WO2015013547A1

WO2015013547A1 - Use of galnac-t13 as a marker in breast or colon cancer diagnostics

Info

Publication number: WO2015013547A1
Application number: PCT/US2014/048082
Authority: WO
Inventors: Nora Berois; Diego TOUYA; Mario VARANGOT; Eduardo Osinaga
Original assignee: Cedars-Sinai Medical Center
Priority date: 2013-07-24
Filing date: 2014-07-24
Publication date: 2015-01-29

Abstract

The invention is directed to assays and methods for determining the likelihood of breast and/or colon cancer in a subject in need thereof. The assays and methods include detecting GalNAc-T13 and/or determining the expression levels of GalNAc-T13.

Description

USE OF GalNAc-T13 AS A MARKER IN BREAST OR COLON CANCER

DIAGNOSTICS

FIELD OF INVENTION

[0001] The invention is directed to the role of an enzyme of the O-glycosylation pathway in breast and/or colon cancer diagnosis and therapy. Specifically, the invention provides a new molecular target, namely GalNAc-T13, as a diagnostic marker for the detection of breast and/or colon cancer, so as to optimize breast and/or colon cancer therapy in the subject.

BACKGROUND

[0002] There is a need in the art for effective diagnostic markers for breast cancer and colon cancer. Here the inventors demonstrate that expression of GalNAc-T13 is increased in human breast and colon cancer cells and tissue, indicating that GalNAc-T13 may be a new diagnostic marker for breast and colon cancer.

SUMMARY OF THE INVENTION

[0003] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0004] Provided herein is an assay comprising obtaining a sample comprising a tumor cell from a breast and/or colon cancer patient; assaying the sample to determine the level of GalNac-T13; and determining the subject has increased likelihood of breast and/or colon cancer if the level of GalNac-T13 is increased relative to a reference sample, or determining the subject has decreased likelihood of breast and/or colon cancer if the level of GalNac-T13 is the same as or decreased relative to the reference sample.

[0005] Also provided herein is an assay for determining an increased likelihood of breast and/or colon cancer in a subject in need thereof comprising providing a biological sample from a subject having breast and/or colon cancer; providing an antibody that specifically binds to GalNAc-T13; contacting the biological sample with the antibody; and detecting, using immunoassay, the level of antibody binding to GalNAc-T13, wherein the presence of binding in the biological sample from the subject relative to a reference sample is indicative of increased likelihood of breast and/or colon cancer in the subject. In some embodiments, the presence of binding is an increase in binding of the antibody to GalNAc-T13. [0006] Further described herein is an assay for selecting a therapy for a subject having breast and/or colon cancer, and optionally administering the therapy, the assay comprising providing a biological sample from a subject having breast and/or colon cancer; providing an antibody that specifically binds to GalNAc-T13; contacting the biological sample with the antibody; detecting, using immunoassay, the level of antibody binding to GalNAc-T13, wherein an increase in binding in the biological sample from the subject relative to a reference sample is indicative of increased expression of ppGalNAc and increased likelihood of breast and/or colon cancer in the subject; and selecting a therapy comprising prescribing a therapy to the subject if the subject has an increased likelihood of breast and/or colon cancer.

[0007] In some embodiments, detecting the level of antibodies reactive to GalNAc-T13 comprises contacting the sample from the breast and/or colon cancer patient with an antibody or a fragment thereof that specifically binds GalNAc-T13; forming an antibody-protein complex between the antibody and GalNAc-T13 present in the sample; washing the sample to remove the unbound antibody; adding a detection antibody that is labeled and is reactive to the antibody bound to GalNAc-T13 in the sample; washing to remove the unbound labeled detection antibody; and converting the label to a detectable signal, wherein the detectable signal is indicative of the level of GalNAc-T13 in the sample from the patient. In some embodiments, the antibody is further conjugated to an effector component. The effector component may be a detectable moiety selected from the group consisting of a fluorescent label, a radioactive compound, an enzyme, a substrate, an epitope tag, electron-dense reagent, biotin, digonigenin, hapten and a combination thereof. In some embodiments, the detection antibody is labeled by covalently linking to an enzyme, labeled with a fluorescent compound or metal, labeled with a chemiluminescent compound. In an embodiment, the level of GalNAc-T13 is obtained by measuring a light scattering intensity resulting from the formation of an antibody-protein complex formed by a reaction of GalNAc-T13 in the sample with the antibody, wherein the light scattering intensity of at least 10% above a control light scattering intensity indicates the likelihood of breast and/or colon cancer.

[0008] Also described herein is a method for treating a subject having breast and/or colon cancer, and optionally administering the therapy. The method includes providing a biological sample from a subject having breast and/or colon cancer; providing an antibody that binds to GalNAc-T13; contacting the biological sample with the antibody; detecting, using immunoassay, whether the antibody binds GalNAc-T13, wherein an increase in binding in the biological sample from the subject relative to a reference sample is indicative of increased expression of GalNAc-T13 and increased likelihood of breast and/or colon cancer in the subject; and prescribing a therapy to the subject if the subject has increased likelihood of breast and/or colon cancer.

[0009] In some embodiments, assaying the sample includes detecting the level of nucleic acid encoding GalNac-T13, determining the level of GalNac-T13 protein, or a combination thereof. Detecting the level of nucleic acid encoding GalNAc-T13 may include determining the amount of mR A, encoding GalNac-T13, present in the sample. Detecting the level of GalNAc-T13 protein comprises detecting the level of GalNac-T13 with an antibody specific to GalNac-T13.

[0010] In some embodiments, the antibody is a monoclonal antibody. The monoclonal antibody may specifically bind, for example, the epitope LLPALR of GalNAc-T13. In an embodiment, the antibody is T 13.5 monoclonal antibody.

[0011] In some embodiments, the biological samples include but are not limited to tissue, blood, plasma or a combination thereof. In an embodiment, the subject is human.

[0012] In one embodiment, the reference value is the mean or median expression level of GalNAc-T13 in a population of subjects that do not have breast and/or colon cancer. In another embodiment, the reference value is the expression level of GalNAc-T13 from the subject in a sample obtained from a different time point.

