WO2010071787A1 - Marqueurs du cancer du pancréas et leurs utilisations - Google Patents

Marqueurs du cancer du pancréas et leurs utilisations Download PDF

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Publication number
WO2010071787A1
WO2010071787A1 PCT/US2009/068235 US2009068235W WO2010071787A1 WO 2010071787 A1 WO2010071787 A1 WO 2010071787A1 US 2009068235 W US2009068235 W US 2009068235W WO 2010071787 A1 WO2010071787 A1 WO 2010071787A1
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Prior art keywords
pancreatic
qsoxl
tumor
antibody
plasma
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PCT/US2009/068235
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English (en)
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Douglas F. Lake
Kwasi Antwi
Galen Hostetter
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Arizona Board Regents, A Body Corporate Acting For And On Behalf Of Arizona State University
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Publication of WO2010071787A1 publication Critical patent/WO2010071787A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • Pancreatic cancer 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 be 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.
  • the present invention provides methods for assessing the probability of a pancreatic tumor in a subject, comprising
  • the present invention provides methods for monitoring efficacy of treatment of a pancreatic tumor in a subject, comprising
  • FIG. 1 Size Exclusion Chromatography of Plasma Peptidome.
  • a calibration plot (log MW versus retention volume) was generated from running bovine serum albumin, ovalbumin, ribonuclease, aprotinin, and vitamin B12 through the size exclusion column (figure IA, inset). The plot yielded an R 2 value of 0.998, which indicates good linearity between MW and retention volume.
  • Figure IB represents the size-exclusion chromatogram of 0.45 ⁇ m filtered normal donor plasma, which was not different from chromatogram of pancreatic cancer patient plasma. The retention times as well as the measured molecular weights of all peaks in the plasma chromatogram are summarized in the table adjacent to the chromatogram.
  • FIG. 1 Stability of Plasma Peptidome. Plot showing stability of ⁇ 3 kDa plasma peptidome over time. Peak areas were obtained from size exclusion chromatogram as shown in figure IB. 24rt and 24ct represent whole blood stored at room temperature and cool temperature (4 0 C), respectively. Note that peptides are classified as plasma components with molecular weight ⁇ 3 kDa and proteins are classified as components with molecular weights >10 kDa. Graph represents 1 of 3 different experiments.
  • FIG. 3 Classification of peptidome proteins according to Gene Ontology algorithm (39) (ebi.ac.uk/GOA, version 55.0). Peptides corresponding to a total of 259 different parent proteins were identified among 42 normal donors and 21 pancreas cancer patients. Top 25 were re-classified into 16 listed in the pie chart (e.g. cytosol and cytoplasm were placed into the same category, etc.) Figure 4. Tandem mass spectra of QSOXl peptide (+2 charge state). Mass spectra from (A) peptide identified from ⁇ 3kDa plasma peptidome and (B) Chemically re- synthesized peptide. Y and b ions are labeled with arrows.
  • B Peri-pancreatic lymph node from a patient with ductal adenocarcinoma. Arrow indicates staining of a micrometastasis in the lymph node.
  • C High magnification of cytoplasmic QSOXl staining of ductal adenocarcinoma 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.
  • 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 the presence of the one or more peptides 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.
  • the inventors have identified peptides from QSOXl and SerpinF2 in patients with pancreatic cancer.
  • 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 QSOXl and/or SerpinF2 can be used as a predictor of which patients might progress to develop ductal adenocarcinoma of the pancreas (DAP).
  • the pancreatic tumor may be of any type; in one embodiment, the pancreatic tumor is an adenocarcinoma, such as a ductal adenocarcinoma.
  • the pancreatic tumor is 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: 12), and the accession number for the short form is NP_001004128 (SEQ ID N0:13).
  • SerpinF2 is Serine protease inhibitor 2, also called 2 anti-plasmin (SEQ ID NO: 14).
  • the protein accession number on the NCBI database is NP_000925.
  • 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.
