METHODS AND COMPOSITIONS FOR DETECTING PANCREATIC CANCER

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 13, 2021, is named 121940_00204_SL.txt and is 15,531,112 bytes in size.

FIELD OF THE INVENTION

The present invention relates to non-invasive methods for the diagnosis and prognosis of pancreatic cancer. In some embodiments, such methods and compositions relate to particular biomarkers and combinations thereof.

BACKGROUND OF THE INVENTION

Disorders associated with the gastrointestinal (GI) and hepatobiliary tracts and the organs/tissues associated with the gastrointestinal tract include cancers such as gastric cancer, esophageal cancer, liver cancer, and pancreatic cancer. Pancreatic cancer (e.g., pancreatic adenocarcinoma), in particular, is a malignant growth of the pancreas that mainly occurs in the cells of the pancreatic ducts. This disease is the ninth most common form of cancer, yet it is the fourth and fifth leading cause of cancer deaths in men and women, respectively. Cancer of the pancreas is almost always fatal, with a five-year survival rate that is less than 3%.

The most common symptoms of pancreatic cancer include jaundice, abdominal pain, and weight loss, which, together with other presenting factors, are often nonspecific in nature. Thus, diagnosing pancreatic cancer at an early stage of tumor growth is often difficult and requires extensive diagnostic work-up, often times incidentally discovered during exploratory surgery. Endoscopic ultrasonography is an example of a non-surgical technique available for diagnosis of pancreatic cancer. However, reliable detection of small tumors, as well as differentiation of pancreatic cancer from focal pancreatitis, is difficult. The vast majority of patients with pancreatic cancer are presently diagnosed at a late stage when the tumor has already extended beyond the pancreas to invade surrounding organs and/or has metastasized extensively. Gold et al., Crit. Rev. Oncology/Hematology, 39:147-54 (2001), incorporated herein by reference in its entirety. Late detection of the disease is common with the majority of patients being diagnosed with advanced disease often resulting in death; only a minority of patients are detected with early stage disease.

Invasive techniques to diagnose disorders and diseases related to the gastrointestinal tract are inconvenient and expose a subject to significant risk. Examples of non-invasive methods to identify patients with disorders of the gastrointestinal tract or associated organs/tissues are described in PCT/US2011/051269 filed Sep. 12, 2011 entitled “NON-INVASIVE METHODS OF DETECTING PANCREATIC CANCER BIOMARKERS” which is incorporated by reference herein in its entirety. Nonetheless, there remains a need for additional methods for the diagnosis and prognosis of disorders such as pancreatic cancer.

SUMMARY OF THE INVENTION

Diagnostic Methods

In one aspect, the present invention is directed to a method of assessing whether a subject is afflicted with pancreatic cancer, the method including determining the level of at least one pancreatic cancer biomarker in a sample derived from said subject; and comparing the level of the pancreatic cancer biomarker with the level of the pancreatic cancer biomarker in a control sample, wherein a difference between the level of the pancreatic cancer biomarker derived from said subject and the pancreatic cancer biomarker in the control sample is an indication that the subject is afflicted with pancreatic cancer.

In various embodiments, the pancreatic cancer biomarker is CA19-9 or a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1-31 and 38-793, or a fragment thereof. In certain embodiments, the pancreatic cancer biomarker is CA19-9 or a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1-19, 47, 49-58, 206, 726, 729, 780 or 793, or a fragment thereof. In a particular embodiment, pancreatic cancer biomarker is a nucleotide sequence encoding the protein or the fragment thereof. In another embodiment, the pancreatic cancer biomarker is CA19-9.

In various embodiments, the sample is selected from the group consisting of a fecal sample, a gastrointestinal lavage fluid, and a combination thereof. In a particular embodiment, the sample is gastrointestinal lavage fluid.

In certain embodiments, the method includes determining the level of at least 2 pancreatic cancer biomarkers and comparing the level of each of the pancreatic cancer biomarkers to the respective level of the pancreatic cancer biomarkers in the control sample. In a particular embodiment, the method includes determining the level of at least 3, 4, 6, 7, 8, 9 or 10 pancreatic cancer biomarkers and comparing the level of each of the pancreatic cancer biomarkers to the respective level of the pancreatic cancer biomarkers in the control sample.

In one embodiment, the subject is a human.

In various embodiments, the method involves administering a lavage fluid and collecting the sample, for example, a gastrointestinal lavage fluid. In a particular embodiment, the lavage fluid is administered orally. In a particular embodiment, the lavage fluid includes an ingredient selected from the group consisting of polyethylene glycol, magnesium sulfate, sodium sulfate, potassium sulfate, magnesium citrate, ascorbic acid, sodium picosulfate, and bisacodyl. For example, the lavage fluid is selected from the group consisting of GOLYTELY, HALFLYTELY, NULYTELY, SUPREP, FLEET'S PHOSPHO-SODA, magnesium citrate, and their generic equivalents. In a particular embodiment, the method further includes partially purging the subject's gastrointestinal system and collecting gastrointestinal lavage fluid.

In one embodiment, the difference is a decrease in the level of the pancreatic cancer biomarker derived from said subject and the pancreatic cancer biomarker in the control sample, and wherein said decrease is an indication that the subject is afflicted with pancreatic cancer. For example, the pancreatic cancer biomarker may be a protein encoded by an amino acid sequence selected from the group consisting of SEQ ID NOs:1-16, 49, 55-58, 206 and 793, or a fragment thereof. In a particular embodiment, the level of the pancreatic cancer biomarker derived from said subject is at least 3 times less than the level of the pancreatic cancer biomarker in the control sample. Alternatively, the level of the pancreatic cancer biomarker derived from said subject is at least 5, 10 or 100 times less than the level of the pancreatic cancer biomarker in the control sample.

In another embodiment, the difference is an increase in the level of the pancreatic cancer biomarker derived from said subject and the pancreatic cancer biomarker in the control sample, and wherein said increase is an indication that the subject is afflicted with pancreatic cancer. For example, the pancreatic cancer biomarker may be a protein encoded by an amino acid sequence selected from the group consisting of SEQ ID NOs:17-19, 47, 726, 729 or 780, or a fragment thereof. Alternatively, the pancreatic cancer biomarker may be CA19-9. In various embodiments, the level of the pancreatic cancer biomarker derived from said subject is at least 3 times more than the level of the pancreatic cancer biomarker in the control sample. In a particular embodiment, the level of the pancreatic cancer biomarker derived from said subject is at least 5, 10 or 100 times more than the level of the pancreatic cancer biomarker in the control sample.

In one embodiment, the pancreatic cancer biomarker is derived from the pancreas. Alternatively, the pancreatic cancer biomarker may be derived from elsewhere in the gastrointestinal tract, for example the intestine.

In certain embodiments, the pancreatic cancer is selected from the group consisting of an exocrine pancreatic cancer, a pancreatic cystic neoplasm and a pancreatic endocrine cancer. For example, the pancreatic cancer may be an exocrine pancreatic cancer selected from the group consisting of pancreatic ductal adenocarcinoma (PDAC), adenosquamous carcinoma, squamous cell carcinoma, giant cell carcinoma, acinar cell carcinoma and small cell carcinoma. In a particular embodiment, the pancreatic cancer is pancreatic ductal adenocarcinoma. Alternatively, the pancreatic cancer may be a pancreatic endocrine tumor selected from the group consisting of insulinomas, glucagonomas, somatostatinomas, gastrinomas, VIPomas and non-secreting islet tumors of the pancreas.

In various embodiments, determining the level of said at least pancreatic cancer biomarker includes performing an immunoassay or a colorimetric assay. For example, the immunoassay may be a Western blot, an enzyme linked immunoabsorbent assay (ELISA), and a radioimmunoassay. In a particular embodiment, the immunoassay is an ELISA.

Alternatively, determining the level of said at least pancreatic cancer biomarker includes performing mass spectrometry.

Alternatively, determining the level of said at least pancreatic cancer biomarker includes applying said sample to a solid phase test strip or a flow-through strip including an agent which selectively binds to said pancreatic cancer biomarker; and detecting said pancreatic cancer biomarker bound to said agent on said solid phase test strip or said flow-through strip.

In particular embodiments, the method further involves comparing the level of the pancreatic cancer biomarker from the subject with the level of at least control polypeptide, or fragment thereof, or a nucleic acid encoding said at least control polypeptide, derived from the sample. For example, the control polypeptide may be a non-pancreatic polypeptide that originates in the gastrointestinal tract. In a particular embodiment, the control polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:27, 32-40, 45, 54, 59 and 59, or a fragment thereof.

Prognostic Methods

In another aspect, the present invention is directed to a method of assessing the progression of pancreatic cancer in a subject afflicted with pancreatic cancer, by determining the level of at least one pancreatic cancer biomarker in a sample derived from said subject; and comparing the level of the pancreatic cancer biomarker with the level of the pancreatic cancer biomarker in a control sample, wherein a decrease in the level of the pancreatic cancer biomarker derived from said subject and the pancreatic cancer biomarker in the control sample is an indication that the pancreatic cancer will progress rapidly; and wherein an increase in the level of the pancreatic cancer biomarker derived from said subject and the pancreatic cancer biomarker in the control sample is an indication that the pancreatic cancer will progress slowly or will regress; optionally, wherein the pancreatic cancer biomarker is CA19-9, a protein encoded by an amino acid sequence selected from the group consisting of SEQ ID NOs:1-31 or 39-793, a fragment thereof, or a nucleotide sequence encoding the protein or fragment thereof. For example, the pancreatic cancer biomarker may be a protein encoded by an amino acid sequence selected from the group consisting of SEQ ID NOs:1-16, 49, 55-58, 206 and 793, a fragment thereof or a nucleotide sequence encoding the protein or the fragment thereof.

In another aspect, the present invention is directed to a method of assessing the progression of pancreatic cancer in a subject afflicted with pancreatic cancer, by determining the level of at least one pancreatic cancer biomarker in a sample derived from said subject; and comparing the level of the pancreatic cancer biomarker with the level of the pancreatic cancer biomarker in a control sample, wherein an increase in the level of the pancreatic cancer biomarker derived from said subject and the pancreatic cancer biomarker in the control sample is an indication that the pancreatic cancer will progress rapidly; and wherein a decrease in the level of the pancreatic cancer biomarker derived from said subject and the pancreatic cancer biomarker in the control sample is an indication that the pancreatic cancer will progress slowly or will regress; optionally, wherein the pancreatic cancer biomarker is CA19-9, a protein encoded by an amino acid sequence selected from the group consisting of SEQ ID NOs:1-31 or 39-793, a fragment thereof, or a nucleotide sequence encoding the protein or fragment thereof. For example, the pancreatic cancer biomarker may be CA19-9 or a protein encoded by an amino acid sequence selected from the group consisting of SEQ ID NOs:17-19, 47, 726, 729 or 780, a fragment thereof, or a nucleotide sequence encoding the protein or the fragment thereof.

In various embodiments of the foregoing aspects, the sample is selected from the group consisting of a fecal sample, a gastrointestinal lavage fluid, and a combination thereof. In a particular embodiment, the sample is gastrointestinal lavage fluid.

In certain embodiments of the foregoing aspects, the method includes determining the level of at least 2 pancreatic cancer biomarkers and comparing the level of each of the pancreatic cancer biomarkers to the respective level of the pancreatic cancer biomarkers in the control sample. In a particular embodiment, the method includes determining the level of at least 3, 4, 6, 7, 8, 9 or 10 pancreatic cancer biomarkers and comparing the level of each of the pancreatic cancer biomarkers to the respective level of the pancreatic cancer biomarkers in the control sample.

In one embodiment, the subject is a human.

In certain embodiments of the foregoing aspects, the decrease is at least 3, 5, 10 or 100 times less than the level of pancreatic cancer biomarker in the control sample. Alternatively, the increase is at least 3, 5, 10 or 100 times more than the level of pancreatic cancer biomarker in the control sample.

Alternatively, determining the level of said at least pancreatic cancer biomarker includes performing mass spectrometry.

Methods of Monitoring Treatment and Method of Treating

In another aspect, the present invention is directed to a method of monitoring the efficacy of treatment of pancreatic cancer in a subject suffering from pancreatic cancer, by determining the level of at least one pancreatic cancer biomarker in a sample derived from said subject, wherein said subject has been previously exposed to treatment for pancreatic cancer; and comparing the level of the pancreatic cancer biomarker with the level of the pancreatic cancer biomarker in a control sample, wherein a decrease in the level of the pancreatic cancer biomarker derived from said subject and the pancreatic cancer biomarker in the control sample is an indication that the treatment is not efficacious; and wherein an increase in the level of the pancreatic cancer biomarker derived from said subject and the pancreatic cancer biomarker in the control sample is an indication that the pancreatic cancer is efficacious; optionally, wherein the pancreatic cancer biomarker is CA19-9, a protein encoded by an amino acid sequence selected from the group consisting of SEQ ID NOs:1-31 or 39-793, a fragment thereof, or a nucleotide sequence encoding the protein or fragment thereof. For example, the pancreatic cancer biomarker may be a protein encoded by an amino acid sequence selected from the group consisting of SEQ ID NOs:1-16, 49, 55-58, 206 and 793, a fragment thereof or a nucleotide sequence encoding the protein or the fragment thereof.

In another aspect, the present invention is directed to a method of monitoring the efficacy of treatment of pancreatic cancer in a subject suffering from pancreatic cancer, by determining the level of at least one pancreatic cancer biomarker in a sample derived from said subject, wherein said subject has been previously exposed to treatment for pancreatic cancer; and comparing the level of the pancreatic cancer biomarker with the level of the pancreatic cancer biomarker in a control sample, wherein an increase in the level of the pancreatic cancer biomarker derived from said subject and the pancreatic cancer biomarker in the control sample is an indication that the treatment is not efficacious; and wherein a decrease in the level of the pancreatic cancer biomarker derived from said subject and the pancreatic cancer biomarker in the control sample is an indication that the pancreatic cancer is efficacious; optionally, wherein the pancreatic cancer biomarker is CA19-9, a protein encoded by an amino acid sequence selected from the group consisting of SEQ ID NOs:1-31 or 39-793, a fragment thereof, or a nucleotide sequence encoding the protein or fragment thereof For example, the pancreatic cancer biomarker may be CA19-9 or a protein encoded by an amino acid sequence selected from the group consisting of SEQ ID NOs:17-19, 47, 726, 729 or 780, a fragment thereof, or a nucleotide sequence encoding the protein or the fragment thereof.

In another aspect, the present invention is directed to a method of treating a subject having pancreatic cancer, by determining the level of at least one pancreatic cancer biomarker in a sample derived from said subject; and comparing the level of the pancreatic cancer biomarker with the level of the pancreatic cancer biomarker in a control sample, wherein a difference between the level of the pancreatic cancer biomarker derived from said subject and the pancreatic cancer biomarker in the control sample is an indication that the subject is afflicted with pancreatic cancer; and exposing said subject to therapeutically effective treatment, thereby treating the subject having pancreatic cancer; optionally, wherein the pancreatic cancer biomarker is CA19-9, a protein encoded by an amino acid sequence selected from the group consisting of SEQ ID NOs:1-31 or 39-793, a fragment thereof, or a nucleotide sequence encoding the protein or fragment thereof.

In various embodiments of the foregoing aspects of the invention, the treatment is selected from the group consisting of surgery, radiation, chemotherapy or a combination thereof. For example, surgery may comprise the Whipple procedure, total pancreatectomy, distal pancreatectomy, surgical biliary bypass, endoscopic stent placement or gastric bypass. Alternatively, treatment may consist of administration of agents for treatment including, for example, tyrosine kinase inhibitors (TKIs) such as Erlotinib.

In one embodiment, the subject is a human.

Alternatively, determining the level of said at least pancreatic cancer biomarker includes performing mass spectrometry.

Kit

In a further aspect, the present invention is directed to a kit for determining the presence, absence or progression of pancreatic cancer in a subject including an agent that selectively binds to at least one pancreatic cancer biomarker.

For example, the pancreatic cancer biomarker may be CA19-9 or a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs:1-31 or 39-793, or a fragment thereof. In a particular embodiment, the pancreatic cancer biomarker is CA19-9 or a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1-19, 47, 49-58, 206, 726, 729, 780 or 793. In a particular embodiment, the pancreatic cancer biomarker is a nucleotide sequence encoding the foregoing protein.

In a particular embodiment, the kit includes at least two agents that selectively bind to at least one pancreatic cancer biomarker. For example, the kit can include at least three, four or five agents that selectively bind to at least one pancreatic cancer biomarker. In a particular embodiments, the agent is an antibody or antigen-binding fragment thereof. In certain embodiments the agent is attached to a solid support, such as a solid phase test strip or a flow-through test strip. In further embodiments, the kit includes a detectable agent which selectively binds to said pancreatic cancer biomarker.

In various embodiments, the kit includes a lavage fluid for oral administration to a subject and, optionally, a vessel for collecting the gastrointestinal lavage fluid from the subject.

Compositions

Some compositions and methods provided herein include an isolated polypeptide consisting essentially of an amino acid sequence selected from the group consisting of a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NOs:1-31 or 39-793 or a fragment thereof, wherein said polypeptide is differentially expressed in cancer.

Some compositions and methods provided herein include an isolated nucleic acid encoding a polypeptide consisting essentially of an amino acid sequence selected from the group consisting of a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NOs:1-31 or 39-793 or a fragment thereof, wherein said polypeptide is differentially expressed in cancer.

Some compositions and methods provided herein include an isolated polypeptide consisting of an amino acid sequence selected from the group consisting of a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NOs:1-31 or 39-793 or a fragment thereof, wherein said polypeptide is differentially expressed in cancer.

Some compositions and methods provided herein include an isolated nucleic acid encoding a polypeptide consisting of an amino acid sequence selected from the group consisting of a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NOs:1-31 or 39-793 or a fragment thereof, wherein said polypeptide is differentially expressed in cancer.

Some compositions and methods provided herein include an isolated agent that selectively binds to an isolated polypeptide consisting essentially of an amino acid sequence selected from the group consisting of a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NOs:1-31 or 39-793 or a fragment thereof, wherein said polypeptide is differentially expressed in cancer. In some embodiments, the agent comprises an antibody or antigen-binding fragment thereof.

Some compositions and methods provided herein include an isolated agent that selectively binds to an isolated polypeptide consisting of an amino acid sequence selected from the group consisting of a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NOs:1-31 or 39-793 or a fragment thereof, wherein said polypeptide is differentially expressed in cancer. In some embodiments, the agent comprises an antibody or antigen-binding fragment thereof.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts the processing of gastrointestinal lavage fluid samples obtained from subjects prior to mass spectrometry analysis, as described in Example 4.

FIG. 2 depicts the processing of the same control sample six times to assess variation in key proteins. The results reflect that the methodology results in data showing little variation and thus, the method is highly reproducible, as described in Example 5.

FIG. 3 depicts a volcano plot of the intensity values prior to “roll up” of proteins in the gastrointestinal lavage fluid of subjects with pancreatic ductal adenocarcinoma in the head of the pancreas versus control, as described in Example 5.

FIG. 4 depicts a volcano plot of the intensity values after “roll up” of proteins in the gastrointestinal lavage fluid of subjects with pancreatic ductal adenocarcinoma in the head of the pancreas versus control, as described in Example 5.

DETAILED DESCRIPTION

The present invention is based, at least in part, on the unexpected discovery that particular pancreatic cancer biomarkers, for example, proteins secreted from the pancreas or other non-pancreatic sources in the gastrointestinal tract, are found at modified levels, for example, at decreased or increased levels, in gastrointestinal lavage fluid or fecal matter of a subject having pancreatic cancer. Indeed, the inventors have identified that gastrointestinal lavage fluid or fecal matter provide a unique opportunity to assess the presence of pancreatic cancer in a non-invasive, rapid and efficient manner.

As a result, the present invention provides methods for diagnosing pancreatic cancer by assessing levels of pancreatic cancer biomarkers in gastrointestinal lavage fluid or fecal matter derived from a subject.

Moreover, the present invention is further predicated, at least in part, on the discovery that relative changes in the levels of proteins or polypeptides that originate from the pancreas, and other sources, compared to relative changes in the levels of particular proteins or polypeptides that originate from other gastrointestinal (GI) systems can be used to detect pancreatic cancer. Accordingly, the levels of particular proteins or polypeptides originating from non-pancreatic sources can be useful as control levels for assessing whether a subject is suffering from pancreatic cancer.

Definitions

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear. However, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms, for example, those characterized by “a” or “an”, shall include pluralities. In this application, the use of “or” means “and/or”, unless stated otherwise. Furthermore, the use of the term “including,” as well as other forms of the term, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one unit unless specifically stated otherwise.

The term “comprising” as used herein is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein “consisting essentially of” refers to a peptide or polypeptide which includes an amino acid sequence of the proteins provided herein, for example, SEQ ID NOs:1-793, along with additional amino acids at the carboxyl and/or amino terminal ends where the additional amino acids do not materially alter the ability of the peptide or polypeptide to be diagnostically useful for the relevant type or types of cancer. For example, in some embodiments, a peptide or polypeptide “consisting essentially of” a particular sequence may include an amino acid sequence of the proteins provided herein, for example SEQ ID NOs:1-793, along with no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 additional amino acid(s) at the carboxyl and/or amino terminal ends of a polypeptide provided herein, for example, one of SEQ ID NOs:1-793.

All numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims in any application claiming priority to the present application, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

As used herein, the term “subject” refers to human and non-human animals, including veterinary subjects. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rabbits, sheep, dog, cat, horse, cow, chickens, amphibians, and reptiles. In a preferred embodiment, the subject is a human.

The terms “cancer” or “tumor” are well known in the art and refer to the presence, e.g., in a subject, of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells may exist alone within a subject, or may be non-tumorigenic cancer cells, such as leukemia cells. As used herein, the term “cancer” includes pre-malignant as well as malignant cancers.

As used herein, “pancreas” in reference to an organ refers to a collection of a plurality of cell types held together by connective tissue, such that the plurality of cells include but are not limited to acini calls, ductal cells and islet cells. The “acini” produce many of the enzymes, such as lipase, which are needed to digest food in the duodenum. The enzymes produced by the acini are carried to the duodenum by small channels called ducts. Typically, ductal cells are held in place by connective tissue in close proximity to vascular cells and nerve cells. Islets of Langerhans are typically embedded between exocrine acini units of the pancreas. Examples of islet endocrine cells are Alpha cells that secrete glucagon which counters the action of insulin while Beta cells secrete insulin, which helps control carbohydrate metabolism.

As used herein, a subject who is “afflicted with pancreatic cancer” is one who is clinically diagnosed with such a cancer by a qualified clinician (for example, by the methods of the present invention), or one who exhibits one or more signs or symptoms (for example, reduced levels of a pancreatic cancer biomarker in gastrointestinal lavage fluid or fecal matter) of such a cancer and is subsequently clinically diagnosed with such a cancer by a qualified clinician (for example, by the methods of the present invention). A non-human subject that serves as an animal model of pancreatic cancer may also fall within the scope of the term a subject “afflicted with pancreatic cancer.”

As used herein, the term “pancreatic cancer” refers to the art recognized disease and includes cancers that originate in the tissue that comprises a pancreas. In various embodiments, the pancreatic cancer is an exocrine pancreatic cancer, a pancreatic cystic neoplasm or a pancreatic endocrine tumor.

In a particular embodiment, the pancreatic cancer is an exocrine pancreatic cancer selected from the group consisting of pancreatic ductal adenocarcinoma (PDAC), adenosquamous carcinoma, squamous cell carcinoma, giant cell carcinoma, acinar cell carcinoma and small cell carcinoma.

In a particular embodiment, the pancreatic cancer is a ductal adenocarcinoma, e.g., resectable pancreatic ductal adenocarcinoma (PDAC), which arises within the exocrine component of the pancreas. As used herein, “adenocarcinoma” refers to a cancerous tumor as opposed to an “adenoma” which refers to a benign (non-cancerous) tumor made up of cells that form glands (collections of cells surrounding an empty space). As used herein, “pancreatic ductal adenocarcinoma cell” refers to a cancerous cell that had the capability to form or originated from the ductal lining of the pancreas. A pancreatic ductal adenocarcinoma cell may be found within the pancreas forming a gland, or found within any organ as a metastasized cell or found within the blood stream of lymphatic system. As used herein, “ductal cell”, in reference to a pancreas, refers to any cell that forms or has the capability to form or originated from the ductal lining of ducts within and exiting from the pancreas.

In another embodiment, the pancreatic cancer is a pancreatic endocrine tumor, also known as islet cell tumors, pancreas endocrine tumors (PETs) and pancreatic neuroendocrine tumors (PNETs), which arises from islet cells. In a particular embodiment, the pancreatic cancer is an endocrine pancreatic cancer selected from the group consisting of insulinomas (i.e., arising from insulin-producing cells), glucagonomas (i.e., arising from glucagon-producing cells), somatostatinomas (i.e., arising from somatostatin-making cells), gastrinomas (i.e., arising from a gastrin-producing cells), VIPomas (arising from vasoactive intestinal peptide-making cells) and non-secreting islet tumors of the pancreas.