[0013] In various embodiments, the assay described may further include prescribing a therapy to the subject if the subject has increased likelihood of breast or colon cancer. The therapies may be any one or more of surgery, radiation, chemotherapy, immunotherapy, vaccine, or a combination thereof.

[0014] Also described herein is an assay for determining an increased likelihood of breast or colon cancer in a subject in need thereof. The assay includes providing a biological sample from a subject having breast or colon cancer; providing an antibody that specifically binds to GalNAc-T13, wherein the antibody is the T13.5 monoclonal antibody that binds to LLPALR epitope on GalNAc-T13; contacting the biological sample with the antibody; and detecting, using immunoassay, the level of antibody binding to GalNAc-T13, wherein the presence of binding in the biological sample from the subject relative to a reference sample is indicative of increased likelihood of cancer in the subject. In some embodiments detecting the level of antibodies reactive to GalNAc-T13 includes contacting the sample from the cancer patient with an antibody or a fragment thereof that specifically binds GalNAc-T13; forming an antibody-protein complex between the antibody and GalNAc-T13 present in the sample; washing the sample to remove the unbound antibody; adding a detection antibody that is labeled and is reactive to the antibody bound to GalNAc-T13 in the sample; washing to remove the unbound labeled detection antibody; and converting the label to a detectable signal, wherein the detectable signal is indicative of the level of GalNAc-T13 in the sample from the patient. In some embodiments, the reference value may be the mean or median GalNAc-T13 expression level from a population of subjects that do not have cancer. In additional embodiments, the reference value may be the expression level of GalNAc-T13 from the subject in a sample obtained from a different time point. The antibody may be further conjugated to an effector component. The effector component may be a detectable moiety selected from the group consisting of a fluorescent label, a radioactive compound, an enzyme, a substrate, an epitope tag, electron-dense reagent, biotin, digonigenin, hapten and a combination thereof. The detection antibody may be labeled by covalently linking to an enzyme, labeled with a fluorescent compound or metal, labeled with a chemiluminescent compound. In some embodiments, the level of GalNAc-T13 is obtained by measuring a light scattering intensity resulting from the formation of an antibody-protein complex formed by a reaction of GalNAc-T13 in the sample with the antibody, wherein the light scattering intensity of at least 10% above a control light scattering intensity indicates the likelihood of cancer. In an embodiment, the presence of binding is an increase in binding of the antibody to GalNAc-T13 relative to the reference sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Exemplary embodiments are illustrated in the referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive

[0016] Figure 1, depicts in accordance with an embodiment of the invention, the GalNAc- T13 expression in MCF-7 breast cancer cells. Lane 1 shows MCF-7 human breast cancer cell line; Lane 2 is the negative control; Lane 3 is the BM neuroblastoma cell line (positive control).

[0017] Figure 2, depicts in accordance with an embodiment of the invention, the GalNAc- T13 expression in breast cancer tissue. [0018] Figure 3, depicts in accordance with an embodiment of the invention, the GalNAc- T13 expression in human colon cancer.

DETAILED DESCRIPTION OF THE INVENTION

[0019] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Allen et al, Remington: The Science and Practice of Pharmacy 22^nd ed., Pharmaceutical Press (September 15, 2012); Horny ak et al, Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3^rd ed., revised ed., J. Wiley & Sons (New York, NY 2006); Smith, March 's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7^th ed., J. Wiley & Sons (New York, NY 2013); Singleton, Dictionary of DNA and Genome Technology 3^rd ed., Wiley-Blackwell (November 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed. , Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2^nd ed., Cold Spring Harbor Press (Cold Spring Harbor NY, 2013); Kohler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 Jul, 6(7):511-9; Queen and Selick, Humanized immunoglobulins, U. S. Patent No. 5,585,089 (1996 Dec); and Riechmann et al, Reshaping human antibodies for therapy, Nature 1988 Mar 24, 332(6162):323-7.

[0020] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0021] "Beneficial results" may include, but are in no way limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition, preventing the disease condition from developing, lowering the chances of a patient developing the disease condition and prolonging a patient's life or life expectancy. In some embodiments, the disease condition is cancer. [0022] "Subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. In certain embodiments, the mammal is a human subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.

[0023] "Treatment" and "treating," as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition, prevent the pathologic condition, pursue or obtain beneficial results, or lower the chances of the individual developing the condition even if the treatment is ultimately unsuccessful. Those in need of treatment include those already with the condition as well as those prone to have the condition or those in whom the condition is to be prevented. Examples of cancer treatment include, but are not limited to, active surveillance, observation, surgical intervention, chemotherapy, immunotherapy, radiation therapy (such as external beam radiation, stereotactic radiosurgery (gamma knife), and fractionated stereotactic radiotherapy (FSR)), focal therapy, systemic therapy, vaccine therapies, viral therapies, molecular targeted therapies, or a combination thereof.

[0024] "Tumor," as used herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0025] "Chemotherapy resistance" as used herein refers to partial or complete resistance to chemotherapy drugs. For example, a subject does not respond or only partially responds to a chemotherapy drug. A person of skill in the art can determine whether a subject is exhibiting resistance to chemotherapy.

[0026] "Chemotherapeutic drugs" or "chemotherapeutic agents" as used herein refer to drugs used to treat cancer including but not limited to Adriamycin, Albumin-bound paclitaxel (nab- paclitaxel), Actinomycin, Alitretinoin, All-trans retinoic acid, anthracyclines, Azacitidine, Azathioprine, Bevacizumab, Bexatotene, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cetuximab, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Erlotinib, Etoposide, Fluorouracil, Gefitinib, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Ipilimumab, Irinotecan, Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone, Ocrelizumab, Ofatumumab, Oxaliplatin, Paclitaxel, Panitumab, Pemetrexed, Rituximab, Tafluposide, Taxol, Taxotere, Teniposide, Tioguanine, Topotecan, Tretinoin, Valrubicin, Vemurafenib, Vinblastine, Vincristine, Vindesine, Vinorelbine, Vorinostat, Romidepsin, 5- fluorouracil (5-FU), 6-mercaptopurine (6-MP), Cladribine, Clofarabine, Floxuridine, Fludarabine, Pentostatin, Mitomycin, ixabepilone, Estramustine, or a combination thereof.