  • the one or more peptides are selected from the group consisting of NEQEQPLGQWHLS (QSOXl) (SEQ ID NO: 1), NEQEQPLGQWH (QSOXl) (SEQ ID NO: T), 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 tissue sample may be any suitable sample from which tumor-derived peptides may be obtained.
  • the tissue sample is selected from the group consisting of plasma, serum, urine, saliva, cells from fine needle aspirate, and pancreatic juice.
  • the tissue sample comprises blood plasma.
  • Methods for preparing blood plasma are well known in the art; such methods include those described below.
  • 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.
  • techniques that can be used in the analysis include mass spectrometry (MS), two dimensional gel electrophoresis, ELISAs, antigen capture assays (including dipstick antigen capture assays) and mass spec immunoassay (MSIA).
  • 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.
  • 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.
  • MS 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.
  • the analysis is performed on a sample of peptides of 3 IcD or less isolated from the tissue sample.
  • use of an antigen capture assay preferably comprises use of ligands that selectively bind to a peptide selected from the group consisting of NEQEQPLGQ WHS (QSOXl) (SEQ ID NO: 1), 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 N0: % 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).
  • selective 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.
  • a complex solution such as a sample of isolated peptides of 3 kD or less from a tissue sample.
  • 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.
  • the subject can be any mammal, preferably a human.
  • the subject is identified as having one or more risk factors for pancreatic cancer prior to analysis of the blood sample.
  • 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 (PMN), 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.
  • PMN intraductal papillary muci
  • 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.
  • early treatment interventions chemotherapy, radiation therapy, etc.
  • 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.
  • the methods of the invention lead to earlier prognosis, diagnosis, and/or treatment of pancreatic cancer, which will lead to improved patient prognosis.
  • 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 CA19-9 in the subject's serum or other suitable tissue sample.
  • 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.
  • this aspect of the invention provides a valuable tool for monitoring effectiveness of treatments for pancreatic cancer.
  • the tissue sample may be any suitable sample from which tumor-derived peptides may be obtained.
  • the subject can be any mammal, preferably a human.
  • 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.
  • 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.
  • the analyzing comprises a technique selected from the group consisting of mass spectrometry (MS), two dimensional gel electrophoresis, antigen capture assays (including dipstick antigen capture assays) and mass spec immunoAssay (MSIA)
  • the one or more peptides are selected from the group consisting of N 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)
  • 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 (SerpinF2) (SEQ ID NO: 8), MEPLGRQLTSGP (SeipinF2) (SEQ ID NO: 9), NEQEQPL (SEQ ID NO: 10) (QSOXl), and GQWHLS
  • isolated peptides are useful, for example, as controls for the assays of the invention (ie: to verify identification of peptides in serum), as well as ligands 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 may be recombinantly produced or chemically synthesized.
  • 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 (N ⁇ -amino protected N ⁇ -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.
  • 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.
  • the polypeptides can have peptidomimetic bonds, such as ester bonds, to prepare peptides with novel properties.
  • a peptide may be generated that incorporates a reduced peptide bond, i.e., Ri-CH 2 -NH-R 2 , where Ri and R 2 are amino acid residues or sequences.
  • a reduced peptide bond may be introduced as a dipeptide subunit.
  • polypeptide would be resistant to protease activity, and would possess an extended half-live in vivo.
  • 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.
  • 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.
  • conventional adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, adjuvants, etc.
  • 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 allcanoic 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.
  • 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),
  • the ligands of the invention can be used, for example, in the antigen capture assays of the invention disclosed herein.
  • 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.
  • 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).
  • pre-immune serum is collected prior to the first immunization.
  • a peptide as disclosed herein (with a carrier if desired), together with an appropriate adjuvant, is 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.
  • the animals are bled, the serum collected, and aliquots are stored at about -20° C.
  • Polyclonal antibodies against the polypeptides 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 the polypeptides bound.
