spectrometry. World J. Gastroenterol. 2006, 12, 1536-1544.

33. Pang, R. T., Poon, T. C, Chan, K. C, Lee, N. L., Chiu, R. W., Tong, Y. K., Wong, R. M., Chim, S. S., Ngai, S. M., Sung, J. J., Lo, Y. M., Serum proteomic fingerprints of adult patients with severe acute respiratory syndrome. CHn. Chem. 2006, 52, 421-429.

34. Gillette, M. A., Mani, D. R., and Carr, S. A., Place of pattern in proteomic biomarker discovery. J. Proteome Res. 2005, 4, 1143— 1154.

35. Coombes, K. R., Morris, J. S., Hu, J., Edmonson, S. R., and Baggerly, K. A., Serum proteomics profiling-a young technology begins to mature. Nat. Biotechnol. Prog.

2005, 23, 291-292.

36. Diamandis, E. P., Mass spectrometry as a diagnostic and a cancer biomarker discovery tool: opportunities and potential limitations. MoI. Cell. Proteomics 2004, 3, 367-378. 37. Check, E., Proteomics and cancer: running before we can walk? Nature 2004, 429, 496-497.

38. Ransohoff, D. F., Opinion: bias as a threat to the validity of cancer molecular- marker research. Nat. Rev. Cancer 2005, 5, 142-149. 39. Abmayr, S. M.; Tingting, Y.; Parmely, T.; J.L., W., Preparation of Nuclear and Cytoplasmic Extracts from Mammalian Cells. . Curr. Protoc. MoI. Biol. 2006, Chapter 12, (Unit 12.1).

40. Klammer, A. A.; MacCoss, M. J., Effects of modified digestion schemes on the identification of proteins from complex mixtures. J Proteome Res 2006, 5, (3), 695-700.

41. Biankin, A. V., Kench, J. G., Biankin, S. A., Lee, C. S., Morey, A. L., Dijkman, F. P., Coleman, M. J., Sutherland, R. L., Henshall, S. M., Pancreatic intraepithelial neoplasia in association with intraductal papillary mucinous neoplasms of the pancreas: implications for disease progression and recurrence. Am. J. Surg. Pathol. 2004, 28, 1184- 1192.

42. Hruhan, R. H., Takaori, K., Klimstra, D. S., Adsay, N. V., Albores-Saavedra, J., Biankin, A. V., Biankin, S. A., Compton, C, Fukushima, N., Furukawa, T., Goggins, M., Kato, Y., Klόppel, G., Longnecker, D. S., Lϋttges, J., Maitra, A., Offerhaus, G. J., Shimizu, M., Yonezawa, S., An illustrated consensus on the classification of pancreatic intraepithelial neoplasia and intraductal papillary mucinous neoplasms. Am. J. Surg. Pathol. 2004, 28, 977-987.

43. Thorpe, C, Hoober, K. L., Raje, S., Glynn, N. M., Burnside, J., Turi, G. K., Coppock, D. L., Sulfhydryl oxidases: emerging catalysts of protein disulfide bond formation in eukaryotes. Arch. Biochem. Biophys. 2002, 405, 1-12. 44. Coppock, D. L.; Cina-Poppe, D.; Gilleran, S., The quiescin Q6 gene (QSCN6) is a fusion of two ancient gene families: thioredoxin and ERVl. Genomics 1998, 54, (3), 460-

8.

45. Coppock, D. L.; Thorpe, C, Multidomain flavin-dependent sulfhydryl oxidases.

Antioxidants & redox signaling 2006, 8, (3-4), 300-11. 46. Morel, C, Adami, P., Musard, J. F., Duval, D., Radom, J., Jouvenot, M.,

Involvement of sulfhydryl oxidase QSOXl in the protection of cells against oxidative stress-induced apoptosis. Exp. Cell Res. 2007, 313, 3971-3982.

(47) Rau, J. C, Beaulieu, L. M., Huntington, J. A., and Church, F. C. . (2007) Serpins in thrombosis, hemostasis and fibrinolysis. J. Thromb. Haemost. 1, 102-115.