As used herein, the term “pancreatic cancer biomarker” refers to a protein or non-proteinaceous substance which is differentially present in gastrointestinal lavage fluid or fecal matter in subjects afflicted with pancreatic cancer as compared to subjects without pancreatic cancer. In particular embodiments, the protein is derived from the pancreas. In other embodiments, the protein is derived from non-pancreatic sources in the gastrointestinal tract, e.g., the intestine. In various embodiments, the pancreatic cancer biomarker is a protein selected from the group consisting of SEQ ID NOs:1-31 or 39-793. In a particular embodiment, the pancreatic cancer biomarker is a protein selected from the group consisting of SEQ ID NOs:1-19, 47, 49, 55-58, 206, 726, 729, 780 or 793. As used herein, isoforms and mature forms of the proteins specifically identified herein are also intended to be encompassed by the methods of the present invention. In addition, fragments of the proteins specifically identified herein are also intended to be encompassed by the methods of the present invention. As used herein, the term “fragment” refers to a fragment of a protein that preserves at least the structure, e.g., a portion of the amino acid sequence, or at least one function, e.g., activity, of the protein from which it is derived.

Alternatively, the pancreatic cancer biomarker may refer to a non-proteinaceous substance. For example, the pancreatic cancer may be CA19-9. As used herein, CA19-9, also known as carbohydrate antigen 19-9, cancer antigen 19-9 or sialylated Lewis (a) antigen) is a tumor marker often assayed in serum or blood.

The “level” of pancreatic cancer biomarker, as used herein, refers to the level of the pancreatic cancer biomarker in gastrointestinal lavage fluid or fecal matter as determined using a method for the measurement of levels of protein or non-proteinaceous substances. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitation reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), solution phase assay, immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, and electrochemiluminescence immunoassay (exemplified below), and the like. In a preferred embodiment, the level is determined using an ELISA based assay.

The term “sample” as used herein refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. In preferred embodiments, the sample is a biological fluid containing a pancreatic cancer biomarker. Biological fluids are typically liquids at physiological temperatures and may include naturally occurring fluids present in, withdrawn from, expressed or otherwise extracted from a subject or biological source. Certain biological fluids derive from particular tissues, organs or localized regions and certain other biological fluids may be more globally or systemically situated in a subject or biological source. Examples of biological fluids include gastrointestinal lavage fluid, fecal matter, blood, serum and serosal fluids, plasma, semen, pancreatic fluid, bile, lymph, urine, cerebrospinal fluid, saliva, ocular fluids, cystic fluid, tear drops, sputum, mucosal secretions of the secretory tissues and organs, vaginal secretions, gynecological fluids, ascites fluids such as those associated with non-solid tumors, fluids of the pleural, pericardial, peritoneal, abdominal and other body cavities, fluids collected by bronchial lavage and the like. In a particular embodiment, the sample is gastrointestinal lavage fluid or fecal matter.

In certain embodiments, the sample is a biological fluid formed of a liquid solution contacted with a subject or biological source. In a particular embodiment, the sample is a gastrointestinal lavage fluid.

In one embodiment, the sample is removed or obtained from the subject, for example, according to the methods described herein. In another embodiment, the sample is present within the subject.

In some embodiments, only a portion of the sample is subjected to an assay for determining the level of the pancreatic cancer biomarker, or various portions of the sample are subjected to various assays for determining the level of the pancreatic cancer biomarker. Also, in many embodiments, the sample may be pre-treated by physical or chemical means prior to the assay. For example, in embodiments discussed in more detail in the Examples section, samples, for example, gastrointestinal lavage fluid samples, were subjected to centrifugation, extraction (e.g., chloroform extraction), precipitation (e.g., methanol, chloroform and/or water precipitation), and digestion (e.g., with trypsin) prior to assaying the samples for the pancreatic cancer biomarker protein. Such techniques serve to enhance the accuracy, reliability and reproducibility of the assays of the present invention.

The term “control sample,” as used herein, refers to any clinically relevant control sample, including, for example, a sample from a healthy subject not afflicted with pancreatic cancer, a sample from a subject having a less severe or slower progressing pancreatic cancer than the subject to be assessed, a sample from a subject having some other type of cancer or disease, and the like. A control sample may include a sample derived from one or more subjects. A control sample may also be a sample made at an earlier time point from the subject to be assessed. For example, the control sample could be a sample taken from the subject to be assessed before the onset of pancreatic cancer, at an earlier stage of disease, or before the administration of treatment or of a portion of treatment. The control sample may also be a sample from an animal model, or from a tissue or cell lines derived from the animal model, of the pancreatic cancer. The level of pancreatic cancer biomarker in a control sample that consists of a group of measurements may be determined based on any appropriate statistical measure, such as, for example, measures of central tendency including average, median, or modal values.

The term “control level” refers to an accepted or pre-determined level of pancreatic cancer biomarker which is used to compare with the level of pancreatic cancer biomarker in a sample derived from a subject. In one embodiment, the control level of pancreatic cancer biomarker is based on the level of pancreatic cancer biomarker in sample(s) from a subject(s) having slow disease progression. In another embodiment, the control level of pancreatic cancer biomarker is based on the level in a sample from a subject(s) having rapid disease progression. In another embodiment, the control level of pancreatic cancer biomarker is based on the level of pancreatic cancer biomarker in a sample(s) from an unaffected, i.e., non-diseased, subject(s), i.e., a subject who does not have pancreatic cancer. In yet another embodiment, the control level of pancreatic cancer biomarker is based on the level of pancreatic cancer biomarker in a sample from a subject(s) prior to the administration of a therapy for pancreatic cancer. In another embodiment, the control level of pancreatic cancer biomarker is based on the level of pancreatic cancer biomarker in a sample(s) from a subject(s) having pancreatic cancer that is not contacted with a test compound. In another embodiment, the control level of pancreatic cancer biomarker is based on the level of pancreatic cancer biomarker in a sample(s) from a subject(s) not having pancreatic cancer that is contacted with a test compound. In one embodiment, the control level of pancreatic cancer biomarker is based on the level of pancreatic cancer biomarker in a sample(s) from an animal model of pancreatic cancer, a cell, or a cell line derived from the animal model of pancreatic cancer.

In one embodiment, the control is a standardized control, such as, for example, a control which is predetermined using an average of the levels of pancreatic cancer biomarker from a population of subjects having no pancreatic cancer. In still other embodiments of the invention, a control level of pancreatic cancer biomarker is based on the level of pancreatic cancer biomarker in a non-cancerous sample(s) derived from the subject having pancreatic cancer.

As used herein, “a difference” between the level of pancreatic cancer biomarker in a sample from a subject (i.e., gastrointestinal lavage fluid) and the level of pancreatic cancer biomarker in a control sample refers broadly to any clinically relevant and/or statistically significant difference in the level of pancreatic cancer biomarker in the two samples. In an exemplary embodiment, the difference is determined as set forth in the Examples set forth below.

In other embodiments, the difference must be greater than the limits of detection of the method for determining the level of pancreatic cancer biomarker. It is preferred that the difference be at least greater than the standard error of the assessment method, and preferably a difference of at least about 2-, about 3-, about 4-, about 5-, about 6-, about 7-, about 8-, about 9-, about 10-, about 15-, about 20-, about 25-, about 100-, about 500-, about 1000-fold or greater than the standard error of the assessment method. The difference may be assessed by any appropriate comparison, including any appropriate parametric or nonparametric descriptive statistic or comparison. For example, “an increase” in the level of pancreatic cancer biomarker may refer to a level in a test sample, e.g., gastrointestinal lavage fluid, that is about two, and more preferably about three, about four, about five, about six, about seven, about eight, about nine, about ten or more times more than the level of pancreatic cancer biomarker in the control sample. An increase may also refer to a level in a test sample that is preferably at least about 1.5, and more preferably about two, about three, about four, about five or more standard deviations above the average level of pancreatic cancer biomarker in the control sample. Likewise, “a decrease” in the level of pancreatic cancer biomarker may refer to a level in a test sample that is preferably at least about two, and more preferably about three, about four, about five, about six, about seven, about eight, about nine, about ten or more times less than the level of pancreatic cancer biomarker in the control sample. A decrease may also refer to a level in a test sample that is preferably at least about 1.5, and more preferably about two, about three, about four, about five or more standard deviations below the average level of pancreatic cancer biomarker in the control sample.

Biological Samples

As set forth herein, a sample for use in the methods of the present invention refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. In preferred embodiments the sample is a biological fluid containing a pancreatic cancer biomarker protein. Examples of biological fluids include gastrointestinal lavage fluid, fecal matter, blood, serum and serosal fluids, plasma, semen, pancreatic fluid, bile, lymph, urine, cerebrospinal fluid, saliva, ocular fluids, cystic fluid, tear drops, sputum, mucosal secretions of the secretory tissues and organs, vaginal secretions, gynecological fluids, ascites fluids such as those associated with non-solid tumors, fluids of the pleural, pericardial, peritoneal, abdominal and other body cavities, fluids collected by bronchial lavage and the like.

In a particular embodiment, the sample is a biological fluid originating from the gastrointestinal tract (GI tract). As is well known in the art, the gastrointestinal tract includes the upper gastrointestinal tract and lower gastrointestinal tract. The upper gastrointestinal tract includes the oral or buccal cavity, esophagus, stomach and duodenum. The lower gastrointestinal tract includes the jejunum, ileum and the large intestine and the anus. The large intestine includes the appendix, cecum, colon, and rectum. Organs and tissues associated with the gastrointestinal tract include structures outside the gastrointestinal tract. Examples of such structures include accessory digestive organs such as salivary glands, e.g., parotid salivary glands, submandibular salivary glands, and sublingual salivary glands, pancreas, e.g., exocrine pancreas, gallbladder, bile duct, and liver. More examples of structures associated with the gastrointestinal tract and outside the gastrointestinal tract include the pancreatic duct, biliary tree, and bile duct.

In a particular embodiment, the biological sample is gastrointestinal lavage fluid. In some embodiments, a biological sample includes a gastrointestinal lavage fluid. Generally, a lavage fluid can be orally administered to a subject, the oral lavage fluid passes through the gastrointestinal tract of the subject, and the resulting gastrointestinal lavage fluid is collected from the subject. Alternative lavage methods include direct washing of the cavity with a lavage fluid during surgery or endoscopy or washing via the rectum by means of enemas or colonic irrigation. As noted above, gastrointestinal lavage fluid provides a cleaner sampling of the gastrointestinal tract than the examination of feces/stool samples. Gastrointestinal lavage fluids appear to mitigate variability related to food intake, type and digestive status.

Some embodiments described herein include analysis of a gastrointestinal lavage fluid for detecting a pancreatic cancer biomarker for screening, disease detection, diagnosis, prognosis, response to treatment, selection of treatment and personalized medicine for diseases and pathological conditions of the gastrointestinal tract or associated organs/tissues, such as pancreatic cancer.

Methods for Obtaining a Gastrointestinal Lavage Fluid

In certain embodiments of the present invention, a gastrointestinal lavage fluid sample is obtained from a subject. For example, a gastrointestinal lavage fluid may be obtained as described in International Application No. PCT/US2011/051269, filed on Sep. 12, 2011 and entitled “NON-INVASIVE METHODS OF DETECTING PANCREATIC CANCER BIOMARKERS”, the entire contents of which are hereby incorporated by reference herein. Some methods of obtaining a gastrointestinal lavage fluid include orthograde colonic lavage. Orthograde lavage can include orally administering a lavage composition to a subject, for example, comprising 4 L of a polyethylene glycol/electrolyte solution (U.S. Patent Application Publication No. 20070298008, incorporated by reference in its entirety). Some methods of obtaining a gastrointestinal lavage fluid include antegrade lavage and retrograde lavage.

More methods of obtaining a gastrointestinal lavage fluid include oral administration of lavage compositions. Such lavage composition may include solutions of electrolytes, such as sodium, potassium and magnesium salts of sulfate, bicarbonate, chloride, phosphate or citrate. Some such compositions may also include polyethylene glycol, which can act as a non-absorbable osmotic agent. Generic compositions include polyethylene glycol with an electrolyte solution, optionally also including bisacodyl, or ascorbic acid, and compositions including sulfate salts such as sodium sulfate, magnesium sulfate, or potassium sulfate. In some embodiments, an oral lavage fluid can include magnesium citrate. In some embodiments, an oral lavage fluid can include sodium picosulfate. One example composition of an oral lavage solution comprising polyethylene glycol with an electrolyte solution is GOLYTELY (Braintree Labs. Inc.). GOLYTELY is formulated as follows: polyethylene glycol 59 g, sodium sulfate 5.68 g, sodium bicarbonate 1.69 g, sodium chloride 1.46 g, potassium chloride 0.745 g and water to make up one liter (Davis et al. (1980) Gastroenterology 78:991-995, incorporated by reference in its entirety). Ingestion of GOLYTELY produces a voluminous, liquid stool with minimal changes in the subject's water and electrolyte balance. Another example of an oral lavage composition comprising polyethylene glycol with an electrolyte solution is NULYTELY (Braintree Labs. Inc.). Another exemplary oral lavage composition is HALFLYTELY (Braintree Labs. Inc.) which includes polyethylene glycol with an electrolyte solution and bisacodyl. An exemplary oral lavage composition comprising sulfate salts, such as sodium sulfate, magnesium sulfate, or potassium sulfate is SUPREP (Braintree Labs. Inc.). An exemplary composition of an oral lavage solution comprising polyethylene glycol with an electrolyte solution and ascorbic acid is MOVIPREP (Salix Pharmaceuticals, Inc.).

Polyethylene glycol is effective as an oral lavage composition when large amounts of polyethylene glycol are administered in large volumes of a dilute salt solution. Usually about 250-400 g polyethylene glycol are administered to the subject in about 4 L of an electrolyte solution in water. Oral administration of polyethylene glycol can be used to produce a bowel movement over a period of time, e.g., overnight. The dose required will vary, but from about 10-100 g of polyethylene glycol in 8 oz. of water can be effective. A dose of from about 68-85 g of polyethylene glycol can be effective to produce an overnight bowel movement, without profuse diarrhea. A volume of a solution of polyethylene glycol in an isotonic fluid can be an effective amount of an osmotic laxative. Volumes from about 0.5 L to about 4 L can be effective. Preferably the effective volume is between about 1.5 L and about 2.5 L. Oral administration of 2 L of isotonic solution is effective.

More examples of oral lavage compositions include hypertonic solutions of non-phosphate salts with an osmotic laxative agent such as polyethylene glycol (U.S. Pat. App. No. 20090258090, incorporated by reference in its entirety). Mixtures of sulfate salts that omit phosphates, for example, effective amounts of one or more of the following sulfate salts Na₂SO₄, MgSO₄, and K₂SO₄can be effective (e.g., SUPREP). Some embodiments include about 0.1 g to about 20.0 g Na₂SO₄, and from about 1.0 g to 10.0 g Na₂SO₄may be useful. Dosage amounts of MgSO₄from about 0.01 g to about 40.0 g can be effective. Doses of from about 0.1 g to about 20.0 g Na₂SO₄may also be advantageously used, as well as dosages of 1.0 to 10.0 g. Dosage amounts of K₂SO₄from about 0.01 g to about 20.0 g can be effective to produce purgation, and doses of from about 0.1 g to about 10.0 g and from about 0.5 g to about 5.0 g K₂SO₄may also be useful. Addition of an osmotic laxative agent, such as polyethylene glycol (PEG) may improve the effectiveness of the above salt mixtures. Doses of PEG from about 1.0 g to about 100 g PEG are effective. Doses from about 10.0 g to about 50 g of PEG are also effective, as is a dose of about 34 g. For ease of administration, the above mixture of salts can be dissolved in a convenient volume of water. A volume of less than one liter of water can be well tolerated by most subjects. The mixture can be dissolved in any small volume of water, and volumes of between 100 and 500 ml are useful. The effective dose may be divided and administered to the patient in two or more administrations over an appropriate time period. Generally, administration of two doses of equal portions of the effective dose, separated by 6 to 24 hours, produces satisfactory purgation. Some embodiments include cessation of normal oral intake during a defined period before and during administration of an oral lavage composition.

Some lavage compositions include a laxative, such as bisacodyl. In some embodiments, a laxative can be co-administered to a subject with a lavage composition. As will be understood, such co-administration can include, for example, administration of a laxative up to several hours before administration of a lavage composition to a subject, administration of a laxative with the administration of a lavage composition to a subject, or administration of a laxative up to several hours after administration of a lavage composition to a subject. Examples of laxatives and their effective doses include Aloe, 250-1000 mg; Bisacodyl, about 5-80 mg; Casanthranol, 30-360 mg; Cascara aromatic fluid extract, 2-24 ml; Cascara sagrada bark, 300-4000 mg; Cascada sagrada extract, 300-2000 mg; Cascara sagrada fliuid extract, 0.5-5.0 ml; Castor oil, 15-240 ml; Danthron, 75-300 mg; Dehydrocholic Acid, 250-2000 mg; Phenolphthalein, 30-1000 mg; Sennosides A and B, 12-200 mg; and Picosulfate, 1-100 mg.

More examples of lavage compositions include aqueous solutions of concentrated phosphate salts. The aqueous phosphate salt concentrate produces an osmotic effect on the intra-luminal contents of the gastrointestinal tract. Evacuation of the bowel occurs with a large influx of water and electrolytes into the colon from the body. One exemplary composition comprises 480 g/L monobasic sodium phosphate and 180 g/L dibasic sodium phosphate in stabilized buffered aqueous solution (FLEET'S PHOSPHO-SODA, C. S. Fleet Co., Inc.). Subjects are typically required to take 2-3 oz doses of this composition, separated by a 3 to 12 hour interval for a total of 6 ounces (180 ml).

Gastrointestinal lavage fluid may be collected from a subject before, during, or after a medical or diagnostic procedure. In some embodiments, a subject may collect gastrointestinal lavage fluid, for example, using a receptacle such as a toilet insert which captures the fluid. Enzyme inhibitors and denaturants may be used to preserve the quality of the gastrointestinal lavage fluid. In some embodiments, the pH of the sample may be adjusted to help stabilize the samples. In some embodiments, gastrointestinal lavage fluid samples may be further treated to remove some or all solids and/or bacteria, such as by centrifugation or filtration. In some embodiments, the gastrointestinal tract may not be fully purged by administration of an oral lavage composition. For example, a portion of a complete dose of an oral lavage composition required to fully purge the gastrointestinal tract of a subject can be administered to the subject. In some embodiments, a gastrointestinal lavage fluid can comprise fecal matter. In more embodiments, fecal matter can comprise a gastrointestinal lavage fluid.

Methods for Detecting Pancreatic Cancer Biomarkers

The level of pancreatic biomarker proteins in a sample obtained from a subject may be determined by any of a wide variety of techniques and methods, which transform the pancreatic biomarker proteins within the sample into a moiety that can be detected and quantified. Non-limiting examples of such methods include analyzing the sample using immunological methods for detection of proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods, immunohistological, immunocytological, hybridization using immunofluorescence and/or immunoenzymatic, hydrometry, polarimetry, spectrophotometry (e.g., mass and NMR), chromatography (e.g., gas liquid, high performance liquid, and thin layer), immunoblotting, Western blotting, Northern blotting, electron microscopy, mass spectrometry, e.g., MALDI-TOF and SELDI-TOF, immunoprecipitations, immunofluorescence, immunohistochemistry, enzyme linked immunosorbent assays (ELISAs), e.g., amplified ELISA, quantitative blood based assays, e.g., serum ELISA, quantitative urine based assays, flow cytometry, Southern hybridizations, array analysis, and the like, and combinations or sub-combinations thereof. In some embodiments, nucleic acid encoding pancreatic cancer biomarker proteins may be detected using nucleic acid hybridization methods, such as Southern blotting, Northern blotting, or PCR.

Some embodiments of the methods and compositions provided herein include characterizing a pancreatic cancer biomarker in a sample, such as a sample obtained from the gastrointestinal tract, including a gastrointestinal lavage fluid and/or fecal sample. Characterizing a pancreatic cancer biomarker can include, for example, identifying a pancreatic cancer biomarker, detecting a pancreatic cancer biomarker, and/or quantifying a pancreatic cancer biomarker.

Some embodiments include identifying, determining the presence or absence of a pancreatic cancer biomarker, and/or quantifying a pancreatic cancer biomarker, wherein the pancreatic cancer biomarker comprises a peptide, polypeptide, protein and/or non-proteinaceous biological molecule.

As used in the present specification, the term “polypeptide” and “protein”, used interchangeably herein, refer to a polymer of amino acids without regard to the length of the polymer; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also includes wild-type polypeptides, as well as mutants, truncations, extensions, splice-variants, and other non-native forms of polypeptide that may be present. This term also includes forms of the foregoing that have been subject to enzymatic degradation by proteases or other mechanisms (enzymatic or non-enzymatic) in the subject. For example, a polypeptide may be subject to degradation by a protease to produce a polypeptide fragment of the polypeptide. The protease may be one that is expressed or increased in expression as a result of the health problem or disease of the gastrointestinal tract system. This term also does not specify or exclude chemical or post-expression/translational modifications of the polypeptides, although chemical or post-expression modifications of these polypeptides may be included or excluded as specific embodiments. Therefore, for example, modifications to polypeptides that include the covalent attachment of glycosyl groups (i.e., glycosylation), acetyl groups (i.e., acetylation), phosphate groups (phosphorylation, including, but not limited to, phosphorylation on serine, threonine and tyrosine groups), lipid groups and the like are expressly encompassed by the term polypeptide. Further, polypeptides with these modifications may be specified as individual species to be included or excluded. The natural or other chemical modifications, such as those listed in the examples above, can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini, and may be present in the same or varying degrees at several sites in a gastrointestinal tract polypeptide. Also, a gastrointestinal tract polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formylation of cysteine, formylation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination (see, for instance Creighton, (1993), Posttranslational Covalent Modification of Proteins, W. H. Freeman and Company, New York B. C. Johnson, Ed., Academic Press, New York 1-12; Seifier, et al., (1990) Meth Enzymol 182:626-646; Rattan et al, (1992) Ann NY Acad Sci 663:48-62). Isoforms of the proteins disclosed herein are also intended to be encompassed by the methods of the present invention.

Such pancreatic cancer biomarkers may be characterized by a variety of methods such as immunoassays, including radioimmunoassays, enzyme-linked immunoassays and two-antibody sandwich assays as described herein. A variety of immunoassay formats, including competitive and non-competitive immunoassay formats, antigen capture assays and two-antibody sandwich assays also are useful (Self and Cook, (1996) Curr. Opin. Biotechnol. 7:60-65, incorporated by reference in its entirety). Some embodiments include one or more antigen capture assays. In an antigen capture assay, antibody is bound to a solid phase, and sample is added such that antigen, e.g., a pancreatic cancer biomarker in a fluid or tissue sample, is bound by the antibody. After unbound proteins are removed by washing, the amount of bound antigen can be quantitated, if desired, using, for example, a radioassay (Harlow and Lane, (1988) Antibodies A Laboratory Manual Cold Spring Harbor Laboratory: New York, incorporated by reference in its entirety). Immunoassays can be performed under conditions of antibody excess, or as antigen competitions, to quantitate the amount of antigen and, thus, determine a level of a pancreatic cancer biomarker in a sample

Enzyme-linked immunosorbent assays (ELISAs) can be useful in certain embodiments provided herein. An enzyme such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase or urease can be linked, for example, to an anti-HMGB1 antibody or to a secondary antibody for use in a method of the invention. A horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm. Other convenient enzyme-linked systems include, for example, the alkaline phosphatase detection system, which can be used with the chromogenic substrate p-nitrophenyl phosphate to yield a soluble product readily detectable at 405 nm. Similarly, a β-galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-O-D-galactopyranoside (ONPG) to yield a soluble product detectable at 410 nm, or a urease detection system can be used with a substrate such as urea-bromocresol purple (Sigma Immunochemicals). Useful enzyme-linked primary and secondary antibodies can be obtained from a number of commercial sources such as Jackson Immuno-Research (West Grove, Pa.), as described further herein.

In certain embodiments, a pancreatic cancer biomarker in a sample, such as a sample obtained from the gastrointestinal tract, for example a gastrointestinal lavage fluid or fecal matter, can be detected and/or measured using chemiluminescent detection. For example in certain embodiments, specific antibodies to a particular pancreatic cancer biomarker are used to capture the pancreatic cancer biomarker present in the biological sample, e.g., such as a sample obtained from the gastrointestinal tract, for example, a gastrointestinal lavage fluid or fecal matter, and an antibody specific for the pancreatic cancer biomarker-specific antibodies and labeled with an chemiluminescent label is used to detect the pancreatic cancer biomarker present in the sample. Any chemiluminescent label and detection system can be used in the present methods. Chemiluminescent secondary antibodies can be obtained commercially from various sources such as Amersham. Methods of detecting chemiluminescent secondary antibodies are known in the art.

Fluorescent detection also can be useful for detecting a pancreatic cancer biomarker in certain methods provided herein. Useful fluorochromes include DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red and lissamine. Fluorescein or rhodamine labeled antibodies, or fluorescein- or rhodamine-labeled secondary antibodies.