  • Monoclonal antibodies can be produced by obtaining spleen cells from the animal. (See Kohler and Milstein, Nature 256, 495-497 (1975)).
  • monoclonal antibodies (mAb) of interest are prepared by immunizing inbred mice with a polypeptide as disclosed herein, or portion 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 that allow 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.
  • DMEM Dulbecco's Modified Eagles Medium
  • 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.
  • a peptide as disclosed herein is typically formulated with a pharmaceutically acceptable carrier for parenteral administration.
  • 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 knowledge of those of skill of the art.
  • Antibodies can be fragmented using conventional techniques, and the fragments screened for utility in the same manner as described herein for whole antibodies.
  • 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 1 fragments.
  • 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), NQEQVSPLTLLKLGN (SerpinF2) (SEQ ID NO: 7), NQEQVSPLTLLK (SerpinF2) (SEQ ID NO: 8), MEPLGRQLTSGP (SerpinF2) (SEQ ID NO: 9), NEQEQPL (SEQ ID NO: 10) (QSOXl) (S
  • 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.
  • AU 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.
  • the tissue sample may be any suitable sample from which tumor-derived peptides may be obtained.
  • the tissue sample is selected from the group consisting of plasma, serum, urine, saliva, cells from fine needle aspirate, and pancreatic juice.
  • 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.
  • the analyzing comprises a technique selected from the group consisting of mass spectrometry (MS), two dimensional gel electrophoresis, antigen capture assays (including dipstick antigen capture assays) and mass spec immunoAssay (MSIA).
  • the present invention provides methods for assessing the probability of a pancreatic tumor in a subject, comprising
  • pancreatic tissue section between the antibody and QSOXl or Serpin F2, wherein the presence of immunocomplex formation in the pancreatic tissue section correlates with a probability of a pancreatic tumor in the subject.
  • the methods of this aspect of the invention are useful, for example, in assessing the probability of a pancreatic tumor in a patient.
  • the methods 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.
  • the inventors have demonstrated that immunohistochemical staining of tissue sections from patients with DAP showed remarkable tumor specificity and anti-QSOXl antibodies. It appears that pancreas tumor cells over-produce QSOXl, while normal adjacent pancreatic tissue, including normal ducts, do not express QSOXl.
  • antibody as used herein is intended to include polyclonal antibodies, monoclonal antibodies, and antibody-like proteins (antibody-like molecules) having an antibody structure (at least partially comprising a tetrameric heavy chain-light chain, heavy chain-light chain structure) such as chimeric antibodies, humanized antibodies, and fully human antibodies.
  • any suitable antibody that specifically binds QSOXl or SerpinF2 may be used in the methods of this aspect of the invention.
  • specific binding means that the antibodies recognize one or more epitope within the recited protein under suitable immunohistochemical conditions, but possess little or no detectable reactivity with other proteins under the same conditions, such as those disclosed herein.
  • antibodies against the peptides of the invention may be used The antibodies may be directed against any one or more epitopes within QSOXl or SerpinF2 so long as the antibodies retain specific binding for the protein in immunohistochenical staining.
  • the contacting occurs under conditions suitable to promote specific binding of the antibody to QSOXl or Serpin F2 while minimizing non-specific binding.
  • Any suitable steps wash steps may be used to remove unbound antibody prior to detection.
  • the antibody is labeled with a detectable marker, including but not limited to enzymes (such as peroxidase) that can catalyze a detectale reaction (typically based on color generation) and fluorophores (including but not limited to FITC, rhodamine, Texas Red, fluorescent proteins (GFP, EFP, etc.).
  • a detectable marker including but not limited to enzymes (such as peroxidase) that can catalyze a detectale reaction (typically based on color generation) and fluorophores (including but not limited to FITC, rhodamine, Texas Red, fluorescent proteins (GFP, EFP, etc.).
  • the antibodies are bound by secondary antibodies on which the detectable label is located.
  • pancreatic tissue section can be used, although thin sections are preferred (ie: 4 ⁇ m to 10 ⁇ m).