Claims

We claim:

1. A method for assessing the probability of a pancreatic tumor in a subject, comprising analyzing a tissue sample of the subject for one or more peptides derived from one or both of QSOXl and SerpinF2, wherein an increased amount of the one or more peptides relative to a control correlates with a probability of a pancreatic tumor in the subject.

2. The method of claim 1 wherein the subject is identified as having one or more risk factors for pancreatic cancer prior to analysis of the tissue sample.

3. The method of claim 2, wherein the one or more risk factors are selected from the group consisting of: intraductal papillary mucinous neoplasm (IPMN), jaundice, pain in the upper abdomen, significant weight loss, age 60 years or more, male gender, African ethnicity, smoker, obesity, diabetes, chronic pancreatitis, Heliobacter pylori infection, family history of pancreatic cancer, autosomal recessive ataxia-telangiectasia, autosomal dominantly inherited mutations in the BRCA2 gene, Peutz-Jeghers syndrome due to mutations on the STKl 1 tumor suppressor gene, hereditary non-polyposis colon cancer (Lynch syndrome), familial adenomatous polyposis, familial atypical multiple mole melanoma-pancreatic cancer syndrome (FAMMM-PC) due to mutations in the CDKN2A tumor suppressor gene, gingivitis or periodontal disease, and alcoholism.

4. The method of any one of claims 1-3, wherein the method comprises analyzing the tissue sample of the subject for peptides of 3 kD or less.

5. The method of claim 4, wherein the method comprises isolating peptides of 3 kD or less from the tissue sample.

6. The method of claim 5, wherein the analyzing comprises a technique selected from the group consisting of mass spectrometry (MS), two dimensional gel electrophoresis, Western blotting, immunofluorescence, ELISAs, antigen capture assays (including dipstick antigen capture assays) and mass spec immunoAssay (MSIA).

7. The method of any one of claims 1 -6, wherein the one or more peptides are selected from the group consisting of NEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 1), NEQEQPLGQWH (QSOXl) (SEQ ID NO: 2), EQPLGQWHLS (QSOXl) (SEQ ID NO: 3), AAPGQEPPEHMAELQR (QSOXl) (SEQ ID NO: 4), AAPGQEPPEHMAELQ (QSOXl) (SEQ ID NO: 5), AAPGQEPPEHMAELQRNEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 6), NQEQVSPLTLLKLGN (SerpinF2) (SEQ ID NO: 7), NQEQVSPLTLLK (SerpinF2) (SEQ ID NO: 8), MEPLGRQLTSGP (SerpinF2) (SEQ ID NO: 9), NEQEQPL (SEQ ID NO: 10) (QSOXl), and GQWHLS (SEQ ID NO: 11) (QSOXl).

8. The method of any one of claims 1-7, where assessing the probability of a pancreatic tumor comprises determining a probability that the subject will develop a pancreatic tumor.

9. The method of any one of claims 1 -7, where assessing the probability of a pancreatic tumor comprises determining a probability that the subject has a pancreatic tumor.

10. A method for monitoring efficacy of treatment of a pancreatic tumor in a subject, comprising analyzing a tissue sample of a subject being treated for a pancreatic tumor for one or more peptides derived from one or both of QSOXl and SerpinF2, wherein the analyzing is carried out after one or more tumor treatments, and wherein a decrease in amount of the one or more peptides in the tissue sample after tumor treatment compared to an amount of the one or more peptides in a tissue sample from the subject after diagnosis of the pancreatic tumor but before tumor treatment began correlates with an effective tumor treatment.

11. The method of claim 11 , wherein the method comprises analyzing the tissue sample of the subject for peptides of 3 kD or less.

12. The method of claim 11 , wherein the method comprises isolating peptides of 3 kD or less from the tissue sample.

13. The method of claim 12, wherein the analyzing comprises a technique selected from the group consisting of mass spectrometry (MS), two dimensional gel electrophoresis, Western blotting, immunofluorescence, ELISAs, antigen capture assays (including dipstick antigen capture assays) and mass spec immunoAssay (MSIA).