Radioimmunoassays (RIAs) also can be useful in certain methods provided herein. Radioimmunoassays can be performed, for example, with ¹²⁵I-labeled primary or secondary antibody (Harlow and Lane, (1988) Antibodies A Laboratory Manual Cold Spring Harbor Laboratory: New York, incorporated by reference in its entirety).

A signal from a detectable reagent can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation, such as a gamma counter for detection of ¹²⁵I; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength. Where an enzyme-linked assay is used, quantitative analysis of the amount of a pancreatic cancer biomarker can be performed using a spectrophotometer such as an EMAX Microplate Reader (Molecular Devices; Menlo Park, Calif.) in accordance with the manufacturer's instructions. The assays of the invention can be automated or performed robotically, if desired, and that the signal from multiple samples can be detected simultaneously.

In some embodiments, capillary electrophoresis based immunoassays (CEIA), which can be automated if desired, may be used to detect and/or measure the pancreatic cancer biomarker. Immunoassays also can be used in conjunction with laser-induced fluorescence as described, for example, in Schmalzing and Nashabeh, Electrophoresis 18:2184-93 (1997), and Bao, J. Chromatogr. B. Biomed. Sci. 699:463-80 (1997), each incorporated by reference in its entirety. Liposome immunoassays, such as flow-injection liposome immunoassays and liposome immunosensors, also can be used to detect pancreatic cancer biomarkers or to determine a level of a pancreatic cancer biomarker according to certain methods provided herein (Rongen et al., (1997) J. Immunol. Methods 204:105-133, incorporated by reference in its entirety).

Sandwich enzyme immunoassays also can be useful in certain embodiments. In a two-antibody sandwich assay, a first antibody is bound to a solid support, and the antigen is allowed to bind to the first antibody. The amount of a pancreatic cancer biomarker is quantitated by measuring the amount of a second antibody that binds to it.

In an exemplary sandwich assay, an agent that selectively binds to a pancreatic cancer biomarker can be immobilized on a solid support. A capture reagent can be chosen to directly bind the pancreatic cancer biomarker or indirectly bind the pancreatic cancer biomarker by binding with an ancillary specific binding member which is bound to the pancreatic cancer biomarker. In addition, the capture reagent may be immobilized on the solid phase before or during the performance of the assay by means of any suitable attachment method. Typically, the capture site of the present invention is a delimited or defined portion of the solid phase such that the specific binding reaction of the capture reagent and analyte is localized or concentrated in a limited site, thereby facilitating the detection of label that is immobilized at the capture site in contrast to other portions of the solid phase. In a related embodiment, the capture reagent can be applied to the solid phase by dipping, inscribing with a pen, dispensing through a capillary tube, or through the use of reagent jet-printing or other techniques. In addition, the capture zone can be marked, for example, with a dye, such that the position of the capture zone upon the solid phase can be visually or instrumentally determined even when there is no label immobilized at the site.

Another exemplary embodiment of a sandwich assay format includes methods wherein a sample is mixed with a labeled first specific binding pair member for the pancreatic cancer biomarker and allowed to traverse a lateral flow matrix, past a series of spatially separated capture zones located on the matrix (See e.g., U.S. Pat. No. 7,491,551, incorporated by reference in its entirety). The sample may be mixed with the labeled first specific binding pair member prior to addition of the sample to the matrix. Alternatively, the labeled first specific binding pair member may be diffusively bound on the matrix on a labeling zone at a point upstream of the series of capture zones. Optionally, the sample is added directly to the labeling zone. Preferably, the sample is added to a sample receiving zone on the matrix at a point upstream of the labeling zone and allowed to flow through the labeling zone. The labeled first specific binding pair member located within the labeling zone is capable of being freely suspendible in the sample. Therefore, if analyte is present in the sample, the labeled first specific binding pair member will bind to the pancreatic cancer biomarker and the resulting pancreatic cancer biomarker-labeled first specific binding pair member complex will be transported to and through the capture zones. The extent of complex formation between the pancreatic cancer biomarker and the labeled specific binding pair member is directly proportional to the amount of pancreatic cancer biomarker present in the sample. A second specific binding pair member capable of binding to the pancreatic cancer biomarker-first specific binding pair member complex is immobilized on each of the capture zones. This second specific binding pair member is not capable of binding the labeled specific binding pair member unless the labeled specific binding pair member is bound to the pancreatic cancer biomarker. Thus, the amount of labeled specific binding pair member that accumulates on the capture zones is directly proportional to the amount of pancreatic cancer biomarker present in the sample.

In some embodiments, an assay includes the use of binding agent immobilized on a solid support to bind to and remove a target polypeptide from the remainder of the sample. The bound target polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent/polypeptide complex. Such detection reagents may comprise, for example, a binding agent that specifically binds to the target polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, protein A or a lectin. In such embodiments, the binding agent can comprise an antibody or antigen-binding fragment thereof specific to a polypeptide or fragment thereof descried herein. Alternatively, a competitive assay may be utilized in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the binding agent is indicative of the reactivity of the sample with the immobilized binding agent. Suitable polypeptides for use within such assays include full length proteins provided herein and polypeptide portions thereof such as SEQ ID NOs:1-793, for example, SEQ ID NOs:1-19, 47, 49-58, 206, 726, 729, 780 or 793, to which the binding agent binds.

The solid support may be any material known to those of ordinary skill in the art to which the binding agent may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane or flow-through format or test strip. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681. The binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature. In the context of the present invention, the term “immobilization” refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the agent and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of binding agent ranging from about 10 ng to about 10 and preferably about 100 ng to about 1 is sufficient to immobilize an adequate amount of binding agent.

Covalent attachment of binding agent to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent. For example, the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).

In certain embodiments, the assay is a two-antibody sandwich assay. This assay may be performed by first contacting an antibody that has been immobilized on a solid support, commonly the well of a microtiter plate, with the sample, such that target polypeptides within the sample are allowed to bind to the immobilized antibody. Unbound sample is then removed from the immobilized polypeptide-antibody complexes and a detection reagent (preferably a second antibody capable of binding to a different site on the polypeptide) containing a reporter group is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.

More specifically, once the antibody is immobilized on the support as described above, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art may be used, such as bovine serum albumin or TWEEN 20. (Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is then incubated with the sample, and target polypeptide is allowed to bind to the antibody. The sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation. In general, an appropriate contact time (i.e., incubation time) is a period of time that is sufficient to detect the presence of target polypeptide within a sample obtained from an individual with breast cancer. Preferably, the contact time is sufficient to achieve a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound polypeptide. Those of ordinary skill in the art will recognize that the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.

Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% TWEEN 20. The second antibody, which contains a reporter group, may then be added to the solid support. Reporter groups are well known in the art. The detection reagent is then incubated with the immobilized antibody-polypeptide complex for an amount of time sufficient to detect the bound detection reagent. An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time. Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.

To determine the level of a polypeptide described herein e.g., SEQ ID NOs:1-793 and, in particular, SEQ ID NOs: 1-19, 47, 49-58, 206, 726, 729, 780 or 793, the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value. In one embodiment, the cut-off value for the detection of a cancer is the average mean signal obtained when the immobilized antibody is incubated with samples from patients without the cancer. In general, a sample generating a signal that is three standard deviations above or below the predetermined cut-off value is considered positive for the cancer. For example, an increased level of certain polypeptides described herein e.g., SEQ ID NOs:17-19, 47, 726, 729 or 780, may be indicative of the presence of cancer or the stage of cancer, such as pancreatic cancer. Similarly, a reduced level of certain polypeptides described herein e.g., SEQ ID NOs:1-16, 49, 55-58, 206 or 793, may be indicative of the presence of cancer or the stage of cancer. In some embodiments, the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p. 106-7. Briefly, in this embodiment, the cut-off value may be determined from a plot of pairs of true positive rates (i.e., sensitivity) and false positive rates (100%-specificity) that correspond to each possible cut-off value for the diagnostic test result. The cut-off value on the plot that is the closest to the upper left-hand corner (i.e., the value that encloses the largest area) is the most accurate cut-off value, and a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive. Alternatively, the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate.

In a related embodiment, the assay is performed in a flow-through or test strip format, wherein the binding agent is immobilized on a membrane, such as nitrocellulose. In the flow-through test, target polypeptides within the sample bind to the immobilized binding agent as the sample passes through the membrane. A second labeled binding agent then binds to the binding agent-polypeptide complex as a solution containing the second binding agent flows through the membrane. The detection of bound second binding agent may then be performed as described herein. In the test strip format, one end of the membrane to which binding agent is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing second binding agent and to the area of immobilized binding agent. The amount of immobilized antibody indicates the presence, or absence or progression or stage of a cancer. Typically, the concentration of second binding agent at that site generates a pattern, such as a line, that can be read visually. In general, the amount of binding agent immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of polypeptide that would be sufficient to generate a positive signal in the two-antibody sandwich assay, in the format discussed above. Preferred binding agents for use in such assays are antibodies and antigen-binding fragments thereof. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 and more preferably from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount of biological sample.

Quantitative Western blotting also can be used to detect a pancreatic cancer biomarker or to determine a level of pancreatic cancer biomarker in a method provided herein. Western blots can be quantitated by well known methods such as scanning densitometry. As an example, protein samples are electrophoresed on 10% SDS-PAGE Laemmli gels. Primary murine monoclonal antibodies, for example, against a pancreatic cancer biomarker are reacted with the blot, and antibody binding confirmed to be linear using a preliminary slot blot experiment. Goat anti-mouse horseradish peroxidase-coupled antibodies (BioRad) are used as the secondary antibody, and signal detection performed using chemiluminescence, for example, with the Renaissance chemiluminescence kit (New England Nuclear; Boston, Mass.) according to the manufacturer's instructions. Autoradiographs of the blots are analyzed using a scanning densitometer (Molecular Dynamics; Sunnyvale, Calif.) and normalized to a positive control. Values are reported, for example, as a ratio between the actual value to the positive control (densitometric index). Such methods are described, for example, in Parra et al., J. Vasc. Surg. 28:669-675 (1998), incorporated herein by reference in its entirety.

As described herein, immunoassays including, for example, enzyme-linked immunosorbent assays, radioimmunoassays and quantitative western analysis, can be useful in some embodiments for detecting a pancreatic cancer biomarker or determining a level of a pancreatic cancer biomarker. Such assays typically rely on one or more antibodies. As would be understood by the skilled artisan, methods described herein can be used to readily distinguish proteins with alternative forms of post-translation modifications, e.g., phosphorylated proteins, and glycosylated proteins.

Some embodiments of the methods and compositions provided herein include generating agents that selectively bind to pancreatic cancer biomarkers. In some embodiments, such agents include an antibody or antigen-binding fragment thereof. Methods of generating polyclonal antibodies and monoclonal antibodies are well known in the art. The antibodies or active fragments thereof may be obtained by methods known in the art for production of antibodies or functional portions thereof. Such methods include, but are not limited to, separating B cells with cell-surface antibodies of the desired specificity, cloning the DNA expressing the variable regions of the light and heavy chains and expressing the recombinant genes in a suitable host cell. Standard monoclonal antibody generation techniques can be used wherein the antibodies are obtained from immortalized antibody-producing hybridoma cells. These hybridomas can be produced by immunizing animals with HSCs or progeny thereof, and fusing B lymphocytes from the immunized animals, preferably isolated from the immunized host spleen, with compatible immortalized cells, preferably a B cell myeloma.

In embodiments where the pancreatic cancer biomarker is a polypeptide associated with one or more iron atoms, antibodies which differentially bind to the iron-associated polypeptide relative to the same polypeptide without iron can be prepared. Antibodies which differentially bind to metal-associated polypeptides relative to the same polypeptide without metal and methods for making such antibodies have been described, for example, in HALLAB, et al., In vitro Reactivity to Implant Metals Demonstrates a Person Dependent Association with both T-Cell and B-Cell Activation, J. Biomed Mater Res A, 2010 February; 92(2):667-682; KONG, et al., Preparation of specific monoclonal antibodies against chelated copper ions, Biol Trace Elem Res., 2012 March; 145(3):388-395; LIU, et al., Preparation and characterization of monoclonal antibody specific for copper-chelate complex, J Immunol Methods., 2013 Jan. 31; 387(1-2):228-236; XIANG, et al., A competitive indirect enzyme-linked immunoassay for lead ion measurement using mAbs against the lead-DTPA complex, Environ Pollut., 2010 May; 158(5):1376-1380; YANG, et al., Detection of antibodies against corrosion products in patients after Co—Cr total joint replacements, J Biomed Mater Res., 1994 November; 28(11):1249-1258; ZHANG, et al., Development of ELISA for detection of mercury based on specific monoclonal antibodies against mercury-chelate, Biol Trace Elem Res., 2011 December; 144(1-3):854-864; and ZHU, et al., Preparation of specific monoclonal antibodies (MAbs) against heavy metals: MAbs that recognize chelated cadmium ions, J Agric Food Chem., 2007 Sep. 19; 55(19):7648-7653, each of which is incorporated by reference in its entirety.

Pancreatic cancer biomarkers, such as protein pancreatic cancer biomarkers, can be characterized, isolated, purified, or obtained for use in generating antibodies by a variety of methods. Proteins, polypeptides and peptides can be isolated by a variety of methods well known in the art, such as protein precipitation, chromatography (e.g., reverse phase chromatography, size exclusion chromatography, ion exchange chromatography, liquid chromatography), affinity capture, and differential extractions.

Isolated proteins can undergo enzymatic digestion or chemical cleavage to yield polypeptide fragments and peptides. Such fragments can be identified and quantified. A particularly useful method for analysis of polypeptide/peptide fragments and other pancreatic cancer biomarkers is mass spectrometry (U.S. Pat. App. No. 20100279382, incorporated by reference in its entirety). A number of mass spectrometry-based quantitative proteomics methods have been developed that identify the proteins contained in each sample and determine the relative abundance of each identified protein across samples (Flory et al., Trends Biotechnol. 20:S23-29 (2002); Aebersold, J. Am. Soc. Mass Spectrom. 14:685-695 (2003); Aebersold, J. Infect. Dis. 187 Suppl 2:S315-320 (2003); Patterson and Aebersold, Nat. Genet. 33 Suppl, 311-323 (2003); Aebersold and Mann, Nature 422:198-207 (2003); Aebersold, R. and Cravatt, Trends Biotechnol. 20:S1-2 (2002); Aebersold and Goodlett, Chem. Rev. 101, 269-295 (2001); Tao and Aebersold, Curr. Opin. Biotechnol. 14:110-118 (2003), each incorporated by reference in its entirety). Generally, the proteins in each sample are labeled to acquire an isotopic signature that identifies their sample of origin and provides the basis for accurate mass spectrometric quantification. Samples with different isotopic signatures are then combined and analyzed, typically by multidimensional chromatography tandem mass spectrometry. The resulting collision induced dissociation (CID) spectra are then assigned to peptide sequences and the relative abundance of each detected protein in each sample is calculated based on the relative signal intensities for the differentially isotopically labeled peptides of identical sequence.

More techniques for identifying and quantifying pancreatic cancer biomarkers include label-free quantitative proteomics methods. Such methods include: (i) sample preparation including protein extraction, reduction, alkylation, and digestion; (ii) sample separation by liquid chromatography (LC or LC/LC) and analysis by MS/MS; (iii) data analysis including peptide/protein identification, quantification, and statistical analysis. Each sample can be separately prepared, then subjected to individual LC-MS/MS or LC/LC-MS/MS runs (Zhu W. et al., J. of Biomedicine and Biotech. (2010) Article ID 840518, 6 pages, incorporated by reference in its entirety). An exemplary technique includes LC-MS in which the mass of a peptide coupled with its corresponding chromatographic elution time as peptide properties that uniquely define a peptide sequence, a method termed the accurate mass and time (AMT) tag approach. Using LC coupled with Fourier transform ion cyclotron resonance (LC-FTICR) MS to obtain the chromatographic and high mass accuracy information, peptide sequences can be identified by matching the AMT tags to previously acquired LC-MS/MS sequence information stored in a database. By taking advantage of the observed linear correlation between peak area of measured peptides and their abundance, these peptides can be relatively quantified by the signal intensity ratio of their corresponding peaks compared between MS runs (Tang, K., et al., (2004) J. Am. Soc. Mass Spectrom. 15:1416-1423; and Chelius, D. and Bondarenko, P. V. (2002) J. Proteome Res. 1: 317-323, incorporated by reference in their entireties). Statistics tools such as the Student's t-test can be used to analyse data from multiple LC-MS runs for each sample (Wiener, M. C., et al., (2004) Anal. Chem. 76:6085-6096, incorporated by reference in its entirety). At each point of acquisition time and m/z, the amplitudes of signal intensities from multiple LC-MS runs can be compared between two samples to detect peptides with statistically significant differences in abundance between samples.

As will be understood, a variety of mass spectrometry systems can be employed in the methods for identifying and/or quantifying a polypeptide/peptide fragments. Mass analyzers with high mass accuracy, high sensitivity and high resolution include ion trap, triple quadrupole, and time-of-flight, quadrupole time-of-flight mass spectrometeres and Fourier transform ion cyclotron mass analyzers (FT-ICR-MS). Mass spectrometers are typically equipped with matrix-assisted laser desorption (MALDI) or electrospray ionization (ESI) ion sources, although other methods of peptide ionization can also be used. In ion trap MS, analytes are ionized by ESI or MALDI and then put into an ion trap. Trapped ions can then be separately analyzed by MS upon selective release from the ion trap. Fragments can also be generated in the ion trap and analyzed. Sample molecules such as released polypeptide/peptide fragments can be analyzed, for example, by single stage mass spectrometry with a MALDI-TOF or ESI-TOF system. Methods of mass spectrometry analysis are well known to those skilled in the art (see, e.g., Yates, J. (1998) Mass Spect. 33:1-19; Kinter and Sherman, (2000) Protein Sequencing and Identification Using Tandem Mass. Spectrometry, John Wiley & Sons, New York; and Aebersold and Goodlett, (2001) Chem. Rev. 101:269-295, each incorporated by reference in its entirety).

For high resolution polypeptide fragment separation, liquid chromatography ESI-MS/MS or automated LC-MS/MS, which utilizes capillary reverse phase chromatography as the separation method, can be used (Yates et al., Methods Mol. Biol. 112:553-569 (1999), incorporated by reference in its entirety). Data dependent collision-induced dissociation (CID) with dynamic exclusion can also be used as the mass spectrometric method (Goodlett, et al., Anal. Chem. 72:1112-1118 (2000), incorporated by reference in its entirety).

Once a peptide is analyzed by MS/MS, the resulting CID spectrum can be compared to databases for the determination of the identity of the isolated peptide. Methods for protein identification using single peptides have been described previously (Aebersold and Goodlett, Chem. Rev. 101:269-295 (2001); Yates, J. Mass Spec. 33:1-19 (1998), David N. et al., Electrophoresis, 20 3551-67 (1999), each incorporated by reference in its entirety). In particular, it is possible that one or a few peptide fragments can be used to identify a parent polypeptide from which the fragments were derived if the peptides provide a unique signature for the parent polypeptide. Moreover, identification of a single peptide, alone or in combination with knowledge of a site of glycosylation, can be used to identify a parent glycopolypeptide from which the glycopeptide fragments were derived. As will be understood, methods that include MS can be used to characterize proteins, fragments thereof, as well as other types of pancreatic cancer biomarkers described herein.

In some embodiments, pancreatic cancer biomarkers include nucleic acids. Nucleic acids can encode a polypeptide or fragment thereof useful to determine the presence or absence of a cancer. As such, pancreatic cancer biomarkers include nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules that correspond to a pancreatic cancer biomarker, including nucleic acids which encode a polypeptide corresponding to a pancreatic cancer biomarkers, and fragments of such nucleic acid molecules, e.g., those suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules. As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

A nucleic acid pancreatic cancer biomarker can be amplified using cDNA, mRNA, or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to all or a portion of a nucleic acid pancreatic cancer biomarker can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

In another preferred embodiment, a nucleic acid pancreatic cancer biomarker comprises a nucleic acid molecule that has a nucleotide sequence complementary to a nucleic acid which is differentially expressed in cancer or a fragment thereof. For example, the pancreatic cancer biomarker may comprise a nucleic acid encoding a polypeptide of any one of SEQ ID NOs:1-31 or 39-793, for example, SEQ ID NOs:1-19, 47, 49-58, 206, 726, 729, 780 or 793, or a fragment comprising at least 10, at least 20, at least 30, at least 40, at least 50 or more consecutive nucleotides thereof. A nucleic acid molecule which is complementary to a pancreatic cancer biomarker nucleotide sequence is one which is sufficiently complementary to the pancreatic cancer biomarker nucleotide sequence that it can hybridize to the pancreatic cancer biomarker nucleotide sequence thereby forming a stable duplex.

In some embodiments, a fragment of a polynucleotide sequence will be understood to include any nucleotide fragment having, for example, at least about 5 successive nucleotides, at least about 12 successive nucleotides, at least about 15 successive nucleotides, at least about 18 successive nucleotides, or at least about 20 successive nucleotides of the sequence from which it is derived. An upper limit for a fragment can include, for example, the total number of nucleotides in a full-length sequence encoding a particular polypeptide. A fragment of a polypeptide sequence will be understood to include any polypeptide fragment having, for example, at least about 5 successive residues, at least about 12 successive residues, at least about 15 successive residues, at least about 18 successive residues, or at least about 20 successive residues of the sequence from which it is derived. An upper limit for a fragment can include, for example, the total number of residues in a full-length sequence of a particular polypeptide.

Moreover, a nucleic acid pancreatic cancer biomarker can comprise all or only a portion of a nucleic acid sequence which is differentially expressed in cancer. For example, the pancreatic cancer biomarker may comprise a nucleic acid encoding a polypeptide of SEQ ID NOs:1-31 or 39-793, for example, SEQ ID NOs:1-19, 47, 49-58, 206, 726, 729, 780 or 793, or a fragment comprising at least 10, at least 20, at least 30, at least 40, at least 50 or more consecutive nucleotides thereof. Such nucleic acids can be used, for example, as a probe or primer. The probe/primer typically is used as one or more substantially purified oligonucleotides. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 7, preferably about 15, more preferably about 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 or more consecutive nucleotides of a nucleic acid.

Probes based on the sequence of a nucleic acid pancreatic cancer biomarker can be used to detect transcripts or genomic sequences corresponding to one or more pancreatic cancer biomarkers. The probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as part of a diagnostic test kit for identifying a biological sample, such as fluids, cells or tissues, which mis-express the protein, such as by measuring levels of a nucleic acid molecule encoding the protein in a sample of a fluid or cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding the protein has been mutated or deleted. Embodiments also include nucleic acid pancreatic cancer biomarkers that differ, due to degeneracy of the genetic code, from the nucleotide sequence of nucleic acids encoding a protein that corresponds to a pancreatic cancer biomarker, and thus encode the same protein.

Method for Assessing the Presence, Absence or Progression of Pancreatic Cancer

Some of the methods and composition provided herein include methods for assessing the presence absence, progression or stage of a cancer, in particular pancreatic cancer, in a subject. Some such embodiments include determining the level of at least one pancreatic cancer biomarker in a sample from said subject. In some embodiments, the pancreatic cancer biomarker comprises at least one polypeptide or fragment thereof or at least one nucleic acid encoding the polypeptide. In some embodiments, the polypeptide is selected from any polypeptide provided herein, for example, SEQ ID NOs:1-31 or 39-793, for example, SEQ ID NOs:1-19, 47, 49-58, 206, 726, 729, 780 or 793.

In some embodiments, a sample is obtained from the gastrointestinal tract of a subject using methods provided herein.

Some embodiments include determining the level in the sample of at least 2 pancreatic cancer biomarkers, at least 3 pancreatic cancer biomarkers, at least 4 pancreatic cancer biomarkers, at least 5 pancreatic cancer biomarkers, at least 6 pancreatic cancer biomarkers, at least 7 pancreatic cancer biomarkers, at least 8 pancreatic cancer biomarkers, at least 9 pancreatic cancer biomarkers, at least 10 pancreatic cancer biomarkers, at least 11 pancreatic cancer biomarkers, at least 12 pancreatic cancer biomarkers, at least 13 pancreatic cancer biomarkers, at least 14 pancreatic cancer biomarkers, at least 15 pancreatic cancer biomarkers, at least 16 pancreatic cancer biomarkers, at least 17 pancreatic cancer biomarkers, at least 18 pancreatic cancer biomarkers, at least 19 pancreatic cancer biomarkers, or at least 20 pancreatic cancer biomarkers.

Some embodiments also include comparing the level of at least one pancreatic cancer biomarker in a sample of a subject with the level of the pancreatic cancer biomarker in a sample from a subject without the cancer. Some embodiments also include comparing the level of at least one pancreatic cancer biomarker in a sample of a subject with the level of the pancreatic cancer biomarker in a sample from a subject with the cancer.