[0027] "Patient outcome" refers to whether a patient survives or dies as a result of treatment. A more accurate prognosis for patients as provided in this invention increases the chances of patient survival.

[0028] "Poor Prognosis" means that the prospect of survival and recovery of disease is unlikely despite the standard of care for the treatment of breast cancer and/or colon cancer, that is, surgery, radiation, chemotherapy. Poor prognosis is the category of patients whose survival is less than that of the median survival.

[0029] "Good Prognosis" means that the prospect of survival and recovery of disease is likely with the standard of care for the treatment of the disease, for example, surgery, radiation, chemotherapy. Good prognosis is the category of patients whose survival is not less than that of the median survival.

[0030] A "recurrence" means that the cancer has returned after initial treatment.

[0031] "Variant" as used herein refers to a mutant GalNAc-T13, a splice variant of GalNAc- T13 or a combination thereof. A mutant of GalNAc-T13 may be a result of an insertion, deletion, missense, nonsense and/or a truncation mutation in the gene encoding GalNAc-T13.

[0032] Being "non-recurrent" or "recurrence-free" means that the cancer is in remission; being recurrent means that the cancer is growing and/or has metastasized, and some surgery, therapeutic intervention, and/or cancer treatment is required to lower the chance of lethality. The "non-recurrent subjects" are subjects who have non-recurrent or recurrence-free disease, and they can be used as the control for recurrent subjects who have recurrent disease or recurrence [0033] O-gylcosylation alterations occur in most carcinomas, resulting in the expression of molecules which may constitute useful targets for diagnosis and therapy. GalNAc-T13 enzyme catalyzes a key step in the initiation of O-glycosylation.

[0034] As described herein, Applicants' data suggests that GaINAc-T13 is a novel marker associated with breast and colon cancer and the invention is based, at least in part, on these findings. The present invention addresses the need for molecular indicators for diagnosis of cancer such as breast and/or colon cancer, for the prognostication of cancer, such as breast and/or colon cancer, for determination of chemotherapy resistance in cancer patients and/or for guiding treatment options in cancer patients. The invention provides processes, assays and methods for determining the presence of breast and/or colon cancer, metastasis of breast and/or colon cancer, chemotherapy resistance in breast and/or colon cancer, degree of aggressiveness of breast and/or colon cancer or a combination thereof in a subject in need thereof, so as to optimize cancer therapy in a subject in need thereof.

[0035] The invention provides assays comprising obtaining a sample comprising a sample (for example, a cancer cells or tissue) from a patient with breast and/or colon cancer and assaying the sample to determine the level of GalNac-T13 in the sample. In some embodiments, an increase in the level of GalNAc-T13 relative to a reference sample is indicative of the presence of breast and/or colon cancer in the subject. In another embodiment, an increase in the level of GalNAc-T13 relative to a reference is indicative of metastasis of breast and/or colon cancer in the subject. In another embodiment, an increase in the level of GalNAc-T13 relative to a reference is indicative of increased likelihood of chemotherapy resistance in the subject with breast and/or colon cancer. In another embodiment, an increase in the level of GalNAc-T13 relative to a reference is indicative of the degree of aggressiveness of breast and/or colon cancer in the subject. In a further embodiment, an increase in the level of GalNAc-T13 relative to a reference is indicative of the presence of breast and/or colon cancer, metastasis of breast and/or colon cancer, chemotherapy resistance in breast and/or colon cancer, degree of aggressiveness of breast and/or colon cancer, or a combination thereof, in the subject.

[0036] Also provided herein is an assay for determining an increased likelihood of breast and/or colon cancer in a subject in need thereof comprising providing a biological sample from a subject having breast and/or colon cancer; providing an antibody that specifically binds to GalNAc-T13; contacting the biological sample with the antibody; and detecting, using immunoassay, the level of antibody binding to GalNAc-T13, wherein the presence of binding in the biological sample from the subject relative to a reference sample is indicative of increased likelihood of breast and/or colon cancer in the subject. In some embodiments, the presence of binding is an increase in binding of the antibody to GalNAc-T13.

[0037] Further described herein is an assay for selecting a therapy for a subject having breast and/or colon cancer, and optionally administering the therapy. The assay includes providing a biological sample from a subject having breast and/or colon cancer; providing an antibody that specifically binds to GalNAc-T13; contacting the biological sample with the antibody; detecting, using immunoassay, the level of antibody binding to GalNAc-T13, wherein an increase in binding in the biological sample from the subject relative to a reference sample is indicative of increased expression of ppGalNAc and increased likelihood of breast and/or colon cancer in the subject; and selecting a therapy comprising prescribing a therapy to the subject if the subject has an increased likelihood of breast and/or colon cancer.

In some embodiments, assaying the sample includes detecting the level of nucleic acid encoding GalNac-T13, determining the level of GalNac-T13 protein, or a combination thereof. Detecting the level of nucleic acid encoding GalNAc-T13 may include determining the amount of mR A, encoding GalNac-T13, present in the sample. Detecting the level of GalNAc-T13 protein comprises detecting the level of GalNac-T13 with an antibody specific to GalNac-T13.

[0038] In some embodiments, the antibody is a monoclonal antibody. The monoclonal antibody may specifically bind, for example, the epitope LLPALR of GalNAc-T13. In an embodiment, the antibody is T 13.5 monoclonal antibody.