  • a pancreatic tissue section from a subject is placed in a fixative, and subsequently embedded in a mounting medium from which appropriate tissue sections are generated using any suitable device, such as a microtome.
  • the tissue section may be subjected to any desired treatments prior to contacting with the antibody, including but not limited to drying, dewaxing, rehydrating, heating, blocking, etc.
  • any desired treatments prior to contacting with the antibody including but not limited to drying, dewaxing, rehydrating, heating, blocking, etc.
  • One exemplary embodiment is provided in detail below.
  • the subject can be any mammal, preferably a human.
  • the subject has one or more risk factors for pancreatic cancer selected from the group consisting of: intraductal papillary mucinous neoplasm (PMN), 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.
  • PMN intraductal papillary mucinous
  • the methods can be used to assess the probability that the subject will develop a pancreatic tumor, or to assess the probability that the subject has a pancreatic tumor, as discussed in detail above.
  • the present invention provides methods for monitoring efficacy of treatment of a pancreatic tumor in a subject, comprising
  • this aspect of the invention provides a valuable tool for monitoring effectiveness of treatments for pancreatic cancer.
  • AU embodiments of the previous aspects of the invention are suitable in this aspect as well, unless the context dictates otherwise.
  • the subject can be any mammal, preferably a human.
  • 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.
  • Plasma is an excellent source of biomarkers to detect disease, but it is a highly complex bodily fluid.
  • LMW low molecular weight
  • LC-MS/MS liquid chromatography-tandem mass spectrometry
  • peptides in the plasma peptidome were identified from the QSOXl and SerpinF2 genes that were present in 19 of 21 patients with ductal adenomocarcinoma of the pancreas (DAP) and 5 of 5 patients with intraductal papillary mucinous neoplasm (PMN), a potentially pre-malignant condition.
  • DAP ductal adenomocarcinoma of the pancreas
  • PMN intraductal papillary mucinous neoplasm
  • 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 with albumin alone constituting about fifty-five percent (1, 4-6). The remaining one percent of the plasma proteome offers insights into normal versus pathological states.
  • a protein 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.
  • proteolytically derived peptides have been reported (6, 19, 24- 28), however, their use for disease diagnosis has been under explored.
  • fibrinogen A-derived peptides have been reported to correlate with hepatocellular carcinoma (29), ovarian carcinoma (30), colorectal carcinoma (31, 32), and severe acute respiratory syndrome (SARS) (33).
  • SARS severe acute respiratory syndrome
  • LC-MS grade water, acetonitrile, formic acid, and ammonium hydroxide were obtained from Fisher Scientific (Fair Lawn, NJ).
  • Ultrafilters with 3 kDa molecular weight cutoff cellulose membrane were obtained from Millipore Corporation (Bedford, MA).
  • HPLC autosampler micro vials were obtained from Agilent Technologies.
  • Evacuated plasma separation tubes containing potassium ethylenediaminetetraacetic acid (EDTA) were obtained from Fisher Scientific (Fair Lawn, NJ).
  • 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 as an anti-coagulant.
  • Blood samples were separated into plasma, lymphocytes and red cells by performing standard Ficoll blood separation. Approximately 3 to 5 ml of plasma were collected and placed into cryovials (Wheaton Science Products, Millville, NJ) and stored at -8O 0 C for analysis. Prior to LC-MS/MS analysis, frozen plasma was thawed on ice, filtered through 0.45 ⁇ m polyvinylidene fluoride (PVDF) membrane ultraf ⁇ lter (Millipore, Bedford, MA) and the filtrate was further passed through a 3 kDa filter with cellulose membrane (Millipore, Bedford, MA). Using NanoDrop spectrophotometer (Thermo Scientific), the concentrations of the 0.45 ⁇ m and 3 kDa filtrates were measured to be 64.5 ⁇ 0.8 mg/ml and 6.4 ⁇ 0.6 mg/ml, respectively.