Some embodiments also include comparing the level of at least one pancreatic cancer biomarker in a sample of a subject with the level of a control molecule. In some embodiments, the levels of a control molecule are determined in the sample from a subject. In some embodiments a control molecule comprises a non-pancreatic polypeptide. In some embodiments a control molecule comprises a non-pancreatic polypeptide that originates from the gastrointestinal tract. In some embodiments the levels of a control molecule are determined in the sample from a subject with cancer. In some embodiments the levels of a control molecule are determined in the sample from a subject without cancer. In some embodiments, the level of at least 1 control molecule is determined in a sample. In some embodiments, the level of at least about 2, 5, 10, or 15 control molecules are determined in a sample. Examples of control molecules include polypeptides and fragments thereof and nucleic acids encoding such polypeptides and fragments thereof, in which the polypeptide comprises, consists essentially of, or consists of SEQ ID NO:27, 32-40, 45, 54, 59 and 59. More examples of control molecules include CEA, and CA19-19.

In some embodiments, an increase in the level of the pancreatic cancer biomarker in a sample from a subject compared to the level of the pancreatic cancer biomarker in a sample from said subject without the cancer is indicative of the presence of the cancer in the subject. In some such embodiments, the pancreatic cancer biomarker can include a polypeptide or a fragment thereof, a nucleic acid encoding the polypeptide or fragment thereof, in which the polypeptide includes SEQ ID NOs: 17-19, 47, 726, 729 or 780.

In some embodiments, an increase in the level of a pancreatic cancer biomarker in a sample compared to the level of the pancreatic cancer biomarker in a sample obtained from a subject without a cancer is indicative of the cancer, in which the increase is at least about a 3-fold increase at least about a 5-fold increase, at least about a 10-fold increase, at least about a 20-fold increase, at least about a 30-fold increase, at least about a 40-fold increase, at least about a 50-fold increase, at least about a 60-fold increase, at least about a 70-fold increase, at least about a 80-fold increase, at least about a 90-fold increase, and at least about a 100-fold increase.

In some embodiments, a decrease in the level of the pancreatic cancer biomarker in a sample from a subject compared to the level of the pancreatic cancer biomarker in a sample from said subject without the cancer is indicative of the presence of the cancer in the subject. In some such embodiments, the pancreatic cancer biomarker can include a polypeptide or a fragment thereof, a nucleic acid encoding the polypeptide or fragment thereof, in which the polypeptide includes SEQ ID NOs:1-16, 49, 55-58, 206 or 793.

In some embodiments, a decrease in the level of a pancreatic cancer biomarker in a sample compared to the level of the pancreatic cancer biomarker in a sample obtained from a subject without a cancer is indicative of the cancer, in which the decrease is at least about a 3-fold decrease at least about a 5-fold decrease, at least about a 10-fold decrease, at least about a 20-fold decrease, at least about a 30-fold decrease, at least about a 40-fold decrease, at least about a 50-fold decrease, at least about a 60-fold decrease, at least about a 70-fold decrease, at least about a 80-fold decrease, at least about a 90-fold decrease, and at least about a 100-fold decrease.

Methods to determine the level of a pancreatic cancer biomarker in a sample are provided herein. In some embodiments, a method for determining the level of a pancreatic cancer biomarker, such as a polypeptide or fragment thereof, can include an immunoassay. Examples of an immunoassay include a Western blot, an enzyme linked immunoabsorbent assay (ELISA), and radioimmunoassay. In some embodiments, a method for determining the level of a pancreatic cancer biomarker, such as a polypeptide or fragment thereof, can include mass spectrometry.

Kits

The present invention further provides a kit for determining the presence, absence, progression, or stage of a cancer in a subject comprising: (a) a lavage fluid for oral administration to a subject; (b) a vessel for collecting the gastrointestinal lavage fluid from the subject; and (c) an agent that selectively binds to at least one polypeptide or fragment thereof or nucleic acid encoding said polypeptide or fragment thereof, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NOs:1-31 or 39-793, for example, SEQ ID NOs:1-19, 47, 49-58, 206, 726, 729, 780 or 793. Such kits can include at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 agents that each selectively bind to a different polypeptide or a nucleic acid encoding said polypeptide or fragment thereof. In some embodiments, the agent comprises an antibody or antigen-binding fragment thereof.

Some embodiments of the methods and compositions provided herein include a kit comprising an agent which selectively binds to at least one polypeptide comprising an amino acid sequence selected from the group consisting of a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NOs:1-31 or 39-793, for example, SEQ ID NOs:1-19, 47, 49-58, 206, 726, 729, 780 or 793, or a fragment thereof, wherein said agent is attached to a solid support. In some embodiments, the kit can include an agent that selectively binds to at least one polypeptide or nucleic acid encoding a polypeptide, wherein said polypeptide is selected from the group consisting of a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NOs:1-16, 49, 55-58, 206 and 793. In some embodiments, the kit can include an agent that selectively binds to at least one polypeptide or nucleic acid encoding a polypeptide, wherein said polypeptide is selected from the group consisting of a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NOs:17-19, 47, 726, 729 or 780. In some embodiments, the kit can include a plurality of agents that bind to different polypeptides comprising an amino acid sequence selected from the group consisting of a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NOs:1-31 or 39-793, for example, SEQ ID NOs:1-19, 47, 49-58, 206, 726, 729, 780 or 793, or a fragment thereof are attached to said solid support. In some embodiments, the solid support comprises a solid phase test strip or a flow-through test strip. In some embodiments, the kit can also include a detectable agent which selectively binds to said polypeptide.

Some embodiments of the methods and compositions provided herein include a kit comprising an agent which selectively binds to at least one nucleic acid encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NOs:1-31 or 39-793, for example, SEQ ID NOs:1-19, 47, 49-58, 206, 726, 729, 780 or 793, or a fragment thereof, wherein said agent is attached to a solid support. In some embodiments, the kit can include an agent that selectively binds to at least one polypeptide or nucleic acid encoding a polypeptide, wherein said polypeptide is selected from the group consisting of a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NOs:1-16, 49, 55-58, 206 and 793. In some embodiments, the kit can include an agent that selectively binds to at least one polypeptide or nucleic acid encoding a polypeptide, wherein said polypeptide is selected from the group consisting of a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NOs:17-19, 47, 726, 729 or 780. In some embodiments, the kit can include a plurality of agents that bind to nucleic acids encoding different polypeptides comprising an amino acid sequence selected from the group consisting of a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NOs:1-31 or 39-793, for example, SEQ ID NOs:1-19, 47, 49-58, 206, 726, 729, 780 or 793, or a fragment thereof are attached to said solid support. In some embodiments, the solid support comprises a solid phase test strip or a flow-through test strip. In some embodiments, the kit can also include a detectable agent which selectively binds to said polypeptide.

The present invention is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited references, including literature references, issued patents and published patent applications, as cited throughout this application are hereby expressly incorporated herein by reference. It should further be understood that the contents of all the figures and tables attached hereto are expressly incorporated herein by reference.

EXAMPLES
Example 1: Identification of Biomarkers Associated with Pancreatic Cancer

Gastrointestinal lavage fluid was obtained from patients with pancreatic cancer and from control patients, after administration of magnesium citrate (MgC) to the patients. Polypeptides were identified in gastrointestinal lavage fluid using mass spectrometry, and further characterized with MASCOT analysis. The presence or absence and/or levels of particular polypeptides were further confirmed using ELISA analyses. In the MASCOT analysis, a score indicates the relative prevalence of a protein or polypeptide, for example, a higher score indicates a greater prevalence for a particular protein or polypeptide in a sample, such that the most prevalent protein or polypeptide in sample will have the highest MASCOT score, and a ranking of “1.” Higher Mascot scores indicate better protein hits and can be correlated to relative protein levels. A score threshold of “>40” was indicative of a p-value significance of <0.05 as determined by the Mascot scoring system based on the search of this database with no enzyme specificity; a score of 40 is consistent with a p<0.01. Standard Mascot scoring was used whereby only the highest score was added for each peptide detected, even if it was sampled during MS/MS multiple times. For all data included, scores were all >40 in at least one sample per protein line. For additional confidence, the numbers of significant peptides were also reported and a minimum criteria of at least 2 peptides was selected. Very few had less than 3 peptides. All significant peptides counted represented different sequences (individual peptides) from their respective proteins. The score and numbers of significant peptides are reported in the format x/y where x is the score and y the number of significant peptides. If a protein was not detected in a particular sample it is listed as “ND”.

Gastrointestinal lavage fluid was collected from patients and analyzed with mass spectrometry (MS) and commercial ELISA. MS Data were acquired on an LTQ-Orbitrap mass spectrometer using input from an LC system. The A solvent contained 3% of B and 0.2% formic acid in water. The B solvent contained 3% of A and 0.2% formic acid in acetonitrile. Solvents were HPLC grade from Fisher. For a 120 min run, the starting solvent was 5% B and remains for 7 min. The gradient was changed to 10% by 13 min, 40% by 83 min, 90% by 103 min, then reduced from 90% to 5% at 111 min. It was then re-equilibrated for the next injection. Three injections were performed for each sample for repeatability determination. The MS was scanned (Orbitrap) over the mass range from 400 m/z to 2000 m/z every second while the LTQ (Trap) acquired up to 5 MSMS (peptide sequence) spectra in parallel. Data were acquired using the standard Thermo Xcalibur software. Peptides were eluted from a C18 LC column using triplicate injections. A search file was created from the triplicate injections from each lavage preparation (patient sample) and converted into a MGF (Mascot Generic Format) file using a combination of Xcalibur and Mascot software packages. Database searching was done using the Mascot search engine (Matrix Science, UK) against the RefSeq database (http://www.ncbi.nlm.nih.gov/RefSeq/) with taxonomy specified as human (Homo sapiens), a mass accuracy of 10 ppm for the parent ion (MS) and 0.6 Da for the fragment ions (MS/MS), and “semitrypsin” selected. The RefSeq database was supplemented by the addition of antibody sequences that are included in the SwissProt protein database, as these antibody sequences are not part of the standard RefSeq listing.

Table 1 provides examples of proteins and polypeptides whose levels were found to have been reduced in pancreatic cancer. In Table 1, the proteins include pancreatic enzymes, such as lipase and amylase, and other pancreatic proteins such as lithostathine. The most dramatic change was observed with pancreatic triacylglycerol lipase precursor which was the most abundant protein in gastrointestinal lavage fluid from control patient, but was not detected (ND) in gastrointestinal lavage fluid from patient with pancreatic cancer.

TABLE 1

Proteins with Reduced Levels in Gastrointestinal

Lavage Fluid of Subjects with Pancreatic Cancer

NCBI

MgC** Pancreatic

SEQ ID
Accession

MgC** Control
Cancer

NO.:
Nos.
Protein name
Ranking
Score
Ranking
Score

1
10835000
pancreatic triacylglycerol
1
5010
—
ND*

lipase precursor

2
4502085
pancreatic alpha-amylase
2
4818
13
1947

precursor

3
10280622
alpha-amylase 2B precursor
3
4581
14
1933

4
4502997
carboxypeptidase A1
4
3974
217
479

precursor

5
40254482
alpha-amylase 1 precursor
5
3675
18
1883

6
54607080
carboxypeptidase B
10
2567
—
ND

preproprotein

7
217416390
carboxypeptidase A2
11
2504
—
ND

precursor

8
236460050
chymotrypsin-like elastase
17
1854
168
534

family member 3A

preproprotein

9
62526043
chymotrypsin-C
19
1649
—
ND

preproprotein

10
15559207
chymotrypsin-like elastase
21
1437
239
462

family member 2A

preproprotein

11
6679625
chymotrypsin-like elastase
24
1276
65
747

family member 3B

preproprotein

12
4506147
trypsin-2 preproprotein
26
1166
59
785

13
4506145
trypsin-1 preproprotein
32
1022
46
890

14
29725633
lithostathine-1-alpha
36
895
103
640

precursor

15
118498350
chymotrypsinogen B2
42
770
—
ND

precursor

16
10835248
lithostathine-1-beta precursor
47
542
—
ND

*ND = not detected

**MgC = magnesium citrate

Table 2 provides examples of proteins and polypeptides whose levels were found to have increased in pancreatic cancer, with the most significant changes being for mucin-2.

TABLE 2

Proteins with Increased Levels in Gastrointestinal

Lavage Fluid of Subjects with Pancreatic Cancer

NCBI

MgC** Pancreatic

SEQ ID
Accession

MgC** Control
Cancer

NO.:
Nos.
Protein name
Ranking
Score
Ranking
Score

17
16306550
selenium-binding
25
1178
17
1887

protein 1

18
83367077
mucin-16
53
502
39
1018

19
116284392
mucin-2
215
223
16
1921

precursor

**MgC = magnesium citrate

Table 3 provides examples of blood/serum proteins identified in gastrointestinal lavage fluid obtained from patients. Generally, blood proteins were found to have a low abundance in gastrointestinal lavage fluid obtained from patients. However, albumin was found to have increased levels in gastrointestinal lavage fluid obtained from patients with pancreatic cancer.

TABLE 3

Blood/Serum Proteins Present in Gastrointestinal

Lavage Fluid of Subjects with Pancreatic Cancer

NCBI

Pancreatic

SEQ ID
Accession

Cancer
Control

NO.:
Nos.
Protein name
Ranking
Score
Ranking
Score

20
4502027
serum albumin
1
4069
20
1638

preproprotein

21
4557871
serotransferrin
11
1252
287
199

precursor

22
115298678
complement C3
117
247
779
130

precursor

23
50363217
alpha-1-antitrypsin
9
1531
16
1940

precursor

24
66932947
alpha-2-
60
452
336
190

macroglobulin

precursor

25
4557321
apolipoprotein A-I
—
—
—
—

preproprotein

26
324021745
vitamin D-binding
420
66
0
0

protein isoform 3

precursor

27
105990532
apolipoprotein B-100
222
118
86
351

precursor

28
4826762
haptoglobin isoform 1
27
795
0
0

preproprotein

29
62739186
complement factor H
1827
29
0
0

isoform a precursor

30
4557485
ceruloplasmin
115
250
2820
40

precursor

31
11321561
hemopexin precursor
113
256
1594
83

Because the levels of certain proteins and polypeptides may vary between different samples, for example, between different patients, and between different samples taken from the same patient at different times, control proteins and polypeptides were identified in gastrointestinal lavage fluid from patients. Table 4 provides example proteins and polypeptides whose levels did not fluctuate significantly between patients with and without pancreatic cancer. The proteins and polypeptides listed in Table 4 include those that originate from the intestine. Some of these proteins that originate from the intestine had an apparent increase in levels in pancreatic cancer, however, this may have been partly due to decreased levels in pancreatic enzymes and other proteins. Preferred control proteins included any with relatively constant levels between patient, and patient types, and included calcium-activated chloride channel regulator 1 precursor; intestinal-type alkaline phosphatase precursor; sucrase-isomaltase intestinal; and maltase-glucoamylase intestinal.

TABLE 4

Proteins not Exhibiting Significant Fluctuation in Gastrointestinal

Lavage Fluid of Subjects with Pancreatic Cancer

NCBI

MgC** Pancreatic

SEQ ID
Accession

MgC** Control
Cancer

NO.:
Nos.
Protein name
Ranking
Score
Ranking
Score

32
157266300
aminopeptidase N precursor
6
3633
25
1589

33
110611231
calcium -activated chloride
9
2731
6
4412

channel regulator 1 precursor

34
157266292
intestinal-type alkaline
91
339
54
830

phosphatase precursor

35
223942069
enteropeptidase precursor
43
656
43
914

36
18765694
dipeptidyl peptidase 4
33
970
36
1125

37
153070264
meprin A subunit beta
27
1164
47
885

precursor

38
153070262
meprin A subunit alpha
31
1030
23
1713

precursor

39
157364974
sucrase-isomaltase intestinal
23
1290
15
1930

40
221316699
maltase-glucoamylase
12
2434
7
3811

intestinal

**MgC = magnesium citrate

Other proteins whose levels were found to either decrease or increase in cancer are shown in Table 5. Alpha-1-antitrypsin may originate from blood while other proteins listed were not typically detected in serum/plasma samples.

TABLE 5

NCBI

MgC** Pancreatic

SEQ ID
Accession

MgC** Control
Cancer

NO.:
Nos.
Protein name
Ranking
Score
Ranking
Score

41
7669492
glyceraldehyde-3-phosphate
7
2793
30
1280

dehydrogenase

42
10334859
creatine kinase U-type
44
656
—

mitochondrial precursor

23
50363217
alpha-1-antitrypsin precursor
16
1940
9
3440

44
110618248
cadherin-related family
54
501
—

member 5 isoform 1 precursor

45
148539840
deleted in malignant brain
57
471
—

tumors 1 protein isoform a

precursor

46
285002214
cadherin-related family
41
785
70
726

member 2 precursor

47
98986445
carcinoembryonic antigen-
222
221
34
1152

related cell adhesion molecule

5 preproprotein

48
4502517
carbonic anhydrase 1
487
162
31
1273

**MgC = magnesium citrate

Example 2: Analysis of Biomarkers in Patients

MS analysis indicating target protein position for gastrointestinal lavage fluid samples from four patients with pancreatic cancer was compared to four normal volunteers. For ELISA analysis, gastrointestinal lavage fluid collected from patients and volunteers was diluted ten-fold with phosphate buffered saline (lx PBS) and analyzed with commercial ELISA methods for some of the proteins and markers detected by MS as well as for known cancer associated antigens. These included pancreatic amylase (ARUP Test #20506, ARUP Laboratories, Salt Lake City, Utah), pancreatic lipase (ARUP Test #20715, ARUP Laboratories, Salt Lake City, Utah), carcinoembryonic antigen (CEA) (ARUP Test #20746, ARUP Laboratories, Salt Lake City, Utah), CA19-9 (ARUP Test #20746, ARUP Laboratories, Salt Lake City, Utah) and trypsin-like immunoreactivity (ARUP Test #70003, ARUP Laboratories, Salt Lake City, Utah). ELISA analyses showed agreement with mass spectrometry where the amounts of pancreatic enzymes in general were reduced and other proteins increased. The results for MS data and ELISA data are summarized in Tables 6 and 7, respectively.

TABLE 6

Mass Spectrometry Analysis

NCBI

MS Posn for
MS Posn for

SEQ ID
Accession

cancer patients
control patients

NO.:
Nos.
Protein name
PC1
PC2
PC3
PC4
C1
C2
C3
C1

1
10835000
pancreatic
ND
ND
ND
ND
2
1
4
1

triacylglycerol

lipase precursor

2
4502085
pancreatic alpha-
ND
ND
63
ND
7
5
1
2

amylase precursor

3
10280622
alpha-amylase 2B
ND
ND
ND
ND
14
8
2
3

precursor

13
4506145
trypsin-1
29
18
12
ND
9
33
9
18

preproprotein

12
4506147
trypsin-2
48
26
17
ND
6
28
15
28

preproprotein

TABLE 7

ELISA Analysis

Concentration
Concentration

NCBI

in cancer
in control

SEQ ID
Accession
Protein name,
patient samples
patient samples

NO.
Nos.
concentration units
PC1
PC2
PC3
PC4
C1
C2
C3
C1

1
10835000
pancreatic lipase,
<4
<4
<4
<4
2793
1060
4040
2525

U/L

2
4502085
pancreatic amylase,
32
31
3
<3
786
392
1240
929

3
10280622
U/L

12
4506145
trypsin-like
113
193
74
3
110
178
380
1586

13
4506147
immunoreactivity,

ng/mL

47
98986445
carcinoembryonic
807
526
157
1311
84
80
28
34

497
40255013
antigen, ng/mL

729
121114300

726
296317312

N/A
None
CA19-9, U/mL
43
83
43
34
28
23
8
6

(Glycan,

not

protein)

Example 3: Collection of Samples

GLF samples were collected from normal volunteers and analyzed by MS. Samples taken early in the bowel cleansing process (following initial induction of copious diarrhea) were compared to samples taken the end of the bowel preparation. The analysis showed that early sample collection yielded results (with respect to protein MS position) similar to the samples collected at the end of the bowel preparation. Thus a full bowel preparation, while desirable to remove stool material, may not be required in particular methods.

Example 4: General Materials and Methods: Sample Collection, Preparation, Processing and Analysis

Control Samples

Control samples were obtained from the Gastrointestinal Laboratory University of South Alabama Medical Center by aspiration of residual gastrointestinal lavage fluid (gastrointenstinal lavage fluid) from the bowels of patients at the beginning of the colonoscopy procedure. Control samples were from routine colonoscopies that were found to be free from adenomas or colorectal cancer and were prepared for colonoscopy using SuPrep (Braintree Laboratories, Braintree, Mass.) per manufacturer's instructions or Polyethylene glycol electrolyte solution (PEG-ELS). Approximately 30 ml of gastrointestinal lavage fluid was aspirated into a mucus trap placed in-line with the endoscope. Immediately after collection, gastrointestinal lavage fluid was transferred to a labeled conical centrifuge tube containing a protease-inhibitor tablet (Complete tablet; Roche, Mannheim, Germany) and stored at −20° C. for no more than 48 hours prior to processing.

Sample Preparation

After collection, samples were thawed and immediately centrifuged at 1000 rpm (˜200×g) for 25 minutes to remove large particulates and cells (FIG. 1). The supernatant was then centrifuged again at 14,000×g for 25 minutes to remove small particulates and bacteria. All centrifugation steps were performed in an Eppendorf model 5804 R centrifuge at 4° C.

Protein Isolation

As further set forth in Example 1, three hundred microliters of each sample was extracted three times with 1 ml of chloroform to remove lipid material and polyethylene glycol. After the final extraction, the aqueous layer was centrifuged at maximum speed for five minutes and 100 μl of the aqueous layer was taken from the top and transferred to a new Eppendorf tube. To precipitate the proteins from the sample, 400 μl of methanol was added to the 100 μl of sample. The sample was centrifuged briefly in a tabletop centrifuge to collect the pellet and 200 μl of chloroform was added to solubilize phospholipids in the methanol layer followed by 300 μl of water to dissolve excess salts and water-soluble pigments. The mixture was vortexed and then centrifuged for five minutes at 13,000×g. This causes the protein to partition at the interface between the aqueous layer, which contains the salts and pigments; and the organic layer, which contains the lipids.

The aqueous layer (about 750 μl) was carefully removed without disturbing the interface and discarded. After this, the protein at the interface was forced to pellet with the addition of 300 μl of methanol. The mixture was vortexed briefly and centrifuged at 13,000×g for five minutes. The supernatant was discarded and the pellet was dried in a speed vac (Savant, Thermo) for ten minutes. The protein pellet was resuspended in a 20 μl of 8 M urea, 10 mM TCEP, 5 mM EDTA, and 0.1 M ABC solution. Once the pellet was completely dissolved, the mixture was diluted with 60 μl of 50 mM ABC/10 mM TCEP and digested with 2 μl of 10 mM sequencing grade trypsin (Promega) overnight on a shaker at 600 rpm at 37° C.

The digest was diluted into an LC vial by adding 75 μl of the digest to 20 μl of water and 75 μl of this mixture was injected onto the C₁₈pre-column (5 μm; 5 by 0.3 mm; Zorbax; Agilent Technologies) connected to an Agilent 1200 series nano-liquid chromatography (nano-LC) pump and thermostated auto-injector (Agilent Technologies, Santa Clara, Calif.). Solvent A was 2% acetonitrile and 0.05% TFA in water, and solvent B consisted of 2% water and 0.05% TFA in acetonitrile. A flow rate of 200 μl/minute was maintained throughout the run. For the first 13 minutes, 2% solvent B was used to load the sample onto a C₁₈pre-column and wash it. From time 15 to 21 minutes the peptides were eluted from the column with 40% B and this fraction is collected. This was followed by a column wash with 90% B from time 22 to 30 minutes and re-equilibration to 2% B in the final minute. The entire run time was 31 minutes. The A280 peak area of the eluted peptide peak was used as an estimate of how much protein was retrieved.

The eluted peptides were dried in a speed vac (Savant, Thermo) and re-dissolved in an amount of 0.1% TFA equal to 1/100 of the area of the A280 peak in with a minimum volume of 50 μl and a maximum volume of 500 μl in order to normalize protein concentrations in the injected samples.

Mass Spectrometry

Samples were injected in triplicate into an Agilent 1200 series nano-liquid HPLC coupled to a linear ion trap/Orbitrap hybrid MS (LTQ-Orbitrap (Thermo)). The HPLC mobile phases consisted of 3% acetonitrile and 0.2% formic acid in water (solvent A), and 3% water and 0.2% formic acid in acetonitrile (solvent B). A flow rate of 4 μl/minute of 5% solvent B was used to load the sample onto a C₁₈pre-column (5 μm; 5 by 0.3 mm; Zorbax; Agilent Technologies), and a flow rate of 1 μl/minute was used to elute the sample from the pre-column onto a separating Hypersil Gold C₁₈chromatography column (30 mm by 0.18 mm; Thermo Fisher Scientific). The linear solvent gradient was slowly ramped to 40% B over 70 min in order to elute the peptides from the column and then to 90% B over the final 20 min to wash the column. The total run time (pre-column and resolving chromatography) for each sample injection was 2 hours. During the 70 minute peptide elution at 40% B, eluted peptides were injected into the nanoflow source of the LTQ for MS-analysis. The LTQ-Orbitrap acquired one MS-only scan (Orbitrap) at a resolution of 60,000, while acquiring up to 5 MS-MS scans (LTQ), with a consistent cycle time of approximately 1 s, using the Xcalibur software program (Thermo Fisher Scientific). Peptide masses selected for fragmentation were then added to an exclusion list (within 10 ppm) to prevent repeated sequencing of abundant peptides for five minutes.