[0039] Also described herein is an assay for determining an increased likelihood of breast or colon cancer in a subject in need thereof. The assay includes providing a biological sample from a subject having breast or colon cancer; providing an antibody that specifically binds to GalNAc-T13, wherein the antibody is the T13.5 monoclonal antibody that binds to LLPALR epitope on GalNAc-T13; contacting the biological sample with the antibody; and detecting, using immunoassay, the level of antibody binding to GalNAc-T13, wherein the presence of binding in the biological sample from the subject relative to a reference sample is indicative of increased likelihood of cancer in the subject. In some embodiments detecting the level of antibodies reactive to GalNAc-T13 includes contacting the sample from the cancer patient with an antibody or a fragment thereof that specifically binds GalNAc-T13; forming an antibody-protein complex between the antibody and GalNAc-T13 present in the sample; washing the sample to remove the unbound antibody; adding a detection antibody that is labeled and is reactive to the antibody bound to GalNAc-T13 in the sample; washing to remove the unbound labeled detection antibody; and converting the label to a detectable signal, wherein the detectable signal is indicative of the level of GalNAc-T13 in the sample from the patient. In some embodiments, the reference value may be the mean or median GalNAc-T13 expression level from a population of subjects that do not have cancer. In additional embodiments, the reference value may be the expression level of GalNAc-T13 from the subject in a sample obtained from a different time point. The antibody may be further conjugated to an effector component. The effector component may be a detectable moiety selected from the group consisting of a fluorescent label, a radioactive compound, an enzyme, a substrate, an epitope tag, electron-dense reagent, biotin, digonigenin, hapten and a combination thereof. The detection antibody may be labeled by covalently linking to an enzyme, labeled with a fluorescent compound or metal, labeled with a chemiluminescent compound. In some embodiments, the level of GalNAc-T13 is obtained by measuring a light scattering intensity resulting from the formation of an antibody-protein complex formed by a reaction of GalNAc-T13 in the sample with the antibody, wherein the light scattering intensity of at least 10% above a control light scattering intensity indicates the likelihood of cancer. In an embodiment, the presence of binding is an increase in binding of the antibody to GalNAc-T13 relative to the reference sample.

[0040] The assays of the invention may further comprise selecting and/or administering a therapy to treat, reduce, inhibit or reduce the severity of cancer in the subject. In some embodiments, for example in subjects with increased likelihood of chemotherapy resistance, selecting the therapy includes prescribing a first therapy to the subject if the subject has decreased likelihood of chemotherapy resistance or prescribing a second therapy to the subject if the subject has increased likelihood of chemotherapy resistance. In some embodiments, the first therapy is any one or more of surgery, radiation, chemotherapy, immunotherapy, vaccine, or a combination thereof. In some embodiments, the second therapy may be non-chemotherapy comprising therapy and may be any one or more of surgery, radiation, immunotherapy, vaccine, or a combination thereof. In additional embodiments, the second therapy is any one or more of surgery, radiation, chemotherapy, immunotherapy, vaccine, or a combination thereof, wherein chemotherapy comprises administering to the subject one or more chemotherapeutic agents that have not been used previously to treat the subject or administering a chemotherapeutic agent that has been previously administered to the subject but at a dose higher than previously administered. In some embodiments, the cancer is breast or colon cancer.

[0041] In some embodiments, the second therapy may include selecting non-chemotherapy comprising cancer therapy for treatment of cancer in the subject. In some embodiments, the assay further comprises selecting non-chemotherapy-comprising cancer therapy for the subject when the expression of GalNAc-T13 in the sample from the subject is increased compared to the reference sample based on the recognition that chemotherapy may not be effective in a subject whose cancer has increased expression of GalNAc-T13. In further embodiments, the second therapy may include selecting chemotherapy-comprising cancer therapy when the expression of GAalNAc-T13 in the sample from the subject is the same as or decreased compared to the reference sample based on the recognition that chemotherapy may be effective in the subject whose cancer has decreased expression of GalNAc-T13.

[0042] The invention further provides an isolated sample obtained from a human subject comprising an abnormal level of GalNAc-T13. In some embodiments, the sample is any one or more of tissue, blood, plasma, urine or a combination thereof.

[0043] The invention also provides combinations of an isolated sample obtained from a human subject that includes an abnormal level of GalNAc-T13 and a reagent which reacts with the GalNAc-T13. In an embodiment, the reagent comprises a label to produce a signal indicative of the presence of the abnormal level of the GalNAc-T13 in the isolated sample. In some embodiments, the label is any one or more of a radiolabel, a chromophore, a fluorophore or a combination thereof. In various embodiments, the reagent is any one or more of a GalNAc-T 13 -specific nucleic acid, a GalNAc-T 13 -specific monoclonal antibody, a GalNAc-T13-enyme-specific substrate, a small molecule, a lipid or a combination thereof.

[0044] The invention also provides a system that includes an isolated sample obtained from a human subject, comprising an abnormal level of GalNAc-T13 and a reagent to react with the GalNAc-T13. In an embodiment, the reagent comprises a label to produce a signal indicative of the presence of the abnormal level of the GalNAc-T13 in the isolated sample. In some embodiments, the label is any one or more of a radiolabel, a chromophore, a fluorophore or a combination thereof. In various embodiments, the reagent is any one or more of a GalNAc- T13-specific nucleic acid, a GalNAc-T 13 -specific monoclonal antibody, a GalNAc-T13- enyme-specific substrate, a small molecule, a lipid or a combination thereof. [0045] In various embodiments of the processes, assays and methods described herein, the subject is human. In some embodiments, the subject has undergone neoadjuvant therapy. . In some embodiments, analyzing the level of GalNAc-T13 in a sample obtained from the subject includes measuring the nucleic acid levels that encode GalNAc-T13, the protein levels of GalNAc-T13, or a combination thereof. In some embodiments, the sample from the subject is obtained before, during or after cancer treatment. In an embodiment, the subject has breast and/or colon cancer. In various embodiments, samples from the subject are obtained from tissue, blood, plasma or a combination thereof.

Analysis of GalNAc-T13 expression

[0046] In various embodiments of the processes, assays and methods described herein, assaying the GalNAc-T13 comprises measuring the amount of nucleic acid encoding GalNAc-T13 present in the sample, measuring the amount of GalNAc-T13 protein protein present in the sample, or a combination thereof.