  • PVDF polyvinylidene fluoride
  • 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 10 males and 16 females whose ages were between 34 and 84 years old.
  • Blood samples were collected into plasma separation tubes containing EDTA and shipped to us within 24 hours at ambient temperature (except in summer when cold packs were used). Blood samples were processed using Ficoll separation using the same methods as healthy donor plasma.
  • Blood plasma constituents were resolved on the HPLC system using high performance Superdex 75 10/300 GL column (GE Healthcare Biosciences, Piscataway, NJ) with optimum separation range for globular proteins (M 1 -) of 3000- 70000 and exclusion limit of 1.0 x 10 s .
  • the mobile phase contained 0.1 M sodium acetate at pH 7.2.
  • the pH of mobile phase was adjusted using ammonium hydroxide.
  • the plasma components were eluted at 0.4 ml/min and detected at wavelengths of 214 and 280 nm using a diode array detector. The analysis time for each plasma sample was 80 minutes.
  • Peaks that corresponded to MWCO greater than 10 kDa were considered “high molecular weight,” whereas peaks that corresponded to MWCO less than 5 kDa were considered “low molecular weight.”
  • the calibration plot used for measuring molecular weight of plasma components was generated from running a solution containing 8.0 mg/ml bovine serum albumin (67000 Da), 2.5 mg/ml ovalbumin (43000 Da), 5.0 mg/ml ribonuclease (13700 kDa), 2.0 mg/ml aprotinin (6512 Da), and 0.1 mg/ml vitamin B12 (1355 Da).
  • Plasma samples from pancreatic cancer patients and normal subjects were filtered through centricon centrifugal filter devices with MWCO of 3 kDa using Microspeedfuge SFRl 3K centrifuge (Savant Instruments, Farmingdale, NY) at 10000xg. The temperature of the centrifuge was always kept at 4 0 C. The filtrates were either analyzed immediately or transferred into siliconized tubes (VWR Scientific) and stored in -20 0 C until analysis. The filtrates were resolved on Agilent HPLC-Chip system, which uses Agilent 1100 Series HPLC-Chip Cube Interface (Agilent Technologies).
  • the Agilent HPLC-Chip integrates enrichment column (ZORBAX 300SB-C 18, 40 nl, 5 ⁇ m), analytical column (ZORBAX 300SB- Cl 8, 75 ⁇ m x 43 mm, 5 ⁇ m), and a nanospray emitter on a single and reusable microfluidic chip.
  • the Agilent 1100 Series system comprises of a capillary HPLC pump system, a nano-HPLC pump system, and a micro well-plate autosampler that was kept at 4 0 C throughout the experiment. The micro well-plate autosampler injected each sample while the capillary HPLC system pumped the mobile phase that carried the sample from the autosampler onto the enrichment column.
  • 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 300 nl/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 32O 0 C, respectively.
  • Mass spectrometer (MS) full scans were acquired from 350 to 2000 m/z in the ultra scan 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 reference sequence protein database using Spectrum Mill algorithm (Agilent Technologies).
  • MS/MS spectra with at least four detected peaks and sequence tag length greater than three were considered for sequence interpretation.
  • Precursor mass tolerance of ⁇ 2.5 Da was used.
  • 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.
  • SPI scored peak intensity
  • a false positive rate of less than 7% was obtained by running our MS/MS spectra against a decoy database that was generated by reshuffling sequences in the NCBI reference sequence protein database.
  • Synthetic peptides Unmodified peptides were synthesized in-house on a Symphony peptide synthesizer (Protein Technologies, Arlington, AZ). Peptide purity was estimated to be greater than 95%. Mass spectrometric analysis was used to confirm amino acid composition of peptides.