Example 5: Data Analysis and Results

MS/MS peptide sequence data obtained from the LTQ-Orbitrap from a representative control gastrointestinal lavage fluid sample collected during colonoscopy and prepared using the standard method described in FIG. 1 above were converted to mascot generic format files (.mgf) and ID matches identified using the Mascot search engine (http://www.matrixscience.com). Protein identifications (with a threshold of 95% confidence) were determined by the Mascot software program. All files were searched against a custom database generated by combining the NCBI RefSeq database with SwissProt Ig sequences (02-08-12-33712 sequences; 18670280 residues) using taxonomy: human, enzyme specificity: semi-trypsin, and a mass accuracy of 10 ppm for precursor ions and 0.6 DA for MS/MS data.

Table 8 shows the top 19 hits in order of Mascot Score, which is determined by how closely the data matches the theoretical data generated for that peptide sequence. The higher the score the more accurate the match as well as the more abundant the protein is in the sample.

TABLE 8

SEQ

NCBI

ID

Accession
Mascot

NO:
Rank
No.
Score
Protein Name

2
1
4502085
1212
Pancreatic alpha amylase precursor

(homo sapiens)

3
2
10280622
1093
Alpha-amylase 2B precursor

(homo sapiens)

49
3
148536848
1057
Bile salt activated lipase precursor

(homo sapiens)

1
4
10835000
1053
Pancreatic triacylglycerol lipase

precursor (homo sapiens)

6
5
54607080
501
Carboxypeptidase B preprotein

(homo sapiens)

8
6
236460050
395
Chymotrypsin-like elastase family

member 3A preprotein

(homo sapiens)

50
7
193806374
364
RecName: Full = Ig mu chain

C region

51
8
113584
354
RecName: Full = Ig alpha-1 chain

C region

13
9
4506145
335
Trypsin-1 preprotein

(homo sapiens)

52
10
31377806
323
Polymeric immunoglobulin

receptor precursor

(homo sapiens)

4
11
4502997
297
Carboxypeptidase A1 precursor

10
12
15559207
280
Chymotrypsin-like elastase family

member 2A preprotein

(homo sapiens)

53
13
218512088
273
RecName: Full = Ig alpha-2 chain

C region

54
14
341913702
269
PREDICTED: deleted in malignant

brain tumors 1 protein isoform 2

12
15
4506147
218
Trypsin-2 preprotein

(homo sapiens)

11
16
6679625
206
Chymotrypsin-like elastase family

member 3B preprotein

(homo sapiens)

55
17
118498341
196
Chymotrypsinogen B precursor

(homo sapiens)

23
18
50363217
166
Alpha-1-antitrypsin precursor

(homo sapiens)

7
19
217416390
165
Carboxypeptidase A2 precursor

(homo sapiens)

Mass, Time, and Intensity Data

The intensity of detected peptides was calculated based on MS data using an approach similar to the Accurate Mass Tag (AMT) method developed by Smith and coworkers (Conrads, T. P. et al., (2000) Anal. Chem. 72, 3349-3354; Strittmatter, E. F. et al., (2003) J. Am. Soc. Mass Spectrom. 14, 980-991). A program called DifProWare, a Web-based platform developed at the University of South Alabama (available at http://mciproteomics.usouthal.edu/difproware/) (Tucker, A. M. et al., (2011) Appl. Environ. Microbiol. 77, 4712-4718), was used to generate the mass, time, and intensity data for analysis. Briefly, MS/MS peptide sequence data were converted to mascot generic format files (.mgf) and matches identified using the Mascot search engine (http://www.matrixscience.com). Protein identifications (with a threshold of 95% confidence) were determined by the Mascot software program. All files were searched against a custom database generated by combining the NCBI RefSeq database with SwissProt Ig sequences (02-08-12-33712 sequences; 18670280 residues) using taxonomy: human, enzyme specificity: semi-trypsin, and a mass accuracy of 10 ppm for precursor ions and 0.6 DA for MS/MS data.

The MS-only data were examined using the ReSpect algorithm (Positive Probability, Ltd., Isleham, United Kingdom). This algorithm deconvolves detected peaks, converts electrospray mass spectra to zero-charge spectra, and corrects baselines, improving signal-to-noise ratios. The raw MS-only isotopic data are processed, generating a file containing deconvoluted mass, time, intensity, and probability statistics. Peptides were only accepted for analyses if they had an isotopic profile agreement confidence level of >95%. The Mascot ID information for each peptide as well as its mass, time, and intensity data in each sample being compared is combined within DifProWare and the resulting file is a comma-separated spreadsheet file associating peptide mass, time, intensity, and ID data.

Peptide to Protein Rollup

Protein abundances were calculated from the individual peptide abundances using the Rollup algorithm implemented in DanteR 0.2 (Taverner, T. et al., (2012) Bioinformatics 28, 2404-2406; Polpitiya, A. D. et al., (2008) Bioinformatics 24, 1556-1558) running under R 32-bit version 2.15.2 (R Development Core Team. (2013) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org) under Windows 7. DanteR is an open source software package that was developed by Tom Taverner and Ashoka Polpitiya at the Pacific Northwest National Laboratory to analyze proteomic data generated using the accurate mass and time tag approach.

This process combines intensity information from individual peptides into a single “intensity” for their identified protein. A brief summary of the process is as follows: For each group of peptides belonging to a single protein, the peptide with the highest overall abundance across all samples is chosen as a reference peptide. All peptides belonging to that protein are then expressed as a ratio to the reference value. The median ratio for each peptide across all samples is also calculated and the median ratio is subtracted from each peptide ratio. Outliers are then detected using Grubb's test and removed, and the median value of remaining selected peptide intensities is used to calculate the protein intensity.

The rollup was performed with the following parameters: rolling up using NCBI Accession number, minimum presence of at least one peptide at 50%, mode median, minimum dataset presence of three peptides, minimum number of peptides required for Grubb's test of 5, and p-value cutoff for Grubb's test at 0.05. The resulting spreadsheet of identified proteins and relative abundances was used in the subsequent statistical analyses.

Comparison of Home Collected Versus Clinic Collected Samples

In order to prove that major proteins were unaffected by the difference in the collection method between the home collected samples and the clinic collected samples, an experiment was performed comparing 44 matched pairs of samples. In one set, samples are self-collected by the subject via the toilet collection container method in which a hat is placed on a toilet seat for collection of gastrointestinal lavage fluid and transferred to tube with inhibitor immediately prior to colonoscopy (“hat samples”). In another set, samples are collected during colonoscopy through an endoscope (“scope samples”). Protein intensity values of the 44 “hat” and “scope” samples were obtained from LC-MS/MS data using the peptide to peptide rollup procedure described above and were compared using the Mann Whitney U test. The p-values are shown in Table 9 for three of the major proteins in gastrointestinal lavage fluid: carboxypeptidase B, pancreatic tracylglycerol lipase and chymotrypsin-like elastase family member 2A, demonstrating the differences were not significant.

TABLE 9

Comparison of Hat vs. Scope Collections

SEQ
NCBI

ID
Accession

NO.
Nos.
# Peptides
Protein ID
P value

6
54607080
30
Carboxypeptidase B
0.90368

1
10835000
41
Pancreatic triacylglycerol lipase
0.40868

10
15559207
15
Chymotrypsin-like elastase
0.84777

family member 2A

Overall comparisons of changes in individual hat and scope pairs were no greater than changes seen in replicates of the same samples. Therefore, the collection method does not affect the data and the two methods may be used interchangeably and compared.

Reproducibility of Methodology

One control sample was processed six times according to the standard methods described previously and the ratios of the intensities of the indicated proteins were analyzed between all pairs of replicates (36 combinations) (FIG. 2). A ratio of 1=identical. Bars show the 5-95% range of the ratios. Analytical replicates did not vary from each other by more than −20%. The data demonstrated that the profiles of key proteins as shown in FIG. 2 showed little variation, and that the method is highly reproducible. 2-sigma confidence level is shown.

Comparison of Abundance of Proteins in GLF of Subjects with Resectable Pancreatic Ductal Adenocarcinoma Versus Abundance of Proteins in GLF of Healthy Subjects

PDAC gastrointestinal lavage fluid samples were collected from 27 cases of resectable PDAC patients in pre-op prior to surgery. Patients had been bowel prepped with two bottles of magnesium citrate solution the previous night and had not eaten or drank since midnight the night before the sample was taken. Patients were asked to defecate into a collection container that fits over the toilet, and the gastrointestinal lavage fluid was transferred to a labeled conical centrifuge tube containing a protease inhibitor tablet (Complete tablet; Roche, Mannheim, Germany) and transported to the laboratory immediately on ice.

The average rankings of the top pancreatic proteins in gastrointestinal lavage fluid were compared between these 27 PDAC patients and 121 control gastrointestinal lavage fluid samples collected at colonoscopy as described previously using the ranking of protein abundance as determined by Mascot as described previously above. The ranking of the pancreatic proteins was significantly decreased in the PDAC group as compared to the control group (p<1.0E-09) (Table 10). Average intensities calculated using the rollup algorithm as described above were also compared and the fold change indicated.

TABLE 10

Relative abundance of proteins detected in the GLF between

healthy control N = 121 and resectable PDAC (N =

27) cases out of >300 detectable proteins (ND = non-detected)

Intensity

Fold

SEQ
NCBI

Decrease

ID
Accession

Healthy
PDAC
PDAC/

NO.
Nos.
Protein ID
Rank
Rank
Healthy

1
10835000
Pancreatic triacylglycerol lipase
1
391
226

2
4502085
Pancreatic alpha-amylase
2
21
7

3
10280622
Alpha-amylase 2B
3
22
18

4
4502997
Carboxypeptidase A1
4
160
10

56
56549662
Alpha-amylase 1
5
20
10

6
54607080
Carboxypeptidase B
10
163
25

7
217416390
Carboxypeptidase A2
11
ND
27

8
236460050
Chymotrypsin-like elastase
17
124
38

family member 3A

9
62526043
Chymostrypsin-C
19
ND
53

10
15559207
Chymotrypsin-like elastase
21
126
6

family member 2A

11
6679625
Chymotrypsin-like elastase
24
107
7

family member 3B

*12 of the PDAC cases showed no evidence of ductal dilation

Analysis of Amylase and Lipase Via Spectrometry and ELISA

GLF samples obtained from three control samples obtained by colonoscopy and three of the PDAC samples obtained prior to surgery were diluted 10× in PBS and analyzed for amylase and lipase using standard ELISA methods which measure units of enzyme per liter. The data demonstrated a greater than 250 fold decrease in lipase and a greater than 3.7 fold decrease in amylase between the PDAC and control samples. Furthermore the MS data and the ELISA data were concordant. MS values are denoted with Mascot scores, determined as described previously above.

TABLE 11

Comparison of Amylase and Lipase Assessed via Mass Spectrometry

(MS) vs. ELISA in Healthy and PDAC samples

Test

Amylase
Lipase 78974

Subject #
Assay
78914 (μ/L)
(μ/L)

Healthy
1
ELISA
1240
4040

MS
1212
1053

2
ELISA
929
2525

MS
1774
2297

3
ELISA
4114
2020

MS
2333
2386

PDAC
4
ELISA
32
<4

MS
0
0

5
ELISA
199
<4

MS
447
0

6
ELISA
250
<4

MS
251
0

7
ELISA
<3
<4

MS
0
0

Comparison of Pancreatic Proteins in Pancreatic Juice as Compared to in Gastrointestinal Lavage Fluid

The average rankings of the top pancreatic proteins in pancreatic juice collected directly from the pancreatic duct during surgery in six PDAC patients (labeled “pc”), and one patient determined to have an intraductal papillary mucinous neoplasm (labeled “IPMN 75”) (which is a benign lesion than may progress to PDAC if left untreated) were compared to pancreatic juice from three patients found to have benign pancreatic cysts at surgery (labeled “cyst”). Samples were compared as described previously using the ranking of protein abundance as determined by Mascot as described previously above. The ranking of the pancreatic proteins was significantly decreased in the pancreatic juice from the PDAC group as demonstrated previously in gastrointestinal lavage fluid. This shows that pancreatic proteins are reduced in both the direct pancreatic secretions as well as the gastrointestinal lavage fluid. The proteins were still present in the benign IPMN and in the benign cyst cases. Results are depicted in Table 12.

TABLE 12

Comparison of Pancreatic Proteins in Pancreatic Juice as Compared to in Gastrointestinal Lavage Fluid

NCBI

cyst
cyst
cyst
ipmn
pc
pc
pc
pc
pc
pc

SEQ ID
Accession

55
61
69
75
29
5
30
44
47
70

NO.
Nos.
Protein name
Posn
Posn
Posn
Posn
Posn
Posn
Posn
Posn
Posn
Posn

49
148536848
bile salt-activated
4
3
5
3
60
30
ND
162
ND
9

lipase precursor

1
10835000
pancreatic
3
4
7
7
ND
81
ND
58
ND
24

triacylglycerol

lipase precursor

12
4506147
trypsin-2
13
5
14
6
23
12
34
24
11
21

preproprotein

13
4506145
trypsin-1
6
8
8
5
21
13
25
22
4
17

preproprotein

55
118498341
chymotrypsinogen
8
9
15
8
36
14
ND
16
26
20

B precursor

57
106507261
pancreatic lipase-
16
11
49
13
ND
ND
ND
85
ND
91

related protein 2

precursor

793
342672030
trypsin-3 isoform 3
18
12
22
14
ND
ND
ND
ND
ND
ND

preproprotein

8
236460050
chymotrypsin-like
11
14
18
9
225
ND
ND
26
28
23

elastase family

member 3A

preproprotein

2
4502085
pancreatic alpha-
10
15
12
12
46
ND
ND
31
3
28

amylase precursor

10
15559207
chymotrypsin-like
20
16
23
30
ND
ND
ND
ND
ND
ND

elastase family

member 2A

preproprotein

3
10280622
alpha-amylase 2B
14
19
13
15
ND
ND
ND
ND
7
ND

precursor

4
4502997
carboxypeptidase
21
20
31
21
ND
99
ND
78
42
ND

A1 precursor

6
54607080
carboxypeptidase
17
22
16
22
37
15
ND
19
22
31

B preproprotein

23
50363217
alpha-1-antitrypsin
23
23
10
33
12
20
26
12
2
18

precursor

11
6679625
chymotrypsin-like
22
24
30
16
ND
ND
ND
ND
27
ND

elastase family

member 3B

preproprotein

9
62526043
chymotrypsin-C
27
28
55
36
ND
ND
ND
ND
ND
ND

preproprotein

7
217416390
carboxypeptidase
31
29
ND
31
ND
ND
ND
ND
ND
ND

A2 precursor

58
58331211
chymotrypsin-like
29
35
ND
53
ND
ND
ND
ND
ND
ND

elastase family

member 2B

preproprotein

Assessment of Gastrointestinal Lavage Fluid Samples from Subjects with Pancreatic Ductal Adenocarcinoma in Head of Pancreas

A second group of PDAC patient gastrointestinal lavage fluid samples were obtained. Patients with pancreatic masses detected using imaging were recruited to the study. gastrointestinal lavage fluid samples were collected after detection of the mass but prior to surgery. Those who were subsequently found to have pancreatic ductal adenocarcinoma in the head of the pancreas (n=6) were selected for comparison to the controls. The patient was provided with a kit to take home that included a dose of SuPrep bowel preparation solution (Braintree Laboratories, Braintree, Mass.), a collection container that fits over the toilet, a labeled conical centrifuge tube containing a protease inhibitor tablet (Complete tablet; Roche, Mannheim, Germany), and a disposable pipette for transfer of sample from toilet collection container to conical tube. The patient collected a sample of clear gastrointestinal lavage fluid and shipped it frozen on ice to the laboratory for analysis. The sample was prepared in the same manner as the previously obtained controls that were collected at colonoscopy as described previously. Data were processed using the standard approach described previously, with the exception of a 2 group ANOVA (t-test) in DanteR being used for comparison instead of the Mann Whitney U test. The intensity values of the individual peptides prior to “rollup” into protein values between 81 control samples that had been bowel-prepared with SuPrep and the 6 PDAC head samples that were also bowel-prepared using SuPrep were compared (FIG. 3). Of the 27,318 peptides were analyzed, 619 were significantly decreased in the PDAC cases (p<0.01). In contrast, 2227 peptides were significantly increased in the PDAC cases (p<0.01). Many of the peptides were unidentified and may contain post-translational modifications or mutations that may cause mass shifts.

The peptide intensity data was “rolled up” into protein intensity data as described above. Intensities of all proteins were compared between the 81 control samples that had been bowel-prepared using SuPrep and the 6 PDAC head samples that were also bowel-prepared using SuPrep using a 2 group ANOVA (t-test) in DanteR (FIG. 4). The data demonstrated that 25 peptides significantly decreased and 33 peptides significantly increased in the PDAC cases.

Table 13 depicts the rolled up intensity values of proteins present in the 6 PDAC head as compared to the 81 control samples. Log 2 Fold changes and p-values as determined by protein level ANOVA are shown.

TABLE 13

PDAC in the Head of the Pancreas

SEQ
NCBI

ID
Accession

Log_2

NO.
Nos.
Protein
change
P value

59
189083692
Fructose-1:6-
5.39
4.23E−06

bisphosphatase 1

20
4502027
Serum Albumin Preprotein
3.43
.0003

10
15559207
Chymotrypsin-like
−5.36
.0008

elastase family

member 2A preprotein

60
11225609
Angiotensin-converting
3.75
.0024

enzyme 2

precursor

16
6679625
Chymotrypsin-like
−1.82
.0025

elastase family

member 3B preprotein

62
93141226
Xaa-Pro aminopeptidase 2
3.99
.0065

precursor

58
58331211
Chymotrypsin-like
−6.15
.0189

elastase family

member 2B preprotein

6
54607080
Carboxypeptidase B
−2.18
.0189

preprotein

40
221316699
Maltase-glucoamylase:
1.81
.0398

intestinal

A similar experiment was performed with respect to gastrointestinal lavage fluid obtained from 3 subjects with neuroendocrine tumors present in the tail of the pancreas and compared to the 81 control samples, per the methods and analysis described above. Table 14 depicts the rolled up intensity values of proteins present in the gastrointestinal lavage fluid obtained from these subjects as compared to the 81 control samples. Log 2 Fold changes and p-values as determined by protein level ANOVA are shown.

TABLE 14

Neuroendocrine Cancer in the Tail of the Pancreas

SEQ
NCBI

ID
Accession

Log_2
P

NO.
Nos.
Protein
change
value

62
93141226
Xaa-Pro aminopeptidase 2
8.42
2.26E−05

precursor

63
308736985
Mucin 13 precursor
11.66
.0002

[homo sapiens]

64
4503273
Angiotensin-converting
2.79
.0002

enzyme isoform 1

precursor

40
221316699
Maltase-glucoamylase:
3.89
.0016

intestinal

56
56549662
Alpha-amylase 1 precursor
4.65
.0024

3
10280622
Alpha amylase 2B precursor
4.51
.0051

2
4502085
Pancreatic alpha-amylase
6.30
.0059

precursor

9
62526043
Chymotrypsin-C-preprotein
5.52
.0060

41
7669492
Glyceraldehyde-3 phosphate
4.29
.0075

dehydrogenase

66
132814467
Glutamyl aminopeptidase
6.91
.0091

As set forth in Tables 13 and 14, some pancreatic proteins were significantly decreased in PDAC cases but increased or unchanged in neuroendocrine cases.

Table 15 depicts the mascot positions (ranks) of major pancreatic enzymes, intestinal proteins, and serum proteins (Albumin and AAT) compared between the average of 6 PDAC head samples and the average of the 3 neuroendocrine tail pancreatic cancer samples, collected and processed as described above.

TABLE 15

SEQ
NCBI

Tail

ID
Accession

Head
Neuroen-

NO.
Nos.
Protein
PDAC
docrine

Pancreatic Enzymes

2
4502085
Pancreatic alpha-amylase
10
1

precursor

3
10280622
Alpha-amylase 2B precursor
0
2

5
40254482
Alpha-amylase 1 precursor
12
3

1
10835000
Pancreatic triacylglycerol
8
4

lipase precursor

8
236460050
Chymotrypsin-like
29
9

elastase family

member 3A preprotein

11
6679625
Chymotrypsin-like
44
17

elastase family

member 3B preprotein

13
4506145
Trypsin-1 preprotein
47
18

Intestinal Proteins

39
157364974
Sucrose-isomaltase intestinal
11
7

32
157266300
Aminopeptidase N precursor
51
8

40
221316699
Maltase glucoamylase
7
12

intestinal

33
110611231
Calcium-activated chloride
14
1

channel regulator 1 precursor

Albumin and AAT

20
4502027
Serum albumin preprotein
2
36

23
50363217
Alpha-1-antitrypsin precursor
3
6

Table 16 provides a complete list of proteins that change between PDAC and control cases. Table 16 reflects changes in more than just pancreatic enzymes.

TABLE 16

Complete List of Proteins that Change between PDAC and Control Cases

NCBI

SEQ ID
Accession

Head
Tail

NO:
No.
Protein name
Posn
Score
Posn
Score

2
4502085
pancreatic alpha-amylase precursor
10
3554
1
5890

[Homo sapiens]

3
10280622
alpha-amylase 2B precursor [Homo sapiens]
0
0
2
5626

5
40254482
alpha-amylase 1 precursor [Homo sapiens]
12
3320
3
5557

1
10835000
pancreatic triacylglycerol lipase
8
4689
4
3575

precursor [Homo sapiens]

68
154146262
IgGFc-binding protein precursor [Homo sapiens]
5
5354
5
2765

23
50363217
alpha-1-antitrypsin precursor [Homo sapiens]
3
5912
6
2464

39
157364974
sucrase-isomaltase intestinal [Homo sapiens]
11
3542
7
2267

32
157266300
aminopeptidase N precursor [Homo sapiens]
51
824
8
2088

8
236460050
chymotrypsin-like elastase family
29
1272
9
1959

member 3A preproprotein [Homo sapiens]

53
218512088
RecName: Full = Ig alpha-2 chain C region
4
5447
10
1878

51
113584
RecName: Full = Ig alpha-1 chain C region
1
7047
11
1626

40
221316699
maltase-glucoamylase intestinal [Homo sapiens]
7
5117
12
1599

33
110611231
calcium-activated chloride channel
14
2356
13
1528

regulator 1 precursor [Homo sapiens]

50
193806374
RecName: Full = Ig mu chain C region
13
2856
14
1499

69
125145
RecName: Full = Ig kappa chain C region
9
3684
15
1383

52
31377806
polymeric immunoglobulin receptor
6
5266
16
1089

precursor [Homo sapiens]

11
6679625
chymotrypsin-like elastase family
44
904
17
983

member 3B preproprotein [Homo sapiens]

13
4506145
trypsin-1 preproprotein [Homo sapiens]
47
873
18
942

6
54607080
carboxypeptidase B preproprotein
17
1696
19
871

[Homo sapiens]

10
15559207
chymotrypsin-like elastase family
60
749
20
826

member 2A preproprotein [Homo sapiens]

9
62526043
chymotrypsin-C preproprotein [Homo sapiens]
65
723
21
822

12
4506147
trypsin-2 preproprotein [Homo sapiens]
48
866
22
821

55
118498341
chymotrypsinogen B precursor [Homo sapiens]
20
1492
23
776

70
291045225
titin isoform N2-A [Homo sapiens]
39
933
24
736

71
291045230
titin isoform novex-2 [Homo sapiens]
38
940
25
705

64
4503273
angiotensin-converting enzyme isoform
52
820
26
690

1 precursor [Homo sapiens]

72
119220571
pancreatic secretory granule membrane
1256
162
27
671

major glycoprotein GP2 isoform 2

precursor [Homo sapiens]

49
148536848
bile salt-activated lipase precursor
21
1478
28
624

[Homo sapiens]

36
18765694
dipeptidyl peptidase 4 [Homo sapiens]
40
931
29
597

73
256222411
filamin-B isoform 1 [Homo sapiens]
86
561
30
552

37
153070264
meprin A subunit beta precursor [Homo sapiens]
37
984
31
522

74
21489959
immunoglobulin J chain precursor
26
1435
32
502

[Homo sapiens]

46
285002214
cadherin-related family member 2
16
1748
33
488

precursor [Homo sapiens]

38
153070262
meprin A subunit alpha precursor
33
1147
34
481

[Homo sapiens]

75
4507725
transthyretin precursor [Homo sapiens]
15
1773
35
409

20
4502027
serum albumin preproprotein [Homo sapiens]
2
6899
36
407

18
83367077
mucin-16 [Homo sapiens]
72
639
37
394

76
121039
RecName: Full = Ig gamma-1 chain C region
41
915
38
386

77
148833506
obscurin isoform b [Homo sapiens]
94
546
39
374

17
16306550
selenium-binding protein 1 [Homo sapiens]
31
1190
40
373

4
4502997
carboxypeptidase A1 precursor [Homo sapiens]
18
1672
41
373

78
118572606
hemicentin-1 precursor [Homo sapiens]
109
505
42
366

79
341913700
PREDICTED: deleted in malignant brain tumors
0
0
43
363

1 protein isoform 1 [Homo sapiens]