14. The method of any one of claims 11-13, wherein the one or more peptides are selected from the group consisting of NEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 1), NEQEQPLGQWH (QSOXl) (SEQ ID NO: 2), EQPLGQWHLS (QSOXl) (SEQ ID NO: 3), AAPGQEPPEHMAELQR (QSOXl) (SEQ ID NO: 4), AAPGQEPPEHMAELQ (QSOXl) (SEQ ID NO: 5), AAPGQEPPEHMAELQRNEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 6), NQEQVSPLTLLKLGN (SerpinF2) (SEQ ID NO: 7), NQEQVSPLTLLK (SerpinF2) (SEQ ID NO: 8), MEPLGRQLTSGP (SerpinF2) (SEQ ID NO: 9), NEQEQPL (SEQ ID NO: 10) (QSOXl), and GQWHLS (SEQ ID NO: 11) (QSOXl).

15. An isolated peptide, selected from the group consisting of NEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 1), NEQEQPLGQWH (QSOXl) (SEQ ID NO: 2), EQPLGQWHLS (QSOXl) (SEQ BD NO: 3), AAPGQEPPEHMAELQR (QSOXl) (SEQ ID NO: 4), AAPGQEPPEHMAELQ (QSOXl) (SEQ ID NO: 5), AAPGQEPPEHMAELQRNEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 6),

NQEQVSPLTLLKLGN (SerpinF2) (SEQ ID NO: 7), NQEQVSPLTLLK (SerpinF2) (SEQ ID NO: 8), MEPLGRQLTSGP (SerpinF2) (SEQ ID NO: 9), NEQEQPL (SEQ ID NO: 10) (QSOXl), and GQWHLS (SEQ ID NO: 11) (QSOXl).

16. An isolated ligand, wherein the ligand selectively binds to a peptide selected from the group consisting of NEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 1), NEQEQPLGQWH (QSOXl) (SEQ ID NO: 2), EQPLGQWHLS (QSOXl) (SEQ ID NO: 3), AAPGQEPPEHMAELQR (QSOXl) (SEQ ID NO: 4), AAPGQEPPEHMAELQ (QSOXl) (SEQ ID NO: 5), AAPGQEPPEHMAELQRNEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 6), NQEQVSPLTLLKLGN (SerpinF2) (SEQ ID NO: 7),

NQEQVSPLTLLK (SerpinP2) (SEQ ID NO: 8), MEPLGRQLTSGP (SerpinF2) (SEQ ID NO: 9), NEQEQPL (SEQ ID NO: 10) (QSOXl), and GQWHLS (SEQ ID NO: 11) (QSOXl).

17. The isolated ligand of claim 16, wherein the ligand is selected from the group consisting of antibodies, antibody fragments, and aptamers.

18. A method for detecting the presence of a peptide selected from the group consisting of NEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 1), NEQEQPLGQWH (QSOXl) (SEQ ID NO: 2), EQPLGQWHLS (QSOXl) (SEQ ID NO: 3), AAPGQEPPEHMAELQR (QSOXl) (SEQ ID NO: 4), AAPGQEPPEHMAELQ (QSOXl) (SEQ ID NO: 5), AAPGQEPPEHMAELQRNEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 6), NQEQVSPLTLLKLGN (SerpinF2) (SEQ ID NO: 7), NQEQVSPLTLLK (SerpinF2) (SEQ ID NO: 8), MEPLGRQLTSGP (SerpinF2) (SEQ ID NO: 9), NEQEQPL (SEQ ID NO: 10) (QSOXl), and GQWHLS (SEQ ID NO: 11) (QSOXl), comprising

(a) contacting a sample to be tested with a ligand according to claim 16 or 17, under conditions in which the ligand selectively binds to its respective peptide to produce a binding complex; and

(b) detecting presence of the binding complex, wherein the presence of the binding complex indicates that the peptide is present in the test sample.