[0047] In various embodiments of the processes, assays and methods described herein, analyzing the sample includes detecting the level of GalNAc-T13 with an antibody specific to GalNAc-T13. In various embodiments, the antibody is any one or more of a monoclonal antibody or fragment thereof, a polyclonal antibody or a fragment thereof, chimeric antibodies, humanized antibodies, human antibodies, and a single chain antibody. In an embodiment, the antibody is a monoclonal antibody. An example of a monoclonal antibody that may be used is the T13.5 monoclonal antibody that binds the sequence LLPALR of GalNAc-T13.

[0048] In some embodiments of the processes, assays and methods described herein, analyzing the sample includes measuring the levels mRNA that encode GalNAc-T13, present in the sample with a polynucleotide capable of hybridizing with mRNA specific for GalNAc- T13 under stringent hybridization conditions.

[0049] Techniques that may be used to assess the amount of nucleic acid encoding GalNAc- T13, present in the sample include but are not limited to in situ hybridization (e.g., Angerer (1987) Meth. Enzymol 152: 649). Preferred hybridization-based assays include, but are not limited to, traditional "direct probe" methods such as Southern blots or in situ hybridization {e.g., FISH and FISH plus SKY), and "comparative probe" methods such as comparative genomic hybridization (CGH), e.g., cDNA-based or oligonucleotide-based CGH. The methods can be used in a wide variety of formats including, but not limited to, substrate (e.g. membrane or glass) bound methods or array-based approaches. Probes that may be used for nucleic acid analysis are typically labeled, e.g., with radioisotopes or fluorescent reporters. Preferred probes are sufficiently long so as to specifically hybridize with the target nucleic acid(s) under stringent conditions. The preferred size range is from about 200 bases to about 1000 bases. Hybridization protocols suitable for use with the methods of the invention are described, e.g., in Albertson (1984) EMBO J. 3: 1227-1234; Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142; EPO Pub. No. 430,402; Methods in Molecular Biology, Vol. 33: In situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, N.J. (1994), Pinkel, et al. (1998) Nature Genetics 20: 207-211, and/or Kallioniemi (1992) Proc. Natl Acad Sci USA 89:5321-5325 (1992).

[0050] Methods of "quantitative" amplification are well known to those of skill in the art. For example, quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. Detailed protocols for quantitative PCR are provided in Innis, et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.). Measurement of DNA copy number at microsatellite loci using quantitative PCR anlaysis is described in Ginzonger, et al. (2000) Cancer Research 60:5405-5409. The known nucleic acid sequence for the genes is sufficient to enable one of skill in the art to routinely select primers to amplify any portion of the gene. Fluorogenic quantitative PCR may also be used in the methods of the invention. In fluorogenic quantitative PCR, quantitation is based on amount of fluorescence signals, e.g., TaqMan and sybr green.

[0051] Other suitable amplification methods include, but are not limited to, ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560, Landegren, et al. (1988) Science 241 : 1077, and Barringer et al. (1990) Gene 89: 117), transcription amplification (Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication (GuateUi, et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapter PCR, etc.

[0052] A two-tailed student t-test with unequal variation may be used to measure the differences between the patient's expression of GalNAc-T13 and a normal blood sample, or the patient's own blood (matched control), or a reference generate by computer algorithm pooling many control samples, as described herein. A significant difference may be achieved where the p value is equal to or less than 0.05. GalNAc-T13 mRNA expression may also be used to determine patient's prognosis and response to chemotherapy, where GalNAc-T13 mRNA expression is separated into two groups: those with high GalNAc-T13 expression and those with low or no detectable GalNAc-T13 expression. The groups may be separated by the median GalNAc-T13 expression and plotted over time with a Kaplan-Meier curve.

[0053] Suitable methods for assaying the expression level of GalNAc-T13 include but are not limited to using DNA sequencing, comparative genomic hybridization (CGH), array CGH (aCGH), SNP analysis, mRNA expression assay, RT-PCR, real-time PCR, or a combination thereof. In various embodiments, the assay to detect the nucleic acid encoding or protein levels of, GalNAc-T13, is any one or more of Northern blot analysis, Southern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), radio-immuno assay (RIA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis or a combination thereof. In some embodiments, the level of GalNAcT13 in a subject may be ascertained by measuring the substrate upon which the enzyme GalNAcT13 acts, such that the substrate serves as a surrogate marker for GalNAcT13.

[0054] Antibodies, both polyclonal and monoclonal, can be produced by a skilled artisan either by themselves using well known methods or they can be manufactured by service providers who specialize making antibodies based on known protein sequences. In the present invention, the protein sequences are known and thus production of antibodies against them is a matter of routine.

[0055] For example, production of monoclonal antibodies can be performed using the traditional hybridoma method by first immunizing mice with an antigen which may be an isolated protein of choice or fragment thereof (for example, GalNAc-T13 or a fragment thereof) and making hybridoma cell lines that each produce a specific monoclonal antibody. The antibodies secreted by the different clones are then assayed for their ability to bind to the antigen using, e.g., ELISA or Antigen Microarray Assay, or immuno-dot blot techniques. The antibodies that are most specific for the detection of the protein of interest can be selected using routine methods and using the antigen used for immunization and other antigens as controls. The antibody that most specifically detects the desired antigen and protein and no other antigens or proteins are selected for the processes, assays and methods described herein. [0056] The best clones can then be grown indefinitely in a suitable cell culture medium. They can also be injected into mice (in the peritoneal cavity, surrounding the gut) where they produce an antibody-rich ascites fluid from which the antibodies can be isolated and purified. The antibodies can be purified using techniques that are well known to one of ordinary skill in the art.

[0057] In the methods and assays of the invention, the presence of any GalNAc-T13 or a fragment thereof is determined using antibodies specific for the GalNAc-T13 protein and detecting immunospecific binding of each antibody to its respective cognate marker.

[0058] Any suitable immunoassay method may be utilized, including those which are commercially available, to determine the level GalNAc-T13 measured according to the invention. Extensive discussion of the known immunoassay techniques is not required here since these are known to those of skill in the art. Typical suitable immunoassay techniques include sandwich enzyme-linked immunoassays (ELISA), radioimmunoassays (RIA), competitive binding assays, homogeneous assays, heterogeneous assays, etc. Various known immunoassay methods are reviewed, e.g., in Methods in Enzymology, 70, pp. 30-70 and 166- 198 (1980).