45
148539840
deleted in malignant brain tumors 1 protein
0
0
44
362

isoform a precursor [Homo sapiens]

80
125817
RecName: Full = Ig kappa chain V-III
0
0
45
347

region HAH; Flags: Precursor

81
163659918
sacsin [Homo sapiens]
90
555
46
346

82
151301127
dynein heavy chain 7 axonemal [Homo sapiens]
177
380
47
344

83
297206791
fibrous sheath-interacting protein 2
82
566
48
342

[Homo sapiens]

84
298351714
RecName: Full = Ig lambda-2 chain C regions
22
1470
49
338

85
296080693
glucose-6-phosphate isomerase isoform
243
325
50
336

1 [Homo sapiens]

86
148762969
histone-lysine N-methyltransferase
123
478
51
333

MLL2 [Homo sapiens]

87
156766050
protein AHNAK2 [Homo sapiens]
111
499
52
330

88
61743954
neuroblast differentiation-associated
141
443
53
326

protein AHNAK isoform 1 [Homo sapiens]

89
119395750
keratin type II cytoskeletal 1
98
542
54
320

[Homo sapiens]

90
113722120
G-protein coupled receptor 98 precursor
112
498
55
316

[Homo sapiens]

91
30520377
CUB and zona pellucida-like domain-
0
0
56
316

containing protein 1 precursor

[Homo sapiens]

92
257196151
immunoglobulin-like and fibronectin
159
399
57
312

type III domain-containing protein 1

[Homo sapiens]

93
330688408
nesprin-1 isoform 1 [Homo sapiens]
0
0
58
311

94
226246554
coiled-coil domain-containing protein
80
578
59
311

168 [Homo sapiens]

95
33188445
microtubule-actin cross-linking factor 1
104
520
60
309

isoform a [Homo sapiens]

96
366039979
RING finger protein 213 isoform 3
113
497
61
308

[Homo sapiens]

65
126032348
E3 ubiquitin-protein ligase HERC2
184
369
62
305

[Homo sapiens]

97
291190787
probable E3 ubiquitin-protein ligase
118
484
63
304

MYCBP2 [Homo sapiens]

98
13489087
leukocyte elastase inhibitor [Homo sapiens]
69
689
64
304

99
4504875
kallikrein-1 preproprotein [Homo sapiens]
568
228
65
300

41
7669492
glyceraldehyde-3-phosphate
35
1047
66
300

dehydrogenase [Homo sapiens]

42
10334859
creatine kinase U-type mitochondrial
599
223
67
298

precursor [Homo sapiens]

100
19115954
dynein heavy chain 5 axonemal [Homo sapiens]
125
472
68
292

101
118918407
nesprin-2 isoform 5 [Homo sapiens]
0
0
69
291

102
47078295
adenosine deaminase [Homo sapiens]
58
753
70
290

103
223555935
dynein heavy chain 14 axonemal
193
367
71
289

isoform 1 [Homo sapiens]

104
13654237
DNA-dependent protein kinase catalytic
0
0
72
288

subunit isoform 1 [Homo sapiens]

105
110349721
titin isoform novex-3 [Homo sapiens]
143
441
73
287

106
119395734
breast cancer type 2 susceptibility
239
327
74
286

protein [Homo sapiens]

35
223942069
enteropeptidase precursor [Homo sapiens]
59
752
75
285

107
91199540
dihydrolipoyl dehydrogenase
352
274
76
284

mitochondrial precursor [Homo sapiens]

108
91718902
histone-lysine N-methyltransferase
181
375
77
283

MLL3 [Homo sapiens]

57
106507261
pancreatic lipase-related protein 2
2178
122
78
281

precursor [Homo sapiens]

109
119703749
hydrocephalus-inducing protein
172
386
79
281

homolog isoform a [Homo sapiens]

110
125788
RecName: Full = Ig kappa chain V-II
89
557
80
272

region TEW

111
33350932
cytoplasmic dynein 1 heavy chain 1
136
451
81
272

[Homo sapiens]

112
116063573
filamin-A isoform 1 [Homo sapiens]
399
262
82
271

113
150418007
E3 SUMO-protein ligase RanBP2 [Homo sapiens]
395
262
83
271

114
31657092
ATP-binding cassette sub-family A
183
370
84
271

member 13 [Homo sapiens]

115
150378539
protein piccolo isoform 1 [Homo sapiens]
127
467
85
270

27
105990532
apolipoprotein B-100 precursor
148
424
86
269

[Homo sapiens]

116
54607053
translational activator GCN1 [Homo sapiens]
354
272
87
266

117
256017163
MAX gene-associated protein isoform 1
0
0
88
266

[Homo sapiens]

118
120587023
small subunit processome component 20
502
238
89
265

homolog [Homo sapiens]

119
41322923
plectin isoform 1a [Homo sapiens]
0
0
90
265

120
226529917
triosephosphate isomerase isoform 2
87
560
91
264

[Homo sapiens]

121
18375650
tyrosine-protein phosphatase non-
247
322
92
264

receptor type 13 isoform 4 [Homo sapiens]

122
126131099
probable E3 ubiquitin-protein ligase
115
490
93
264

HERC1 [Homo sapiens]

123
34577049
bullous pemphigoid antigen 1 isoform
121
479
94
263

1eA precursor [Homo sapiens]

61
32967601
ankyrin-3 isoform 1 [Homo sapiens]
156
408
95
260

124
93102379
low-density lipoprotein receptor-related
314
289
96
259

protein 1B precursor [Homo sapiens]

125
118498345
zinc finger homeobox protein 3 isoform
345
277
97
258

A [Homo sapiens]

126
359718912
probable E3 ubiquitin-protein ligase
191
367
98
257

C12orf51 [Homo sapiens]

127
115527120
nebulin isoform 3 [Homo sapiens]
116
486
99
255

128
28559088
laminin subunit alpha-2 isoform a
323
284
100
255

precursor [Homo sapiens]

129
171184451
centrosome-associated protein 350
317
287
101
252

[Homo sapiens]

130
221316593
cadherin-17 precursor [Homo sapiens]
262
316
102
252

131
150378498
uncharacterized protein KIAA1109
74
609
103
252

[Homo sapiens]

34
157266292
intestinal-type alkaline phosphatase
153
413
104
251

precursor [Homo sapiens]

132
332688227
dynein heavy chain 8 axonemal [Homo sapiens]
209
351
105
251

133
62241003
cardiomyopathy-associated protein 5
120
479
106
249

[Homo sapiens]

134
114155133
dynein heavy chain 9 axonemal isoform
155
412
107
249

2 [Homo sapiens]

135
341913678
PREDICTED: cadherin-23-like isoform
197
365
108
247

1 [Homo sapiens]

136
24415404
midasin [Homo sapiens]
114
496
109
247

137
88501738
TRIO and F-actin-binding protein
394
262
110
247

isoform 6 [Homo sapiens]

138
112799847
ryanodine receptor 2 [Homo sapiens]
138
449
111
247

139
122937398
cytoplasmic dynein 2 heavy chain 1
0
0
112
246

isoform 2 [Homo sapiens]

140
122937514
protein unc-13 homolog C [Homo sapiens]
218
347
113
246

141
87196343
PDZ domain-containing protein 2
316
289
114
245

[Homo sapiens]

142
256542310
dynein heavy chain 17 axonemal [Homo sapiens]
166
392
115
243

143
120587025
SH3 and multiple ankyrin repeat
441
250
116
241

domains protein 1 [Homo sapiens]

144
153792694
baculoviral IAP repeat-containing
189
368
117
240

protein 6 [Homo sapiens]

145
270265793
stAR-related lipid transfer protein 9
164
394
118
240

[Homo sapiens]

146
38455402
neutrophil gelatinase-associated
83
565
119
240

lipocalin precursor [Homo sapiens]

147
5031863
galectin-3-binding protein precursor
91
551
120
239

[Homo sapiens]

148
22538387
A-kinase anchor protein 9 isoform 2
201
362
121
239

[Homo sapiens]

149
257743023
nebulin isoform 1 [Homo sapiens]
0
0
122
239

150
55743098
collagen alpha-3(VI) chain isoform 1
214
349
123
239

precursor [Homo sapiens]

151
306922386
adenomatous polyposis coli protein
0
0
124
237

isoform a [Homo sapiens]

152
295986608
immunoglobulin lambda-like
30
1193
125
236

polypeptide 5 isoform 1 [Homo sapiens]

153
121047
RecName: Full = Ig gamma-4 chain C region
102
528
126
234

154
169658378
trinucleotide repeat-containing gene 18
393
263
127
233

protein [Homo sapiens]

155
149363685
uncharacterized protein KIAA0947
901
186
128
232

[Homo sapiens]

156
113204617
ryanodine receptor 1 isoform 2 [Homo sapiens]
0
0
129
231

157
54607139
vacuolar protein sorting-associated
145
429
130
227

protein 13D isoform 1 [Homo sapiens]

158
4501901
aminoacylase-1 isoform a [Homo sapiens]
0
0
131
225

159
1730075
RecName: Full = Ig kappa chain V-IV
103
523
132
224

region Len

160
38788274
nucleosome-remodeling factor subunit
380
265
133
223

BPTF isoform 1 [Homo sapiens]

161
119120894
dmX-like protein 2 isoform 2 [Homo sapiens]
483
243
134
223

162
93141047
collagen alpha-1(XII) chain long
558
229
135
222

isoform precursor [Homo sapiens]

163
14790190
msx2-interacting protein [Homo sapiens]
285
306
136
221

164
194353966
dynein heavy chain 6 axonemal [Homo sapiens]
299
300
137
220

165
197313748
histone-lysine N-methyltransferase
246
323
138
220

SETD2 [Homo sapiens]

166
4502961
collagen alpha-1(VII) chain precursor
126
468
139
219

[Homo sapiens]

167
331284180
nuclear receptor corepressor 2 isoform 3
0
0
140
219

[Homo sapiens]

168
165932370
protocadherin Fat 4 precursor [Homo sapiens]
206
356
141
219

169
198442844
dynein heavy chain 10 axonemal [Homo sapiens]
142
443
142
218

170
87196339
collagen alpha-1(VI) chain precursor
911
185
143
217

[Homo sapiens]

171
223633988
uncharacterized protein KIAA1671
298
300
144
217

[Homo sapiens]

172
16933557
protocadherin-16 precursor [Homo sapiens]
327
283
145
217

173
222144249
dynein heavy chain domain-containing
263
315
146
216

protein 1 isoform 1 [Homo sapiens]

174
66347828
vacuolar protein sorting-associated
240
326
147
216

protein 13C isoform 2A [Homo sapiens]

175
113722133
probable helicase senataxin [Homo sapiens]
963
181
148
216

176
126012573
low-density lipoprotein receptor-related
215
349
149
215

protein 2 precursor [Homo sapiens]

177
118498337
E3 ubiquitin-protein ligase HECTD1
296
301
150
215

[Homo sapiens]

178
4505847
phospholipase A2 precursor [Homo sapiens]
351
274
151
214

179
126116589
fibrocystin-L precursor [Homo sapiens]
280
307
152
214

180
169177000
PREDICTED: LOW QUALITY
157
406
153
214

PROTEIN: hemicentin-2 [Homo sapiens]

181
31563330
A-kinase anchor protein 13 isoform 1
304
293
154
213

[Homo sapiens]

182
79749430
FRAS1-related extracellular matrix
198
364
155
213

protein 2 precursor [Homo sapiens]

183
91208420
protein bassoon [Homo sapiens]
137
450
156
212

184
20336205
transcriptional regulator ATRX isoform
0
0
157
211

2 [Homo sapiens]

185
81295809
pericentrin [Homo sapiens]
135
451
158
211

186
45545421
ectonucleotide pyrophosphatase/
133
453
159
211

phosphodiesterase family member 7

precursor [Homo sapiens]

187
296011010
protein FAM208B [Homo sapiens]
373
267
160
210

188
82659109
E3 ubiquitin-protein ligase UBR4
128
467
161
210

[Homo sapiens]

189
169178458
PREDICTED: LOW QUALITY
170
390
162
209

PROTEIN: hemicentin-2 [Homo sapiens]

190
116805322
filamin-C isoform a [Homo sapiens]
0
0
163
209

191
4557793
neurofibromin isoform 2 [Homo sapiens]
0
0
164
209

192
45827701
protein dopey-2 [Homo sapiens]
548
231
165
209

193
149158690
protein PRRC2A [Homo sapiens]
916
185
166
208

194
35493701
vacuolar protein sorting-associated
253
320
167
207

protein 13B isoform 1 [Homo sapiens]

195
15147337
E3 ubiquitin-protein ligase UBR5
163
395
168
207

[Homo sapiens]

196
139948432
matrix-remodeling-associated protein 5
217
347
169
206

precursor [Homo sapiens]

197
87299628
biorientation of chromosomes in cell
213
350
170
206

division protein 1-like [Homo sapiens]

198
89363017
collagen alpha-2(V) chain preproprotein
744
200
171
206

[Homo sapiens]

199
126131102
fibrocystin isoform 1 precursor [Homo sapiens]
223
342
172
205

200
61676188
E3 ubiquitin-protein ligase HUWE1
292
303
173
204

[Homo sapiens]

201
55770834
centromere protein F [Homo sapiens]
174
384
174
204

202
157266317
serine/threonine-protein kinase ATR
402
261
175
204

[Homo sapiens]

203
105990541
retinal-specific ATP-binding cassette
863
189
176
204

transporter [Homo sapiens]

204
51479173
dynein heavy chain 11 axonemal
196
366
177
204

[Homo sapiens]

205
56550039
histone-lysine N-methyltransferase MLL
168
390
178
204

isoform 2 precursor [Homo sapiens]

206
170296790
trypsin-3 isoform 1 preproprotein
567
228
179
203

[Homo sapiens]

207
51317366
myosin-XVIIIb [Homo sapiens]
319
286
180
202

208
47717123
intersectin-1 isoform ITSN-1 [Homo sapiens]
462
247
181
202

209
4502443
bullous pemphigoid antigen 1 isoform
0
0
182
202

1e precursor [Homo sapiens]

210
62243658
serine/threonine-protein kinase SMG1
219
346
183
202

[Homo sapiens]

211
66346672
vacuolar protein sorting-associated
0
0
184
202

protein 13A isoform C [Homo sapiens]

212
111118976
collagen alpha-1(II) chain isoform 1
369
267
185
201

precursor [Homo sapiens]

213
121583483
1-phosphatidylinositol-3-phosphate 5-
312
290
186
201

kinase isoform 2 [Homo sapiens]

214
75677365
dynein heavy chain 2 axonemal [Homo sapiens]
282
306
187
200

215
27436938
reelin isoform a precursor [Homo sapiens]
0
0
188
200

216
21264602
laminin subunit alpha-5 precursor
457
247
189
200

[Homo sapiens]

217
257467639
uncharacterized protein KIAA0889
1244
163
190
200

isoform 1 [Homo sapiens]

218
110349772
collagen alpha-1(I) chain preproprotein
492
240
192
199

[Homo sapiens]

219
119372317
xin actin-binding repeat-containing
194
366
193
199

protein 2 isoform 1 [Homo sapiens]

220
134268640
alpha-tectorin precursor [Homo sapiens]
404
260
194
199

19
116284392
mucin-2 precursor [Homo sapiens]
75
600
195
199

221
92110053
CUB and sushi domain-containing
274
310
196
198

protein 2 [Homo sapiens]

222
291167749
zinc finger homeobox protein 4 [Homo sapiens]
203
359
198
198

223
223029410
talin-1 [Homo sapiens]
281
307
200
197

224
52426735
ankyrin-2 isoform 1 [Homo sapiens]
160
398
201
196

225
148746189
multiple PDZ domain protein [Homo sapiens]
396
262
202
196

226
109633039
receptor-type tyrosine-protein phosphatase F
709
205
204
196

isoform 2 precursor [Homo sapiens]

227
7656967
cadherin EGF LAG seven-pass G-type
560
229
205
196

receptor 1 precursor [Homo sapiens]

228
239735519
myotubularin-related protein 5 [Homo sapiens]
342
278
206
196

229
45439359
triple functional domain protein
268
313
207
195

[Homo sapiens]

230
41152086
serpin B6 [Homo sapiens]
55
775
208
195

231
256000767
extracellular matrix protein FRAS1
470
245
209
195

isoform 1 precursor [Homo sapiens]

232
19923586
histone-lysine N-methyltransferase H3
300
300
210
195

lysine-36 and H4 lysine-20 specific

isoform b [Homo sapiens]

233
118572613
serine/arginine repetitive matrix protein
465
246
211
194

2 [Homo sapiens]

234
153792012
DNA polymerase zeta catalytic subunit
278
308
213
193

[Homo sapiens]

235
149192855
protein PRRC2B [Homo sapiens]
340
278
214
193

236
67782321
spectrin beta chain erythrocyte isoform
453
248
215
192

a [Homo sapiens]

237
126116596
abnormal spindle-like microcephaly-
250
322
216
192

associated protein isoform 1 [Homo sapiens]

238
242332527
hypothetical protein LOC65250 [Homo sapiens]
353
272
217
192

239
149589008
xaa-Pro dipeptidase isoform 1 [Homo sapiens]
543
232
218
192

240
38788416
laminin subunit alpha-1 precursor
341
278
219
191

[Homo sapiens]

241
150418009
transforming acidic coiled-coil-containing
424
254
220
191

protein 2 isoform a [Homo sapiens]

242
4502951
collagen alpha-1(III) chain preproprotein
613
221
222
191

[Homo sapiens]

243
365192532
myosin-10 isoform 1 [Homo sapiens]
408
259
223
191

244
16445436
bromodomain and WD repeat-containing
495
240
224
191

protein 1 isoform A [Homo sapiens]

245
110349786
Alstrom syndrome protein 1 [Homo sapiens]
131
454
225
190

246
207452735
epiplakin [Homo sapiens]
412
257
226
190

247
153945846
inositol 1 4 5-trisphosphate receptor
307
293
228
189

type 3 [Homo sapiens]

248
221316747
exophilin-5 [Homo sapiens]
419
256
229
188

249
260064009
ubiquitin carboxyl-terminal hydrolase 24
273
310
230
188

[Homo sapiens]

250
310114187
PREDICTED: ankyrin repeat domain-
468
245
231
188

containing protein 36A [Homo sapiens]

251
66346693
protocadherin Fat 1 precursor
185
369
232
188

[Homo sapiens]

252
188536004
zinc finger protein 469 [Homo sapiens]
122
479
233
188

253
157785645
striated muscle preferentially expressed
256
318
234
187

protein kinase isoform 1 [Homo sapiens]

254
38045910
laminin subunit alpha-3 isoform 1
187
369
235
187

precursor [Homo sapiens]

255
26080431
ATPase family AAA domain-containing
360
270
236
187

protein 5 [Homo sapiens]

256
4503355
dedicator of cytokinesis protein 1
413
257
238
186

[Homo sapiens]

257
259013213
CUB and sushi domain-containing
295
301
239
186

protein 1 precursor [Homo sapiens]

258
148886654
sushi von Willebrand factor type A
248
322
240
186

EGF and pentraxin domain-containing

protein 1 precursor [Homo sapiens]

259
54292123
lysosomal-trafficking regulator
228
336
241
186

[Homo sapiens]

260
110735435
collagen alpha-3(V) chain preproprotein
265
314
242
185

[Homo sapiens]

261
122891870
melanoma inhibitory activity protein 3
421
256
243
185

precursor [Homo sapiens]

262
21264565
AT-rich interactive domain-containing
686
208
244
185

protein 1A isoform a [Homo sapiens]

263
38202205
zinc finger FYVE domain-containing
407
259
245
184

protein 26 [Homo sapiens]

264
13787217
protocadherin Fat 2 precursor [Homo sapiens]
241
326
246
184

265
24308169
dynein heavy chain 3 axonemal [Homo sapiens]
212
350
248
183

266
62177127
myosin-XVI isoform 2 [Homo sapiens]
506
237
249
183

267
224028289
tetratricopeptide repeat protein 28
222
342
250
183

[Homo sapiens]

268
126723564
pecanex-like protein 1 [Homo sapiens]
382
265
251
183

269
12667788
myosin-9 [Homo sapiens]
227
337
252
183

270
126012571
basement membrane-specific heparan
186
369
253
182

sulfate proteoglycan core protein

precursor [Homo sapiens]

271
5031587
adenomatous polyposis coli protein 2
397
262
255
181

[Homo sapiens]

272
92091572
dedicator of cytokinesis protein 4
531
233
256
181

[Homo sapiens]

273
10863903
probable E3 ubiquitin-protein ligase
500
239
258
181

TRIP 12 [Homo sapiens]

274
90903231
huntingtin [Homo sapiens]
326
283
259
180

275
156104874
envoplakin [Homo sapiens]
315
289
263
179

276
73747881
zinc finger ZZ-type and EF-hand
308
293
264
179

domain-containing protein 1 [Homo sapiens]

277
150170699
kinesin-like protein KIF26A [Homo sapiens]
444
249
265
179

278
13325064
cadherin EGF LAG seven-pass G-type
286
305
267
178

receptor 2 precursor [Homo sapiens]

279
281485550
fibrillin-1 precursor [Homo sapiens]
632
217
268
178

44
110618248
cadherin-related family member 5
107
511
271
177

isoform 1 precursor [Homo sapiens]

280
54873613
agrin precursor [Homo sapiens]
368
267
272
177

281
169790825
teneurin-4 [Homo sapiens]
313
289
273
177

282
150456444
protein unc-79 homolog [Homo sapiens]
329
282
274
177

283
148762940
protein Daple [Homo sapiens]
724
202
275
177

284
119964726
cation-independent mannose-6-phosphate
236
329
276
177

receptor precursor [Homo sapiens]

285
154354990
ankyrin repeat domain-containing
365
269
277
176

protein 26 isoform 1 [Homo sapiens]

286
62422577
neurobeachin isoform 1 [Homo sapiens]
410
258
279
176

287
148886692
protocadherin Fat 3 precursor [Homo sapiens]
290
303
281
175

14
29725633
lithostathine-1-alpha precursor
71
643
282
174

[Homo sapiens]

288
224458301
protein FAM186A [Homo sapiens]
696
207
284
174

289
7662046
histone-lysine N-methyltransferase
372
267
285
174

MLL4 [Homo sapiens]

290
95147335
inositol 1 4 5-trisphosphate receptor
288
304
286
174

type 2 [Homo sapiens]

291
54112403
chromodomain-helicase-DNA-binding
356
272
287
173

protein 7 [Homo sapiens]

292
191252801
WD repeat- and FYVE domain-
226
339
288
173

containing protein 4 [Homo sapiens]

24
66932947
alpha-2-macroglobulin precursor
32
1169
290
173

[Homo sapiens]

293
89276751
collagen alpha-1(V) chain preproprotein
336
280
291
173

[Homo sapiens]

294
255003833
centrosomal protein of 192 kDa [Homo sapiens]
325
283
294
172

295
50659080
alpha-1-antichymotrypsin precursor
23
1465
295
172

[Homo sapiens]

296
183583553
collagen alpha-5(VI) chain precursor
192
367
296
171

[Homo sapiens]

297
41054864
regulating synaptic membrane exocytosis
423
255
298
171

protein 1 isoform 1 [Homo sapiens]

298
301172750
mucin-5B precursor [Homo sapiens]
93
549
301
171

299
114842389
myosin-7B [Homo sapiens]
357
271
302
171

300
197927452
dynein heavy chain 1 axonemal [Homo sapiens]
165
393
305
170

301
110611228
utrophin [Homo sapiens]
208
353
306
170

302
55749742
HEAT repeat-containing protein 5B
302
297
307
170

[Homo sapiens]

303
117606355
protein furry homolog [Homo sapiens]
271
311
308
170

304
282165704
chromodomain-helicase-DNA-binding
237
329
310
169

protein 8 isoform 1 [Homo sapiens]

305
306922394
zinc finger homeobox protein 2 [Homo sapiens]
249
322
314
168

306
95147555
microtubule-associated protein 1A
272
310
316
168

[Homo sapiens]

307
359385708
uncharacterized protein C10orf92
486
242
317
168

[Homo sapiens]

308
126012562
prolow-density lipoprotein receptor-
348
275
318
168

related protein 1 precursor [Homo sapiens]

309
332634937
myomegalin isoform 9 [Homo sapiens]
426
254
319
168

310
118402590
myosin-XV [Homo sapiens]
230
334
321
168

311
156938343
talin-2 [Homo sapiens]
294
302
323
167

312
126091152
cubilin precursor [Homo sapiens]
403
261
325
167

313
219842266
usherin isoform B [Homo sapiens]
277
308
326
167

314
57222563
cytoskeleton-associated protein 5
409
259
330
167

isoform b [Homo sapiens]