  • Plasma Peptidome Based on the diversity of plasma we initially thought that it would not be possible to distinctively resolve the plasma peptidome ( ⁇ 3 kDa molecules) from other plasma components. However, when 0.45um filtered plasma from normal healthy donors and pancreas cancer patients were run through a Superdex 75 10/300 GL molecular weight sieving column, we observed that ⁇ 3 kDa plasma components can be resolved from all other plasma components larger than 1OkDa (figure IB). SDS-PAGE analysis on peaks at 19.8 and 21.4 minutes showed multiple high molecular weight bands for each peak ranging from 15OkDa to 2OkDa including a very prominent band at ⁇ 67kDa which was likely albumin.
  • peptidome is not as diverse as the 'plasma proteome, we found peptides representing over 259 different proteins in the plasma peptidome among all donors. The origin of these peptides were not just secreted proteins, but from many different compartments of the cell.
  • GO Gene Ontology
  • Gene Ontology annotation of human proteins was downloaded from Gene Ontology Annotation (GOA) Databases (ebi.ac.uk/GOA, version 55.0). This GOA human database contains 33,731 distinct proteins and 172661 GO associations.
  • GO hierarchy information version 52 was downloaded from www.geneontology.com.
  • QSOXl and SerpinF2 are prominent peptides in plasma from patients with pancreatic ductal adenocarcinoma. Many peptides were identified in common among patients with a diagnosis of DAP. However, peptides corresponding to QSOXl and/or SerpinF2 proteins occurred at high frequency (19 of 21) in the LMW fraction of plasma from patients with DAP (Table IA). No peptide from either QSOXl or SerpinF2 was found in plasma from 42 normal donors using the same processing and detection methods. Other peptides such as complement C4A, apolipoprotein A2 and fibrinogen were commonly found in plasma from cancer patients as well as normal donors.
  • IPMN intraductal papillary mucinous neoplasm
  • Table 1 Detection of QSOXl and SerpinF2 peptides in plasma peptidome.
  • A LC- MS/MS analysis of ⁇ 3kDa fraction of plasma from surgically resectable patients diagnosed with ductal adenocarcinoma.
  • B LC-MS/MS analysis of ⁇ 3kDa 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.
  • QSOXl is a 747 amino acid long sulfhydryl oxidase that is thought to participate in redox reactions during intracellular protein folding (42). According to the Uniprot website, two isoforms exist for QSOXl. Conflicting reports on the expression and possible cellular function of QSOXl (43, 44), along with our findings suggest that it should be further studied. In our LC-MS/MS analysis, five peptides corresponding to the C-terminus of the QSOXl protein were identified, two parent peptides and three sequences contained within the parent peptides (Table 2).
  • NEQEQPLGQWHLS (SEQ ID NO:1) (amino acid 631-643) is from the C-terminus of QSOXl and occurred frequently (13 of 21) in patients with DAP and 4 of 5 patients with IPMN.
  • the other parent peptide, AAPGQEPPEHMAELQR 9SEQ ID N0:4) (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, 2 of 21, in DAP and 2 of 5 in IPMN plasmas as shown in Table 2.
  • 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 ⁇ g/ml. Despite reports of 70 ⁇ g/ml SerpinF2 concentration in plasma, we never detected SerpinF2 in the low molecular weight fraction of plasma from normal donors (45). Three peptides corresponding to SerpinF2 protein were identified and derived from the N terminal region of the protein. One of the peptides was contained within the NQEQVSPLTLLKLGN (SEQ ID NO:6) (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 PMN.
  • SerpinF2 is also called ⁇ -2 anti-plasmin. It is the main blood- derived inhibitor of fibrin clot-dissolving plasmin.
  • Anti-QSOXl antibodies did not stain a tissue section from a patient with chronic pancreatitis (figure 5D). 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). Staining for SerpinF2 was not successful. We are developing an IHC procedure to detect SerpinF2 to determine if it can be localized to tumor.
  • the first finding is the stability of the plasma peptidome.
  • the peptidome in whole blood stays relatively intact over a 24 hour time period post phlebotomy and is not "fed” by degradation of larger proteins.
  • we never observed peptides from abundant HMW proteins such as transferrin, immunoglobulins, myoglobulin, rantes, thyroglobulin, and complement proteins with the exception of C4a in the plasma peptidome.