[0059] In the assays of the invention, "sandwich-type" assay formats can be used. Some examples of such sandwich-type assays are described in by U.S. Pat. No. 4,168,146 to Grubb, et al. and U.S. Pat. No. 4,366,241 to Tom, et al. An alternative technique is the "competitive- type" assay. In a competitive assay, the labeled probe is generally conjugated with a molecule that is identical to, or an analog of, the analyte. Thus, the labeled probe competes with the analyte of interest for the available receptive material. Competitive assays are typically used for detection of analytes such as haptens, each hapten being monovalent and capable of binding only one antibody molecule. Examples of competitive immunoassay devices are described in U.S. Pat. No. 4,235,601 to Deutsch, et al, U.S. Pat. No. 4,442,204 to Liotta, and U.S. Pat. No. 5,208,535 to Buechler, et al.

[0060] The antibodies can be labeled. In some embodiments, the detection antibody is labeled by covalently linking to an enzyme, label with a fluorescent compound or metal, label with a chemiluminescent compound. For example, the detection antibody can be labeled with catalase and the conversion uses a colorimetric substrate composition comprises potassium iodide, hydrogen peroxide and sodium thiosulphate; the enzyme can be alcohol dehydrogenase and the conversion uses a colorimetric substrate composition comprises an alcohol, a pH indicator and a pH buffer, wherein the pH indicator is neutral red and the pH buffer is glycine-sodium hydroxide; the enzyme can also be hypoxanthine oxidase and the conversion uses a colorimetric substrate composition comprises xanthine, a tetrazolium salt and 4,5-dihydroxy-l,3-benzene disulphonic acid. In one embodiment, the detection antibody is labeled by covalently linking to an enzyme, label with a fluorescent compound or metal, or label with a chemiluminescent compound.

[0061] Direct and indirect labels can be used in immunoassays. A direct label can be defined as an entity, which in its natural state, is visible either to the naked eye or with the aid of an optical filter and/or applied stimulation, e.g., ultraviolet light, to promote fluorescence. Examples of colored labels which can be used include metallic sol particles, gold sol particles, dye sol particles, dyed latex particles or dyes encapsulated in liposomes. Other direct labels include radionuclides and fluorescent or luminescent moieties. Indirect labels such as enzymes can also be used according to the invention. Various enzymes are known for use as labels such as, for example, alkaline phosphatase, horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactate dehydrogenase and urease. For a detailed discussion of enzymes in immunoassays see Engvall, Enzyme Immunoassay ELISA and EMIT, Methods of Enzymology, 70, 419-439 (1980).

[0062] The antibody can be attached to a surface. Examples of useful surfaces on which the antibody can be attached for the purposes of detecting the desired antigen include nitrocellulose, PVDF, polystyrene, and nylon. The surface or support may also be a porous support (see., e.g., U.S. Patent No. 7,939,342). The assays can be carried out in various assay device formats including those described in U.S. Pat. Nos. 4,906,439; 5,051,237 and 5,147,609 to PB Diagnostic Systems, Inc.

[0063] In some embodiments of the processes, assays and methods described herein, detecting the level of antibodies reactive to GalNAc-T13 includes contacting the sample from the cancer patient with an antibody or a fragment thereof that specifically binds GalNAc-T13, forming an antibody-protein complex between the antibody and GalNAc-T13 present in the sample, washing the sample to remove the unbound antibody, adding a detection antibody that is labeled and is reactive to the antibody bound to GalNAc-T13 in the sample, washing to remove the unbound labeled detection antibody and converting the label to a detectable signal, wherein the detectable signal is indicative of the level of GalNAc-T13 in the sample from the patient. In some embodiments, the effector component is a detectable moiety selected from the group consisting of a fluorescent label, a radioactive compound, an enzyme, a substrate, an epitope tag, electron-dense reagent, biotin, digonigenin, hapten and a combination thereof. In some embodiments, the detection antibody is labeled by covalently linking to an enzyme, labeled with a fluorescent compound or metal, labeled with a chemiluminescent compound. The level of GalNAc-T13 may be obtained by measuring a light scattering intensity resulting from the formation of an antibody-protein complex formed by a reaction of GalNAc-T13 in the sample with the antibody, wherein the light scattering intensity of at least 10% above a control light scattering intensity indicates the likelihood of breast and/or colon cancer, chemotherapy resistance or a combination thereof.

[0064] In various embodiments of the processes, assays and methods of the invention, an increased likelihood of chemotherapy resistance may result in poor prognosis wherein the poor prognosis comprises decreased survival likelihood, shortened life expectancy, or enhanced tumor sternness.

[0065] In various embodiments of the processes, assays and methods of the invention, the process described herein further comprises prescribing a first therapy to the subject if the subject has a good prognosis or prescribing a second therapy, or both the first therapy and the second therapy, to the subject if the subject has a poor prognosis.

Reference Values

[0066] In various embodiments of the processes, assays and methods described herein, the reference value is based on the expression level of GalNAc-T13. In one embodiment, the expression level is in a breast and/or colon cancer cell. In another embodiment, the expression level is in a non-cancer cell. In an additional embodiment, the expression level is in any cell. In some embodiments, the reference value is the mean or median expression level of GalNAc-T13 in a population of subjects that do not have cancer. In other embodiments, the reference value is the mean or median expression level of GalNAc-T13 in a population of subjects that have breast and/or colon cancer and respond to chemotherapy. In some embodiments the reference value that comprises the population of subjects that have breast and/or colon cancer and respond to chemotherapy, show undetectable expression of GalNAc-T13 or show reduced expression of GalNAc-T13. In additional embodiments, the reference value is the expression level of GalNAc-T13 in a sample obtained from the subject from a different (for example, an earlier) time point, such as during diagnosis, before treatment, after treatment or a combination thereof. In some embodiments, the cancer is breast and/or colon cancer.

[0067] In various embodiments, the expression level of GalNAc-T13 in the breast and/or colon cancer subject compared to the reference value is increased by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In various embodiments, the expression level of GalNAc-T13 in the breast and/or colon cancer subject compared to the reference value is increased by at least or about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold or a combination thereof.