315
148596944
C2 domain-containing protein 3 [Homo sapiens]
610
221
332
167

316
126032338
ryanodine receptor 3 isoform 1 [Homo sapiens]
149
424
333
166

317
87116683
zinc finger C3H1 domain-containing
671
211
336
165

protein [Homo sapiens]

318
56676397
ankyrin repeat domain-containing
676
209
337
165

protein 11 [Homo sapiens]

319
109255228
centrosomal protein of 170 kDa isoform
370
267
338
165

alpha [Homo sapiens]

320
122937512
myosin-VIIb [Homo sapiens]
416
257
340
164

321
188528648
tenascin-X isoform 1 precursor [Homo sapiens]
330
281
341
164

322
221219020
WD repeat-containing protein 87
640
215
342
164

[Homo sapiens]

323
49640009
E3 ubiquitin-protein ligase TTC3
371
267
344
164

[Homo sapiens]

324
109637791
transcription factor TFIIIB component
267
314
345
164

B″ homolog [Homo sapiens]

325
37620163
ANKHD1-EIF4EBP3 protein [Homo sapiens]
428
254
346
164

326
21626468
zinc finger protein 638 isoform 1
692
208
347
164

[Homo sapiens]

327
116256356
collagen alpha-4(IV) chain precursor
732
201
352
163

[Homo sapiens]

328
310110158
PREDICTED: otogelin isoform 1 [Homo sapiens]
739
201
353
163

329
71061468
centromere-associated protein E
238
328
356
162

[Homo sapiens]

330
123853
RecName: Full = Ig heavy chain V-III
139
448
357
162

region POM

331
115430237
spectrin beta chain brain 3 isoform
242
325
358
162

sigma1 [Homo sapiens]

332
145309304
cadherin EGF LAG seven-pass G-type
235
330
360
162

receptor 3 precursor [Homo sapiens]

333
144226847
obscurin-like protein 1 isoform 1
513
235
362
161

precursor [Homo sapiens]

334
40217847
U5 small nuclear ribonucleoprotein 200
386
264
363
161

kDa helicase [Homo sapiens]

335
93352554
probable G-protein coupled receptor 179
629
218
365
161

precursor [Homo sapiens]

336
239050813
lipoxygenase homology domain-containing
259
316
366
161

protein 1 isoform 1 [Homo sapiens]

337
21359935
Down syndrome cell adhesion molecule-
668
211
367
161

like protein 1 [Homo sapiens]

338
40805823
collagen alpha-1(XXII) chain precursor
387
264
369
161

[Homo sapiens]

339
19913408
DNA topoisomerase 2-beta [Homo sapiens]
328
282
372
160

340
203098098
protein Shroom3 [Homo sapiens]
347
275
374
160

341
57232740
N-acetylated-alpha-linked acidic
73
612
375
160

dipeptidase-like protein [Homo sapiens]

342
30089962
serine/threonine-protein kinase MRCK
587
225
376
159

alpha isoform B [Homo sapiens]

48
4502517
carbonic anhydrase 1 [Homo sapiens]
670
211
377
159

343
237681119
breast cancer type 1 susceptibility
636
216
378
159

protein isoform 2 [Homo sapiens]

344
112421122
dnaJ homolog subfamily C member 13
566
228
381
159

[Homo sapiens]

345
4507691
transformation/transcription domain-
244
324
382
159

associated protein isoform 2 [Homo sapiens]

346
89191868
von Willebrand factor preproprotein
438
250
385
158

[Homo sapiens]

347
125792
RecName: Full = Ig kappa chain V-II
140
446
387
157

region RPMI 6410; Flags: Precursor

348
29244924
chromodomain-helicase-DNA-binding
324
284
389
157

protein 6 [Homo sapiens]

349
104487006
receptor-type tyrosine-protein phosphatase S
750
200
391
157

isoform 1 precursor [Homo sapiens]

350
48762934
collagen alpha-2(I) chain precursor
224
339
392
157

[Homo sapiens]

351
38679967
acetyl-CoA carboxylase 1 isoform 2
461
247
393
157

[Homo sapiens]

352
34740331
otoferlin isoform a [Homo sapiens]
635
217
395
156

353
134031945
SCO-spondin precursor [Homo sapiens]
332
280
396
156

354
71361682
nuclear mitotic apparatus protein 1
216
347
397
156

[Homo sapiens]

355
71902540
serine-protein kinase ATM [Homo sapiens]
335
280
398
156

356
140560919
myomesin-1 isoform a [Homo sapiens]
293
303
399
156

357
150010558
myosin-15 precursor [Homo sapiens]
565
228
403
155

358
148536825
collagen alpha-1(IV) chain
497
239
404
155

preproprotein [Homo sapiens]

359
38683860
insulin receptor substrate 2 [Homo sapiens]
693
208
405
155

360
32481206
lactase-phlorizin hydrolase
43
909
406
154

preproprotein [Homo sapiens]

361
150170718
zinc finger protein 292 [Homo sapiens]
637
216
407
154

362
148528998
dmX-like protein 1 [Homo sapiens]
458
247
409
154

363
38045888
CUB and sushi domain-containing
283
306
410
154

protein 3 isoform 1 [Homo sapiens]

364
112734845
collagen alpha-1(XX) chain precursor
731
201
411
154

[Homo sapiens]

365
38093637
Nance-Horan syndrome protein isoform
431
252
414
153

1 [Homo sapiens]

366
119220552
protein sidekick-1 isoform 1 [Homo sapiens]
398
262
415
153

367
148612838
uncharacterized protein KIAA2026
375
266
416
152

[Homo sapiens]

368
95147342
chromodomain-helicase-DNA-binding
362
269
419
152

protein 9 [Homo sapiens]

369
110349788
histone-lysine N-methyltransferase
176
381
420
152

ASH1L [Homo sapiens]

370
254826809
prematurely terminated mRNA decay
569
228
423
152

factor-like [Homo sapiens]

371
150417973
supervillin isoform 2 [Homo sapiens]
434
251
425
151

372
148596992
alpha-protein kinase 2 [Homo sapiens]
602
222
426
151

373
209862789
protein MICAL-3 isoform 1 [Homo sapiens]
344
277
427
151

374
38490688
immunoglobulin superfamily member
279
307
430
151

10 isoform 1 precursor [Homo sapiens]

375
134142826
pericentriolar material 1 protein
581
226
432
151

[Homo sapiens]

376
87578396
microtubule-associated protein 2
522
234
434
151

isoform 1 [Homo sapiens]

377
134948558
ankyrin repeat domain-containing
728
202
435
150

protein 12 isoform 1 [Homo sapiens]

378
169658367
BAH and coiled-coil domain-containing
310
291
437
150

protein 1 [Homo sapiens]

379
4502337
zinc-alpha-2-glycoprotein precursor
50
836
438
150

[Homo sapiens]

380
119874201
protein furry homolog-like [Homo sapiens]
255
320
441
150

381
30794488
kinesin-like protein KIF27 [Homo sapiens]
401
261
442
150

382
257467648
microtubule-associated serine/threonine-
119
480
444
149

protein kinase 4 isoform c [Homo sapiens]

383
55956899
keratin type I cytoskeletal 9 [Homo sapiens]
100
528
445
149

384
58331187
T-lymphoma invasion and metastasis-
530
233
447
149

inducing protein 2 isoform a [Homo sapiens]

385
154350241
brefeldin A-inhibited guanine nucleotide-
625
218
448
149

exchange protein 3 [Homo sapiens]

386
262118282
plexin-A1 precursor [Homo sapiens]
690
208
451
148

387
21493045
A-kinase anchor protein 6 [Homo sapiens]
429
253
452
148

388
156105693
peroxisomal proliferator-activated
417
256
453
148

receptor A-interacting complex 285 kDa

protein isoform 1 [Homo sapiens]

389
194440727
dynein heavy chain 12 axonemal
199
363
456
148

isoform 1 [Homo sapiens]

390
335353804
protein SZT2 [Homo sapiens]
433
251
461
148

391
157738645
plexin-A4 isoform 1 precursor [Homo sapiens]
414
257
462
148

392
114431248
basic helix-loop-helix domain-containing
651
213
464
147

protein KIAA2018 [Homo sapiens]

393
371877632
armadillo repeat-containing X-linked
363
269
466
147

protein 4 [Homo sapiens]

394
116256354
collagen alpha-2(IV) chain
715
204
468
147

preproprotein [Homo sapiens]

395
145309309
probable ubiquitin carboxyl-terminal
322
284
469
147

hydrolase FAF-X isoform 3 [Homo sapiens]

396
47059046
protocadherin-23 isoform 1 [Homo sapiens]
547
231
476
147

397
115511036
alpha-protein kinase 3 [Homo sapiens]
746
200
478
146

398
87298937
centriolin [Homo sapiens]
367
267
480
146

399
267844811
neuron navigator 1 isoform 1 [Homo sapiens]
442
249
482
146

400
195972871
1-phosphatidylinositol-4 5-bisphosphate
643
215
486
145

phosphodiesterase eta-1 isoform a

[Homo sapiens]

401
134142062
acetyl-CoA carboxylase 2 precursor
318
286
488
145

[Homo sapiens]

402
156104908
myosin-6 [Homo sapiens]
56
767
490
145

403
31563507
GRIP and coiled-coil domain-containing
638
216
492
145

protein 2 [Homo sapiens]

404
148233596
lipopolysaccharide-responsive and
605
222
495
144

beige-like anchor protein isoform 2

[Homo sapiens]

405
262359929
protein ELYS [Homo sapiens]
188
369
497
144

406
154240686
FYVE RhoGEF and PH domain-
658
213
498
144

containing protein 6 [Homo sapiens]

407
110611226
protein unc-13 homolog B [Homo sapiens]
642
215
499
144

408
291190781
leucine-rich repeat-containing protein
749
200
500
144

16A isoform 1 [Homo sapiens]

409
153791497
rootletin [Homo sapiens]
430
252
501
143

410
122937211
proteasome-associated protein ECM29
710
205
503
143

homolog [Homo sapiens]

411
103472005
antigen KI-67 isoform 1 [Homo sapiens]
229
336
504
143

412
157738667
FYVE and coiled-coil domain-
563
228
507
143

containing protein 1 [Homo sapiens]

413
118600981
probable JmjC domain-containing
542
232
511
142

histone demethylation protein 2C

isoform a [Homo sapiens]

414
61743980
stabilin-2 precursor [Homo sapiens]
515
235
512
142

415
116534898
desmoglein-2 preproprotein [Homo sapiens]
685
209
515
142

416
115527097
serine/threonine-protein kinase MRCK
623
219
516
141

beta [Homo sapiens]

417
194294554
SET-binding protein isoform a [Homo sapiens]
425
254
517
141

418
110624781
myosin-13 [Homo sapiens]
364
269
520
140

419
149363642
coiled-coil domain-containing protein
519
235
522
140

144A [Homo sapiens]

420
112821681
G protein-regulated inducer of neurite
551
231
523
140

outgrowth 1 [Homo sapiens]

421
58530840
desmoplakin isoform I [Homo sapiens]
305
293
525
140

422
74136549
AT-rich interactive domain-containing
627
218
526
139

protein 5B isoform 1 [Homo sapiens]

423
157426887
dedicator of cytokinesis protein 6
538
232
527
139

[Homo sapiens]

424
71143119
signal-induced proliferation-associated
475
243
532
139

1-like protein 3 [Homo sapiens]

425
94681049
WD repeat-containing protein 96
507
237
534
139

[Homo sapiens]

426
146219843
helicase SRCAP [Homo sapiens]
350
274
538
139

427
56676335
telomere-associated protein RIF1
346
276
542
138

isoform 1 [Homo sapiens]

428
53832009
voltage-dependent T-type calcium
630
218
543
138

channel subunit alpha-1H isoform a

[Homo sapiens]

429
148536869
ninein isoform 2 [Homo sapiens]
439
250
544
138

430
170016091
teneurin-2 [Homo sapiens]
275
308
551
137

431
183396804
regulation of nuclear pre-mRNA domain-
665
212
552
137

containing protein 2 [Homo sapiens]

432
205360962
polycystin-1 isoform 2 precursor
269
313
553
137

[Homo sapiens]

433
270133251
amyotrophic lateral sclerosis 2
631
217
559
136

chromosomal region candidate gene 11

protein isoform 1 [Homo sapiens]

434
98986453
myosin-3 [Homo sapiens]
77
586
560
136

435
54112429
dedicator of cytokinesis protein 7
606
222
561
136

[Homo sapiens]

436
119703755
laminin subunit beta-2 precursor
704
206
564
136

[Homo sapiens]

437
148839466
kalirin isoform 1 [Homo sapiens]
254
320
566
136

438
115583670
T-lymphoma invasion and metastasis-
450
248
568
136

inducing protein 1 [Homo sapiens]

439
110611903
myosin-4 [Homo sapiens]
28
1297
570
135

440
217330594
tubulin polyglutamylase TTLL4 [Homo sapiens]
544
232
572
135

441
21361831
partitioning defective 3 homolog
730
202
576
135

isoform 1 [Homo sapiens]

442
38202209
methyl-CpG-binding domain protein 5
703
206
580
135

[Homo sapiens]

443
110347463
transcription factor HIVEP2 [Homo sapiens]
546
232
584
135

444
111118970
collagen alpha-2(XI) chain isoform 1
652
213
589
134

preproprotein [Homo sapiens]

445
190194412
thyroid receptor-interacting protein 11
655
213
591
134

[Homo sapiens]

446
116006951
polycystic kidney disease protein 1-like
590
225
595
134

2 isoform a precursor [Homo sapiens]

447
224451128
protein eyes shut homolog isoform 1
456
247
597
134

[Homo sapiens]

448
115527062
collagen alpha-2(VI) chain isoform 2C2
621
219
600
133

precursor [Homo sapiens]

449
50959205
adenylate cyclase type 9 [Homo sapiens]
578
226
602
133

450
21361458
rho guanine nucleotide exchange factor
333
280
607
133

17 [Homo sapiens]

451
40254442
plexin-B1 precursor [Homo sapiens]
699
207
615
132

452
222352127
protein sidekick-2 precursor [Homo sapiens]
377
266
618
132

453
71565160
structural maintenance of chromosomes
672
210
619
132

protein 1B [Homo sapiens]

454
331284125
E1A-binding protein p400 [Homo sapiens]
521
234
622
132

455
134133288
zinc finger protein 407 isoform 1
600
223
626
131

[Homo sapiens]

456
148806908
fibronectin type III domain-containing
537
232
629
131

protein 1 precursor [Homo sapiens]

457
153945790
myosin-8 [Homo sapiens]
34
1053
631
131

458
170016061
spectrin beta chain brain 4 [Homo sapiens]
152
416
633
131

459
19923084
polycystic kidney disease protein 1-like
385
264
640
130

1 [Homo sapiens]

460
51339291
sterile alpha motif domain-containing
586
225
644
130

protein 9-like [Homo sapiens]

461
93102424
protein FAM179B [Homo sapiens]
525
234
652
129

462
55743096
collagen alpha-1(XIV) chain precursor
473
244
657
129

[Homo sapiens]

463
283837842
protein unc-13 homolog A [Homo sapiens]
550
231
661
129

464
47078218
ATP-binding cassette sub-family A
498
239
666
129

member 2 isoform b [Homo sapiens]

465
51599156
chromodomain-helicase-DNA-binding
306
293
667
129

protein 4 [Homo sapiens]

466
310119144
PREDICTED: rootletin [Homo sapiens]
388
264
672
128

467
119943102
CREB-binding protein isoform b
454
247
683
128

[Homo sapiens]

468
332801082
citron Rho-interacting kinase isoform 1
366
269
685
127

[Homo sapiens]

469
93277088
mediator of RNA polymerase II transcription
738
201
690
127

subunit 12-like protein [Homo sapiens]

470
120587019
zinc finger protein 318 [Homo sapiens]
427
254
693
127

471
156139122
methylcytosine dioxygenase TET1
303
296
697
127

[Homo sapiens]

472
150417986
brefeldin A-inhibited guanine nucleotide-
467
245
698
126

exchange protein 2 [Homo sapiens]

473
46358428
intraflagellar transport protein 172
556
230
700
126

homolog [Homo sapiens]

474
100913220
collagen alpha-1(XVI) chain precursor
411
258
705
126

[Homo sapiens]

475
5902122
spectrin beta chain brain 2 [Homo sapiens]
480
243
708
126

476
149274646
uncharacterized protein KIAA1614
620
219
711
126

[Homo sapiens]

477
47578105
nipped-B-like protein isoform A [Homo sapiens]
232
332
712
125

478
19923191
80 kDa MCM3-associated protein [Homo sapiens]
459
247
713
125

21
4557871
serotransferrin precursor [Homo sapiens]
64
731
717
125

480
90991702
leucine-rich repeat serine/threonine-
503
238
726
124

protein kinase 1 [Homo sapiens]

481
122937400
teneurin-3 [Homo sapiens]
418
256
728
124

482
6715600
Golgin subfamily A member 4 isoform 2
374
266
735
123

[Homo sapiens]

483
92859678
snRNA-activating protein complex
501
238
736
123

subunit 4 [Homo sapiens]

484
333440449
CLIP-associating protein 2 isoform 1
526
234
743
123

[Homo sapiens]

485
120953251
neuron navigator 3 [Homo sapiens]
571
227
748
123

486
157952215
receptor- type tyrosine-protein
422
256
749
123

phosphatase beta isoform a [Homo sapiens]

487
160948599
integrator complex subunit 1 [Homo sapiens]
700
207
754
122

488
89142730
collagen alpha-3(IV) chain precursor
596
224
756
122

[Homo sapiens]

489
18105007
CAD protein [Homo sapiens]
684
209
760
122

490
44771211
mediator of RNA polymerase II
697
207
761
122

transcription subunit 13-like [Homo sapiens]

491
110347427
ubiquitin carboxyl-terminal hydrolase 34
169
390
765
121

[Homo sapiens]

492
21536376
ATP-binding cassette sub-family A
694
207
766
121

member 1 [Homo sapiens]

493
224451124
neurobeachin-like protein 1 [Homo sapiens]
337
280
768
121

494
74048554
protein CASC5 isoform 2 [Homo sapiens]
376
266
775
120

495
21361116
versican core protein isoform 1
455
247
779
120

precursor [Homo sapiens]

496
112382257
inaD-like protein [Homo sapiens]
718
204
780
120

497
40255272
xin actin-binding repeat-containing
205
358
781
120

protein 1 isoform 1 [Homo sapiens]

498
348041302
phosphatidylinositol 4-kinase alpha
617
220
782
120

isoform 1 [Homo sapiens]

499
231573214
E3 ubiquitin-protein ligase listerin
512
236
787
120

[Homo sapiens]

500
4507157
sortilin-related receptor preproprotein
675
209
790
119

[Homo sapiens]

501
150378463
histone acetyltransferase KAT6A
607
222
792
119

[Homo sapiens]

502
6912288
CASP8-associated protein 2 [Homo sapiens]
592
224
796
119

503
50843820
sickle tail protein homolog isoform 1
616
220
800
119

[Homo sapiens]

504
33946282
protein virilizer homolog isoform 1
594
224
801
119

[Homo sapiens]

505
118600961
ral GTPase-activating protein subunit
669
211
802
119

alpha-2 [Homo sapiens]

506
154813199
poly [ADP-ribose] polymerase 14 [Homo sapiens]
384
264
805
119

507
4757960
cadherin-1 preproprotein [Homo sapiens]
129
467
811
118

508
11968023
zinc finger protein 106 homolog [Homo sapiens]
557
230
818
118

509
310110100
PREDICTED: mucin-5AC [Homo sapiens]
200
363
829
117

510
21735548
centrosome-associated protein CEP250
320
285
831
117

[Homo sapiens]

511
239582741
FERM and PDZ domain-containing
653
213
837
117

protein 1 [Homo sapiens]

512
197245440
uncharacterized protein KIAA1107
533
233
839
116

[Homo sapiens]

513
116268127
protein very KIND isoform a [Homo sapiens]
476
243
844
116

514
41872631
fatty acid synthase [Homo sapiens]
585
225
848
116

515
124430752
kinesin-like protein KIF26B [Homo sapiens]
708
205
851
115

516
154354979
unconventionnal myosin-X [Homo sapiens]
589
225
852
115

517
115496169
myosin-7 [Homo sapiens]
49
852
861
115

518
148806881
uncharacterized protein KIAA1462
608
222
862
115

[Homo sapiens]

519
164607133
fer-1-like protein 5 [Homo sapiens]
663
212
865
115

520
32313593
olfactomedin-4 precursor [Homo sapiens]
67
705
866
115

521
222537743
phosphotidylinositol phosphatase
723
202
873
114

PTPRQ precursor [Homo sapiens]

522
341915841
PREDICTED: hypothetical protein
597
224
875
114

LOC100129543 [Homo sapiens]

523
149944526
putative Polycomb group protein
727
202
876
114

ASXL3 [Homo sapiens]

524
19923790
rab3 GTPase-activating protein non-
646
214
880
114

catalytic subunit [Homo sapiens]

525
41393547
neuroblastoma-amplified sequence
471
244
884
114

[Homo sapiens]

526
178557739
complement C4-B preproprotein [Homo sapiens]
720
203
888
114

527
183396787
BCL-6 corepressor isoform c [Homo sapiens]
540
232
890
113

528
223468663
aldo-keto reductase family 1 member
70
667
895
113

B10 [Homo sapiens]

529
16357503
collagen alpha-6(IV) chain isoform B
291
303
897
113

precursor [Homo sapiens]

530
33620745
pre-mRNA cleavage complex 2 protein
664
212
908
113

Pcf11 [Homo sapiens]

531
134276943
separin [Homo sapiens]
555
231
909
113

532
194328738
uncharacterized protein KIAA0556
440
250
911
113

[Homo sapiens]

533
222537754
uncharacterized protein C3orf77 [Homo sapiens]
472
244
921
112

534
56711286
uncharacterized protein KIAA2022
721
203
935
111

[Homo sapiens]

535
54607120
lactotransferrin isoform 1 precursor
42
914
936
111

[Homo sapiens]

536
256773222
uncharacterized protein C12orf35
516
235
940
111

[Homo sapiens]

537
31317272
WD repeat and FYVE domain-
266
314
944
111

containing protein 3 [Homo sapiens]

538
89111135
multidrug resistance-associated protein
741
201
947
111

9 [Homo sapiens]

539
102468717
mediator of RNA polymerase II
603
222
959
110

transcription subunit 13 [Homo sapiens]

540
257196142
piezo-type mechanosensitive ion
622
219
974
110

channel component 1 [Homo sapiens]

541
38372909
lysine-specific demethylase 3B [Homo sapiens]
661
212
982
110

542
59891448
rapamycin-insensitive companion of
717
204
986
109

mTOR [Homo sapiens]

543
153946395
tenascin precursor [Homo sapiens]
509
237
1003
108

544
149944548
neurobeachin-like protein 2 [Homo sapiens]
674
210
1007
108

545
21536371
telomerase protein component 1 [Homo sapiens]
485
242
1016
108

47
98986445
carcinoembryonic antigen-related cell adhesion
36
996
1020
108

molecule 5 preproprotein [Homo sapiens]

546
150417984
ATP-binding cassette sub-family A
554
231
1022
108

member 7 [Homo sapiens]

547
10835063
nucleophosmin isoform 1 [Homo sapiens]
289
304
1036
106

548
237858799
adenylate kinase domain-containing
508
237
1038
106

protein 1 isoform 1 [Homo sapiens]

549
291219891
PH domain leucine-rich repeat-containing
559
229
1039
106

protein phosphatase 1 [Homo sapiens]

550
115334682
SRC kinase signaling inhibitor 1
359
270
1043
106

[Homo sapiens]

551
162287219
protein prune homolog 2 [Homo sapiens]
487
242
1044
106

552
70980549
protein RRP5 homolog [Homo sapiens]
742
200
1045
106

553
33946327
nuclear pore complex protein Nup214
618
220
1046
106

[Homo sapiens]

554
302565871
uncharacterized protein C9orf174
698
207
1055
106

[Homo sapiens]

555
167857790
alpha-1-acid glycoprotein 1 precursor
204
358
1063
106

[Homo sapiens]

556
38348729
uncharacterized protein C9orf93 [Homo sapiens]
580
226
1076
105

557
50658063
structural maintenance of chromosomes
532
233
1083
105

protein 4 [Homo sapiens]

558
139394648
DNA polymerase theta [Homo sapiens]
436
250
1086
105

559
218083800
rho GTPase-activating protein 32
644
215
1106
104

isoform 1 [Homo sapiens]

560
7706457
A-kinase anchor protein 11 [Homo sapiens]
598
223
1110
104

561
19923723
ribosomal protein S6 kinase delta-1
722
203
1116
103

isoform a [Homo sapiens]

562
4502523
voltage-dependent N-type calcium channel
601
223
1122
103

subunit alpha-1B isoform 1 [Homo sapiens]

563
30089940
Golgin subfamily A member 3 isoform 1
619
220
1130
103

[Homo sapiens]

564
122937345
myosin-Vb [Homo sapiens]
733
201
1132
103

565
45387958
protein phosphatase 1 regulatory subunit
504
238
1146
102

26 [Homo sapiens]

566
27436873
E3 ubiquitin-protein ligase SHPRH
448
248
1152
102

isoform b [Homo sapiens]