  • transferrin immunoglobulins
  • myoglobulin myoglobulin
  • rantes thyroglobulin
  • QSOXl and SerpinF2 peptides were detected at time zero and at each time point during 24 hours incubation of whole blood at room temperature (data not shown).
  • One possibility for the lack of proteolysis of small and large proteins post phlebotomy is that blood was collected into EDTA-containing tubes. EDTA may chelate divalent cations necessary for proteases to function. Alternatively, most of the proteolytic activity could occur in vivo before blood is drawn.
  • the second finding is that peptides from QSOXl and SerpinF2 proteins were identified in the low molecular weight fraction of plasma from patients with DAP, but not in normal healthy donors using LC-MS/MS techniques. These results suggest that QSOXl and/or SerpinF2 could be plasma-based biomarkers. Although peptides from QSOXl and SerpinF2 were never found in normal healthy donor plasma, we plan to examine plasma from patients with benign pancreaticobiliary conditions and other neoplasms such as cholangiocarcinoma to determine if the presence of QSOXl and/or SerpinF2 can distinguish cancer from benign conditions and other malignant diseases.
  • QSOXl or SerpinF2 peptides in some patient plasma samples. This suggests that the peptides for QSOXl and SerpinF2 are either not present in some patient tumors or are present at concentrations too dilute to be reliably detected by LC-MS/MS. Although more sampling may reveal their presence, we are also developing antigen capture ELISAs to quantify QSOXl and SerpinF2 in plasma and correlate them with IHC staining intensity.
  • the antigen capture ELISAs can also be used to quantify levels of each biomarker and correlate them with stage of disease or even predict who may be at increased risk for developing DAP in patients whose pre-malignant IPMN stains with anti-QSOXl antibodies.
  • tumor cells secrete the entire protein or a fragment of the protein such that it can be detected in blood. It is interesting that only C-terminal peptides were detected and identified for QSOXl .
  • One explanation for this is that tumor cells cleave the C-terminal end of QSOXl 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 at an unknown location such that our method of examining only the ⁇ 3kDa fraction of plasma does not allow detection of the remainder of the molecule, or larger fragments of the proteolyzed protein.
  • the plasma peptidome is an excellent source to search for potential biomarkers.
  • peptides of interest were stable and did not get degraded even in whole blood after 24 hours in a blood collection tube.
  • we identified peptides corresponding to only 259 different proteins in the plasma peptidome it was diverse enough to detect tumor-derived peptides. This suggests that the plasma peptidome could be a supplemental source of peptides found in the plasma proteome.
  • simplifying plasma by analyzing a LMW fraction allows excellent fractionation by LC and identification of peptides that have thus far been obscured by HAP in plasma.
  • 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 colon and pancreas cancer
  • DAP Ductal Adenocarcinoma of the Pancreas
  • EDTA ethylenediaminetetraacetic acid
  • HAP Highly Abundant Proteins
  • IPMN Intraductal Papillary Mucinous Neoplasm kDa: kilodaltons
  • PSA Prostate Specific Antigen
  • PVDF polyvinylidene fluoride
  • SerpinF2 Serine protease inhibitor 2, also called ⁇ 2 anti-plasmin
  • Example 2 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%).
  • %fPSA free/total prostate-specific antigen ratio

Abstract

L’invention concerne des méthodes et des réactifs pour évaluer la probabilité d’un sujet d’avoir une tumeur pancréatique, et surveiller l’efficacité des traitements des tumeurs pancréatiques.
PCT/US2009/068235 2008-12-17 2009-12-16 Marqueurs du cancer du pancréas et leurs utilisations WO2010071787A1 (fr)

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WO2017072757A1 (fr) 2015-10-25 2017-05-04 Yeda Research And Development Co. Ltd. Anticorps ciblant la quiescine sulfhydryle oxydase (qsox1) et leurs utilisations
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