Therapies

[0068] In accordance with various embodiments of the invention, the therapies described herein may be selected, used and/or administered to treat a cancer patient with breast and/or colon cancer. In various embodiments, the therapy may be any one or more of surgery, radiation, chemotherapy, immunotherapy, vaccine or combinations thereof.

[0069] In some embodiments, in patients with chemotherapy resistant forms of breast or colon cancer, a second therapy is administered if GalNAc-T13 is present in the subject or the levels of GalNAc-T13 have increased in the subject, which may be indicative of chemotherapy resistance in the cancer patient. Second therapy includes surgery, radiation, immunotherapy, vaccine or combinations thereof. In some embodiments, chemotherapy may be included in the second therapy with administering higher dosages of chemotherapeutic drugs, administering combinations of chemotherapeutic drugs or a combination thereof.

[0070] In some embodiments, chemotherapeutic agents may be selected from any one or more of cytotoxic antibiotics, antimetabolities, anti-mitotic agents, alkylating agents, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogues, plant alkaloids, and toxins; and synthetic derivatives thereof. Exemplary compounds include, but are not limited to, alkylating agents: treosulfan, and trofosfamide; plant alkaloids: vinblastine, paclitaxel, docetaxol; DNA topoisomerase inhibitors: doxorubicin, epirubicin, etoposide, camptothecin, topotecan, irinotecan, teniposide, crisnatol, and mitomycin; anti-folates: methotrexate, mycophenolic acid, and hydroxyurea; pyrimidine analogs: 5-fluorouracil, doxifluridine, and cytosine arabinoside; purine analogs: mercaptopurine and thioguanine; DNA antimetabolites: 2'-deoxy-5-fluorouridine, aphidicolin glycinate, and pyrazoloimidazole; and antimitotic agents: halichondrin, colchicine, and rhizoxin. Compositions comprising one or more chemotherapeutic agents (e.g., FLAG, CHOP) may also be used. FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOP comprises cyclophosphamide, vincristine, doxorubicin, and prednisone. In another embodiments, PARP (e.g., PARP-1 and/or PARP-2) inhibitors are used and such inhibitors are well known in the art (e.g., Olaparib, ABT-888, BSI-201, BGP-15 (N-Gene Research Laboratories, Inc.); INO-1001 (Inotek Pharmaceuticals Inc.); PJ34 (Soriano et al., 2001; Pacher et al., 2002b); 3-aminobenzamide (Trevigen); 4-amino-l,8-naphthalimide; (Trevigen); 6(5H)-phenanthridinone (Trevigen); benzamide (U.S. Pat. Re. 36,397); and NU1025 (Bowman et al.).

[0071] In various embodiments, therapies include use of chemotherapeutic agents to treat breast and/or colon cancer. Such agents include but are not limited to Abitrexate, Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), Afatinib, Alimta (Pemetrexed Disodium), Avastin (Bevacizumab), Bevacizumab, Carboplatin, Cisplatin, Crizotinib, Erlotinib Hydrochloride, Folex (Methotrexate), Folex PFS (Methotrexate), Gefitinib, Gilotrif (Afatinib), Gemcitabine Hydrochloride, Gemzar (Gemcitabine Hydrochloride), Iressa (Gefitinib), Methotrexate, Methotrexate LPF, Mexate, Mexate-AQ, Nivolumab, Necitumumab, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pemetrexed Disodium, Platinol (Cisplatin), Platinol- AQ (Cisplatin), Tarceva (Erlotinib Hydrochloride), Taxol (Paclitaxel), Xalkori (Crizotinib) or a combination thereof.

[0072] In various embodiments, therapies include, for example, radiation therapy. The radiation used in radiation therapy can be ionizing radiation. Radiation therapy can also be gamma rays, X-rays, or proton beams. Examples of radiation therapy include, but are not limited to, external-beam radiation therapy, interstitial implantation of radioisotopes (1-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and/or total abdominal and pelvic radiation therapy. For a general overview of radiation therapy, see Hellman, Chapter 16: Principles of Cancer Management: Radiation Therapy, 6th edition, 2001, DeVita et al., eds., J. B. Lippencott Company, Philadelphia. The radiation therapy can be administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source. The radiation treatment can also be administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body close to cancer cells or a tumor mass. Also encompassed is the use of photodynamic therapy comprising the administration of photosensitizers, such as hematoporphyrin and its derivatives, Vertoporfm (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A; and 2BA-2-DMHA.

[0073] In various embodiments, therapies include, for example, immunotherapy. Immunotherapy may comprise, for example, use of cancer vaccines and/or sensitized antigen presenting cells. The immunotherapy can involve passive immunity for short-term protection of a host, achieved by the administration of pre-formed antibody directed against a cancer antigen or disease antigen (e.g., administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen). Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer cell lines.

[0074] In various embodiments, therapies include, for example, hormonal therapy, Hormonal therapeutic treatments can comprise, for example, hormonal agonists, hormonal antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, Cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins), vitamin A derivatives (e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs; antigestagens (e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproterone acetate).

[0075] The duration and/or dose of treatment with anti-cancer therapies may vary according to the particular anti-cancer agent or combination thereof. An appropriate treatment time for a particular cancer therapeutic agent will be appreciated by the skilled artisan. The invention contemplates the continued assessment of optimal treatment schedules for each cancer therapeutic agent, where the genetic signature of the cancer of the subject as determined by the methods of the invention is a factor in determining optimal treatment doses and schedules.

[0076] In various embodiments, the subject for whom predicted efficacy of an anti-cancer therapy is determined, is a mammal (e.g., mouse, rat, primate, non-human mammal, domestic animal such as dog, cat, cow, horse), and is preferably a human. In another embodiment of the methods of the invention, the subject has not undergone chemotherapy or radiation therapy. In alternative embodiments, the subject has undergone chemotherapy or radiation therapy (e.g., such as with cisplatin, carboplatin, and/or taxane). In related embodiments, the subject has not been exposed to levels of radiation or chemotoxic agents above those encountered generally or on average by the subjects of a species. In certain embodiments, the subject has had surgery to remove cancerous or precancerous tissue. In other embodiments, the cancerous tissue has not been removed, e.g., the cancerous tissue may be located in an inoperable region of the body, such as in a tissue that is essential for life, or in a region where a surgical procedure would cause considerable risk of harm to the patient, or e.g., the subject is given the anti-cancer therapy prior to removal of the cancerous tissue.