567
341914961
PREDICTED: FERM and PDZ domain-
479
243
1159
102

containing protein 3 [Homo sapiens]

568
299829223
coiled-coil domain-containing protein
751
200
1167
101

141 [Homo sapiens]

569
7662126
signal-induced proliferation-associated
650
213
1188
100

1-like protein 1 [Homo sapiens]

570
150378549
EH domain-binding protein 1-like
584
225
1193
100

protein 1 [Homo sapiens]

571
168823435
calpain-7-like protein [Homo sapiens]
493
240
1212
100

572
32455273
serine/threonine-protein kinase WNK2
659
213
1216
99

[Homo sapiens]

573
153945715
myosin-Vc [Homo sapiens]
491
241
1227
99

574
33620775
kinectin isoform a [Homo sapiens]
535
232
1229
99

575
341915544
PREDICTED: LOW QUALITY
151
418
1232
99

PROTEIN: mucin-5AC [Homo sapiens]

30
4557485
ceruloplasmin precursor [Homo sapiens]
338
279
1234
99

576
54792138
probable helicase with zinc finger
511
237
1237
99

domain [Homo sapiens]

577
63252863
structure-specific endonuclease subunit
660
213
1241
99

SLX4 [Homo sapiens]

578
150036262
calcium-activated chloride channel
252
321
1247
99

regulator 4 precursor [Homo sapiens]

579
38348727
thyroid adenoma-associated protein
435
251
1256
98

[Homo sapiens]

580
218505835
membrane-associated guanylate kinase
576
227
1264
98

WW and PDZ domain-containing

protein 3 isoform 1 [Homo sapiens]

581
178056552
condensin complex subunit 1 [Homo sapiens]
518
235
1286
97

582
44889475
DENN domain-containing protein 5A
740
201
1299
96

isoform 1 [Homo sapiens]

583
282721063
uncharacterized protein C1orf173
549
231
1317
96

[Homo sapiens]

584
122891862
DENN domain-containing protein 5B
705
206
1335
95

[Homo sapiens]

585
112382250
spectrin beta chain brain 1 isoform 1
258
317
1352
94

[Homo sapiens]

586
156119615
myosin-IXa [Homo sapiens]
505
238
1358
94

587
221139764
PHD and RING finger domain-
494
240
1367
94

containing protein 1 [Homo sapiens]

588
242246985
clathrin heavy chain 2 isoform 1
654
213
1385
93

[Homo sapiens]

589
4502271
sodium/potassium-transporting ATPase
734
201
1413
92

subunit alpha-2 proprotein [Homo sapiens]

590
155030216
sister chromatid cohesion protein PDS5
604
222
1422
92

homolog A isoform 1 [Homo sapiens]

591
47419930
chondroitin sulfate proteoglycan 4
378
266
1428
92

precursor [Homo sapiens]

592
4759146
slit homolog 2 protein precursor
657
213
1442
91

[Homo sapiens]

593
18079216
caskin-1 [Homo sapiens]
562
228
1444
91

594
188528675
slit homolog 1 protein precursor
614
220
1492
90

[Homo sapiens]

595
281485608
trefoil factor 3 precursor [Homo sapiens]
678
209
1501
89

596
105990535
coagulation factor V precursor [Homo sapiens]
679
209
1504
89

597
57863271
uncharacterized protein KIAA0564
628
218
1514
89

isoform a precursor [Homo sapiens]

598
255759952
WD repeat-containing protein 81
695
207
1532
88

isoform 1 [Homo sapiens]

599
171906559
peripheral-type benzodiazepine receptor-
452
248
1533
88

associated protein 1 isoform a [Homo sapiens]

600
115529484
CD 109 antigen isoform 1 precursor
714
204
1543
88

[Homo sapiens]

601
54607035
integrin beta-4 isoform 1 precursor
391
263
1544
88

[Homo sapiens]

602
157426864
zinc finger FYVE domain-containing
634
217
1554
88

protein 16 [Homo sapiens]

603
11321571
slit homolog 3 protein precursor
645
214
1556
87

[Homo sapiens]

604
94400919
WD repeat-containing protein 90 [Homo sapiens]
520
234
1567
87

605
207028821
RNA-binding protein 44 [Homo sapiens]
588
225
1580
86

606
207113160
treacle protein isoform d [Homo sapiens]
574
227
1599
86

607
221219000
inactive phospholipase C-like protein 2
706
206
1645
84

isoform 1 [Homo sapiens]

608
256600196
rap guanine nucleotide exchange factor
682
209
1657
84

6 isoform 1 [Homo sapiens]

609
4557565
DNA excision repair protein ERCC-6
536
232
1679
83

[Homo sapiens]

610
308736994
NACHT and WD repeat domain-
496
239
1703
83

containing protein 1 [Homo sapiens]

611
303304991
centrosomal protein of 152 kDa isoform
361
269
1706
83

1 [Homo sapiens]

612
10835109
myotubularin-related protein 3 isoform c
743
200
1716
83

[Homo sapiens]

613
145701025
multiple epidermal growth factor-like
432
251
1735
82

domains protein 8 precursor [Homo sapiens]

614
160420295
centrosomal protein KIAA1731 [Homo sapiens]
499
239
1798
80

615
28626521
NFX1-type zinc finger-containing
517
235
1816
80

protein 1 [Homo sapiens]

616
31742492
NEDD4-binding protein 2 [Homo sapiens]
713
205
1882
78

617
4758190
dipeptidase 1 precursor [Homo sapiens]
162
395
1883
78

618
24308089
chromodomain-helicase-DNA-binding
564
228
1888
77

protein 5 [Homo sapiens]

619
222136585
protein timeless homolog [Homo sapiens]
572
227
1980
75

620
171846278
leucine-rich repeat serine/threonine-
514
235
1983
75

protein kinase 2 [Homo sapiens]

621
21361241
ephrin type-A receptor 3 isoform a
528
234
2031
74

precursor [Homo sapiens]

622
164565408
pleckstrin homology domain-containing
748
200
2072
73

family G member 2 [Homo sapiens]

623
4505621
phosphatidylethanolamine-binding
233
332
2086
73

protein 1 preproprotein [Homo sapiens]

624
46049114
kinesin-like protein KIF20B [Homo sapiens]
270
312
2117
72

625
67782362
ATP-dependent RNA helicase DHX29
510
237
2156
71

[Homo sapiens]

626
31657140
insulin receptor-related protein
656
213
2247
69

precursor [Homo sapiens]

62
93141226
xaa-Pro aminopeptidase 2 precursor
147
426
2293
68

[Homo sapiens]

627
350529351
protein NLRC5 [Homo sapiens]
477
243
2421
65

628
163792198
latrophilin-3 precursor [Homo sapiens]
729
202
2431
65

629
145046269
rotatin [Homo sapiens]
445
249
2476
64

630
32189398
gastric intrinsic factor precursor
297
301
2510
64

[Homo sapiens]

631
134133226
POTE ankyrin domain family member E
611
221
2624
61

[Homo sapiens]

632
115527082
myosin-1 [Homo sapiens]
19
1529
0
0

633
153791586
myosin-2 [Homo sapiens]
24
1461
0
0

15
118498350
chymotrypsinogen B2 precursor
25
1445
0
0

[Homo sapiens]

22
115298678
complement C3 precursor [Homo sapiens]
27
1360
0
0

634
125819
RecName: Full = Ig kappa chain V-III
45
898
0
0

region HIC; Flags: Precursor

635
4557894
lysozyme C precursor [Homo sapiens]
46
884
0
0

636
125801
RecName: Full = Ig kappa chain V-III
53
812
0
0

region Ti

637
125797
RecName: Full = Ig kappa chain V-III
54
789
0
0

region SIE

638
294956573
RecName: Full = Ig lambda-6 chain C
57
763
0
0

region

54
341913702
PREDICTED: deleted in malignant brain tumors
61
744
0
0

1 protein isoform 2 [Homo sapiens]

639
4505605
regenerating islet-derived protein
62
743
0
0

3-alpha precursor [Homo sapiens]

640
294956599
RecName: Full = Ig lambda-7 chain C
63
735
0
0

region

641
148539842
deleted in malignant brain tumors 1
66
717
0
0

protein isoform b precursor [Homo sapiens]

28
4826762
haptoglobin isoform 1 preproprotein
68
692
0
0

[Homo sapiens]

642
123843
RecName: Full = Ig heavy chain V-III
76
591
0
0

region VH26; Flags: Precursor

643
4503571
alpha-enolase isoform 1 [Homo sapiens]
78
582
0
0

644
40354205
fructose-bisphosphate aldolase B
79
580
0
0

[Homo sapiens]

645
118918403
nesprin-2 isoform 1 [Homo sapiens]
81
567
0
0

646
113204615
ryanodine receptor 1 isoform 1
84
564
0
0

[Homo sapiens]

647
125770
RecName: Full = Ig kappa chain V-I region OU
85
562
0
0

648
4557577
fatty acid-binding protein liver
88
560
0
0

[Homo sapiens]

649
125761
RecName: Full = Ig kappa chain V-I region DEE
92
549
0
0

650
23097308
nesprin-1 isoform 2 [Homo sapiens]
95
543
0
0

651
4504963
lipocalin-1 isoform 1 precursor [Homo sapiens]
96
543
0
0

652
58331253
obscurin isoform a [Homo sapiens]
97
542
0
0

653
126032350
DNA-dependent protein kinase catalytic
99
537
0
0

subunit isoform 2 [Homo sapiens]

654
125756
RecName: Full = Ig kappa chain V-I region AG
101
528
0
0

655
125774
RecName: Full = Ig kappa chain V-I region WEA
105
520
0
0

656
125758
RecName: Full = Ig kappa chain V-I region AU
106
517
0
0

657
125790
RecName: Full = Ig kappa chain V-II
108
511
0
0

region GM607; Flags: Precursor

658
125799
RecName: Full = Ig kappa chain V-III
110
504
0
0

region NG9; Flags: Precursor

659
1170720
RecName: Full = Ig kappa chain V-I region WAT
117
484
0
0

660
123845
RecName: Full = Ig heavy chain V-III
124
474
0
0

region BRO

661
125833
RecName: Full = Ig kappa chain V-IV
130
457
0
0

region JI; Flags: Precursor

662
257743025
nebulin isoform 2 [Homo sapiens]
132
454
0
0

663
123844
RecName: Full = Ig heavy chain V-III
134
453
0
0

region TIL

664
193806361
RecName: Full = Ig gamma-3 chain C
144
433
0
0

region; AltName: Full = HDC; AltName:

Full = Heavy chain disease protein

665
54607141
vacuolar protein sorting-associated
146
426
0
0

protein 13D isoform 2 [Homo sapiens]

666
223633991
pantetheinase precursor [Homo sapiens]
150
419
0
0

667
341914926
PREDICTED: ig heavy chain V-III
154
413
0
0

region VH26-like [Homo sapiens]

668
218512079
RecName: Full = Ig gamma-2 chain C region
158
402
0
0

669
125779
RecName: Full = Ig kappa chain V-I
161
397
0
0

region Walker; Flags: Precursor

670
125766
RecName: Full = Ig kappa chain V-I
167
391
0
0

region HK102; Flags: Precursor

671
125763
RecName: Full = Ig kappa chain V-I region Gal
171
388
0
0

672
341914862
PREDICTED: hypothetical protein
173
385
0
0

LOC100291917 [Homo sapiens]

673
125795
RecName: Full = Ig kappa chain V-III
175
381
0
0

region B6

674
73858568
plasma protease C1 inhibitor precursor
178
377
0
0

[Homo sapiens]

675
341913664
PREDICTED: hypothetical protein
179
377
0
0

LOC642424 [Homo sapiens]

676
188595687
filamin-C isoform b [Homo sapiens]
180
376
0
0

677
341915168
PREDICTED: ig kappa chain V-I region
182
373
0
0

Walker-like [Homo sapiens]

678
256017159
MAX gene-associated protein isoform 2
190
368
0
0

[Homo sapiens]

679
53759122
adenomatous polyposis coli protein
195
366
0
0

isoform b [Homo sapiens]

680
4502067
protein AMBP preproprotein [Homo sapiens]
202
361
0
0

681
47607492
plectin isoform 1c [Homo sapiens]
207
354
0
0

682
310114449
PREDICTED: hypothetical protein
210
351
0
0

LOC131544 [Homo sapiens]

683
341915514
PREDICTED: ig kappa chain V-I region
211
351
0
0

Walker-like [Homo sapiens]

684
125762
RecName: Full = Ig kappa chain V-I region EU
220
346
0
0

685
125811
RecName: Full = Ig kappa chain V-III
221
343
0
0

region VG; Flags: Precursor

686
312032409
aminoacylase-1 isoform d [Homo sapiens]
225
339
0
0

687
283806679
cytoplasmic dynein 2 heavy chain 1
231
333
0
0

isoform 1 [Homo sapiens]

688
21735621
malate dehydrogenase mitochondrial
234
332
0
0

precursor [Homo sapiens]

689
115298657
protein S100-A7 [Homo sapiens]
245
323
0
0

690
27436940
reelin isoform b precursor [Homo sapiens]
251
321
0
0

691
51230412
ral GTPase-activating protein subunit
257
317
0
0

alpha-1 isoform 2 [Homo sapiens]

692
374532817
Golgin subfamily B member 1 isoform 1
260
316
0
0

[Homo sapiens]

693
109826564
neurofibromin isoform 1 [Homo sapiens]
261
316
0
0

694
301897469
beta-enolase isoform 1 [Homo sapiens]
264
315
0
0

695
38683816
ankyrin repeat domain-containing
276
308
0
0

protein 17 isoform b [Homo sapiens]

696
16579888
fructose-16-bisphosphatase 1 [Homo sapiens]
284
306
0
0

697
126362967
nck-associated protein 5 isoform 1
287
305
0
0

[Homo sapiens]

698
38045890
CUB and sushi domain-containing
301
299
0
0

protein 3 isoform 2 [Homo sapiens]

699
331284178
nuclear receptor corepressor 2 isoform 1
309
292
0
0

[Homo sapiens]

700
151301154
mucin-6 precursor [Homo sapiens]
311
291
0
0

701
298919181
nuclear receptor corepressor 1 isoform 3
321
285
0
0

[Homo sapiens]

702
27477095
histone-lysine N-methyltransferase H3
331
281
0
0

lysine-36 and H4 lysine-20 specific

isoform a [Homo sapiens]

703
98985810
collagen alpha-1(XI) chain isoform B
334
280
0
0

preproprotein [Homo sapiens]

704
269954694
inositol 1 4 5-trisphosphate receptor
339
279
0
0

type 1 isoform 3 [Homo sapiens]

705
66346674
vacuolar protein sorting-associated
343
278
0
0

protein 13A isoform A [Homo sapiens]

706
125786
RecName: Full = Ig kappa chain V-II region MIL
349
274
0
0

707
38505274
voltage-dependent T-type calcium channel
355
272
0
0

subunit alpha-1G isoform 7 [Homo sapiens]

708
148536846
voltage-dependent P/Q-type calcium channel
358
271
0
0

subunit alpha-1A isoform 2 [Homo sapiens]

709
5032281
dystrophin Dp427c isoform [Homo sapiens]
379
266
0
0

710
122937195
perilipin-4 [Homo sapiens]
381
265
0
0

711
209969819
protein PRR14L [Homo sapiens]
383
264
0
0

712
341915607
PREDICTED: hypothetical protein
389
263
0
0

LOC144535 [Homo sapiens]

713
33356170
myosin-IXb isoform 1 [Homo sapiens]
390
263
0
0

714
59710093
probable G-protein coupled receptor 112
392
263
0
0

[Homo sapiens]

715
341913933
PREDICTED: hypothetical protein
400
262
0
0

LOC100653084 [Homo sapiens]

716
341914924
PREDICTED: ig heavy chain V-III
405
260
0
0

region VH26-like [Homo sapiens]

717
116089331
transcription factor HIVEP3 isoform a
406
260
0
0

[Homo sapiens]

718
224586880
ras-specific guanine nucleotide-releasing
415
257
0
0

factor 1 isoform 3 [Homo sapiens]

719
113415686
PREDICTED: hypothetical protein
420
256
0
0

LOC285556 [Homo sapiens]

720
311771583
LY75-CD302 fusion protein isoform 1
437
250
0
0

precursor [Homo sapiens]

721
214010175
CLIP-associating protein 1 isoform 3
443
249
0
0

[Homo sapiens]

722
16933542
fibronectin isoform 3 preproprotein
446
249
0
0

[Homo sapiens]

723
31563537
CLIP-associating protein 1 isoform 1
447
249
0
0

[Homo sapiens]

724
73695475
HEAT repeat-containing protein 1
449
248
0
0

[Homo sapiens]

725
5803011
gamma-enolase [Homo sapiens]
451
248
0
0

726
296317312
carcinoembryonic antigen-related cell
460
247
0
0

adhesion molecule 1 isoform 3 precursor

[Homo sapiens]

727
117168250
1-phosphatidylinositol-4 5-bisphosphate
463
246
0
0

phosphodiesterase epsilon-1 isoform 1

[Homo sapiens]

728
363807222
girdin isoform 3 [Homo sapiens]
464
246
0
0

729
121114300
carcinoembryonic antigen-related cell
466
245
0
0

adhesion molecule 3 precursor

[Homo sapiens]

730
125809
RecName: Full = Ig kappa chain V-III
469
245
0
0

region CLL; AltName: Full = Rheumatoid

factor; Flags: Precursor

731
170650694
arf-GAP with GTPase ANK repeat and
474
243
0
0

PH domain-containing protein 2 isoform

PIKE-L [Homo sapiens]

732
116008192
myosin light chain kinase smooth
478
243
0
0

muscle isoform 1 [Homo sapiens]

733
33620769
E3 ubiquitin-protein ligase RBBP6
481
243
0
0

isoform 1 [Homo sapiens]

734
4502895
colipase isoform 1 preproprotein
482
243
0
0

[Homo sapiens]

735
4505941
DNA-directed RNA polymerase II
484
242
0
0

subunit RPB2 [Homo sapiens]

736
154759259
spectrin alpha chain brain isoform 2
488
242
0
0

[Homo sapiens]

737
208022632
girdin isoform 1 [Homo sapiens]
489
241
0
0

738
350276222
neuron navigator 2 isoform 5 [Homo sapiens]
490
241
0
0

739
15890086
collagen alpha-5(IV) chain isoform 2
523
234
0
0

precursor [Homo sapiens]

740
333440451
CLIP-associating protein 2 isoform 2
524
234
0
0

[Homo sapiens]

741
327365361
HEAT repeat-containing protein 5A
527
234
0
0

[Homo sapiens]

742
92091583
myosin-11 isoform SM2B [Homo sapiens]
529
234
0
0

743
215598574
ankyrin-1 isoform 9 [Homo sapiens]
534
233
0
0

744
20336209
transcriptional regulator ATRX isoform
539
232
0
0

1 [Homo sapiens]

745
24308029
dedicator of cytokinesis protein 9
541
232
0
0

isoform a [Homo sapiens]

746
123847
RecName: Full = Ig heavy chain V-III
545
232
0
0

region CAM

747
161169013
neuron navigator 2 isoform 1 [Homo sapiens]
552
231
0
0

748
4557365
Bloom syndrome protein [Homo sapiens]
553
231
0
0

749
46049105
nebulin-related-anchoring protein
561
228
0
0

isoform S [Homo sapiens]

750
4504349
hemoglobin subunit beta [Homo sapiens]
570
228
0
0

751
93204888
spatacsin isoform 1 [Homo sapiens]
573
227
0
0

752
123846
RecName: Full = Ig heavy chain V-III
575
227
0
0

region BUT

753
120953300
leucine-rich repeat and IQ domain-
577
227
0
0

containing protein 1 [Homo sapiens]

754
311771516
myomegalin isoform 8 [Homo sapiens]
579
226
0
0

755
114155142
nucleoprotein TPR [Homo sapiens]
582
226
0
0

756
71274186
uncharacterized protein KIAA1755
583
225
0
0

[Homo sapiens]

757
70780355
ankyrin-1 isoform 2 [Homo sapiens]
591
225
0
0

758
4503689
fibrinogen alpha chain isoform alpha-E
593
224
0
0

preproprotein [Homo sapiens]

759
4506773
protein S100-A9 [Homo sapiens]
595
224
0
0

760
355390328
kinesin-like protein KIF21B isoform 3
609
221
0
0

[Homo sapiens]

761
315709510
protein dopey-1 isoform b [Homo sapiens]
612
221
0
0

762
224831241
myosin-14 isoform 3 [Homo sapiens]
615
220
0
0

763
22094135
histone-lysine N-methyltransferase H3
624
219
0
0

lysine-79 specific [Homo sapiens]

764
300797780
serine/threonine-protein kinase WNK1
626
218
0
0

isoform 3 [Homo sapiens]

765
256818778
protein unc-80 homolog isoform 2
633
217
0
0

[Homo sapiens]

766
125777
RecName: Full = Ig kappa chain V-I region Ni
639
216
0
0

767
21361861
Fanconi anemia group D2 protein
641
215
0
0

isoform a [Homo sapiens]

768
293597572
transient receptor potential cation
647
214
0
0

channel subfamily M member 6 isoform

b [Homo sapiens]

769
41393563
kinesin-like protein KIF1B isoform b
648
214
0
0

[Homo sapiens]

770
267844813
neuron navigator 1 isoform 2 [Homo sapiens]
649
213
0
0

771
269847874
probable ATP-dependent RNA helicase
662
212
0
0

YTHDC2 [Homo sapiens]

772
222352161
probable phospholipid-transporting
666
211
0
0

ATPase VD [Homo sapiens]

773
65287717
eukaryotic translation initiation factor 2-
667
211
0
0

alpha kinase 4 [Homo sapiens]

774
224586815
Golgi apparatus protein 1 isoform 2
673
210
0
0

precursor [Homo sapiens]

775
94966754
elongation factor Tu GTP-binding
677
209
0
0

domain-containing protein 1 isoform 1

[Homo sapiens]

776
115648142
centrosomal protein of 164 kDa
680
209
0
0

[Homo sapiens]

777
46852172
kinesin-like protein KIF13B [Homo sapiens]
681
209
0
0

778
19913410
major vault protein [Homo sapiens]
683
209
0
0

58
58331211
chymotrypsin-like elastase family
687
208
0
0

member 2B preproprotein [Homo sapiens]

779
38044112
CAP-Gly domain-containing linker
688
208
0
0

protein 1 isoform b [Homo sapiens]

780
40255013
carcinoembryonic antigen-related cell
689
208
0
0

adhesion molecule 6 precursor

[Homo sapiens]

781
4504919
keratin type II cytoskeletal 8 isoform 2
691
208
0
0

[Homo sapiens]

782
194097325
fatty acid-binding protein intestinal
701
206
0
0

[Homo sapiens]

783
319803120
testis-expressed protein 14 isoform c
702
206
0
0

[Homo sapiens]

784
354721145
transient receptor potential cation
707
206
0
0

channel subfamily M member 1 isoform

1 [Homo sapiens]

785
151301137
AT-hook-containing transcription factor
711
205
0
0

[Homo sapiens]

786
45827771
enhancer of mRNA-decapping protein 4
712
205
0
0

[Homo sapiens]

787
208609951
neurexin-1-beta isoform alpha2
716
204
0
0

precursor [Homo sapiens]

788
217416354
A-kinase anchor protein SPHKAP
719
203
0
0

isoform 1 [Homo sapiens]

789
30794372
protein polybromo-1 isoform 1 [Homo sapiens]
725
202
0
0

790
24307991
cullin-9 [Homo sapiens]
726
202
0
0

791
50345997
histone acetyltransferase p300 [Homo sapiens]
735
201
0
0

792
149773449
zinc finger protein 862 [Homo sapiens]
736
201
0
0

479
38327601
regulator of G-protein signaling 12
737
201
0
0

isoform 1 [Homo sapiens]

43
42794779
myosin-XVIIIa isoform b [Homo sapiens]
745
200
0
0

67
157419122
laminin subunit alpha-4 isoform 2
747
200
0
0

precursor [Homo sapiens]

The NCBI Accession Numbers for proteins defined by the NCBI protein database has been provided. The sequences of the proteins as reflected by the NCBI Accession Numbers listed throughout the present application are incorporated herein by reference. Where a protein is named in its preprotein or other non-mature form, the mature form of the protein is equally implied including such changes as removal of signal sequences and the addition of post-translational modifications. In all cases, the protein has been named by its gene derived sequence to provide consistency. In addition, isoforms of each of the proteins identified herein are similarly envisioned.

The above description discloses several methods and materials of the present invention. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the invention.

Lengthy table referenced here

US20220128563A1-20220428-T00001

Please refer to the end of the specification for access instructions.

LENGTHY TABLES

The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

	Number	Date	Country
Parent	16046606	Jul 2018	US
Child	17380219		US
Parent	14773969	Sep 2015	US
Child	16046606		US

METHODS AND COMPOSITIONS FOR DETECTING PANCREATIC CANCER

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

International Classifications

Abstract

Description

Claims

RELATED APPLICATIONS

Provisional Applications (1)

Continuations (2)