Samples

[0077] Samples, such as cancer cells, cancerous tissue, plasma and/or blood, could be collected preferably at the time of biopsy for diagnosis of breast and/or colon cancer. This would allow the best chance to design a course of treatment that would best serve the patient. For example, if expression of GalNAc-T13 has increased, the patient may require a more aggressive treatment course compared to another patient with a breast and/or colon cancer that does not have increased expression of GalNAc-T13. It is also possible to obtain cancerous tissue, plasma and/or blood after cancer treatment (e.g., surgery) or during cancer treatment (e.g., radiation, chemotherapy etc.). This would allow for a change in treatment course or decision on the course of treatment with the prospect of recurrence. In various embodiments, the cancer is a breast and/or colon cancer.

[0078] The steps involved in the current invention comprise obtaining either through surgical biopsy or surgical resection, a sample of the patient's breast and/or colon tumor and matching blood sample from the patient. Alternatively, a sample can be obtained through primary patient harvested breast and/or colon tumor stem cells, primary patient breast and/or colon tumor derived cell lines, or archived patient samples in the form of FFPE (Formalin fixed, paraffin embedded) samples, or fresh frozen breast and/or colon tumor samples. This invention also allows for the possibility of retrospectively evaluating the above mentioned parts of this invention (i.e. likelihood of survival, estimated life expectancy and the potential of acquiring this mutation in the future).

[0079] Patient's tumor sample is then used to extract Deoxyribonucleic acid (DNA) using the standard protocol designated "QIAamp DNA Mini and Blood Mini kit" or for FFPE samples "QIAamp DNA FFPE Tissue kit" commercially available from Qiagen®. The above and following procedures require informed consent from patients. [0080] The invention provides a system for determining responsiveness of a cancer cell to chemotherapy wherein the cancer cell is obtained from a cancer patient. The system includes a sample analyzer configured to produce a signal for mRNA encoding GalNAc-T13 present in the cancer cell obtained from the cancer patient and a computer sub-system programmed to calculate, based on the mRNA whether the signal is greater than or not greater than a reference value.

[0081] The invention also provides a system for determining responsiveness of a cancer cell to chemotherapy wherein the cancer cell is obtained from a cancer patient. The system comprises a sample analyzer configured to produce a signal when a GalNAc-T 13 -specific antibody binds GalNAc-T13 in the cancer cell obtained from a cancer patient and a computer sub-system programmed to calculate, based on the antibody binding whether the signal is greater than or not greater than a reference value.

[0082] In some embodiments, the computer sub-system is programmed to compare the mRNA to determine a likelihood of responsiveness of said cancer cell to chemotherapy based on an algorithm that classifies the patient as likely to responds to a chemotherapy-comprising therapy if GalNAc-T13 expression is increased and as unlikely to respond to chemotherapy- comprising therapy if the GalNAc-T13 is not increased.

[0083] The invention further provides a computer program product embodied in a computer readable medium that, when executed on a computer, performs steps comprising detecting GalNAc-T13 expression in a sample comprising a cancer cell obtained from a cancer patient and comparing the GalNAc-T13 expression to a reference value .A diagnostic kit for detecting a likelihood of a cancer patient responding to chemotherapy comprising no more than 10 probes comprising a combination of detectable labeled probes or primers for GalNAc-T13 and a computer program product described herein.

EXAMPLES

[0084] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention. Example 1

Experimental Methods

Production of anti-GalNAc-Tl 3 monoclonal antibody.

[0085] A synthetic peptide specific of GalNAc-T13 was select in the region that displays very high variability among GalNAc-Ts family members (RSLLPALRAVISRNQE, accession number BAC54545, Biosynthesis). BALB/c mice were immunized four times at 2-week intervals with 100 jig of this peptide conjugated to keyhole-limpet hemocyanin. Isolated spleen cells were fused with mouse myeloma cells SP2/0. Antibody titer and supernatant screening were performed by ELISA, using microtiter plates coated with the same peptide sequence conjugated to bovine serum albumin.

Analysis of monoclonal antibody specificity by Surface Plasmon Resonance

[0086] Interactions between the mAb 13.5 and synthetic peptides were analyzed by performing surface plasmon resonance experiments on a BIAcore 3000 instrument (GE Healthcare, Sweden). Purified mAb was coupled to an activated carboxymethylated dextran CM-5 sensor surfaces (SA sensorchip, GE Healthcare, Sweden). The peptides were diluted in HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.005% Surfactant P20, pH 7.4) and were passed over the sensorchip. All experiments were run in duplicates at a 30 μΕ/ηιίη flow rate, a contact time of 180s and a dissociation time of 360s, with the biosensor instrument thermostated at 25 °C. After dissociation the sensor chip was regenerated by injecting 10 mM glycine-HCl (pH 2.5) at the end of each experiment. All data processing was carried out using the BIAevaluation 4.1 software provided by BIAcore.

Example 2

Generation of a monoclonal antibody specific for GalNAc-Tl 3 useful for immunohistochemical studies in paraffin embedded tissues.

[0087] Considering that GalNAc-T13 displays 84% homology compared with GalNAc-Tl we immunized mice with a KLH-conjugated specific motif (RSLLPALRAVISRNQE) of GalNAc-T13, without any homology with GalNAc-Tl sequence. Selection of specific hybridomas was performed by ELISA, screening against BSA-conjugated GalNAc-T13 peptide. One of the mAbs, T13.5, strongly reactive against the synthetic peptide, was used for further characterization. We evaluated the mAb T13.5 reactivity in Western blot using GalNAc-Tl and -T13 expressed in baculovirus. We found that the antibody reacts with GalNAc-T13 but not with GalNAc-Tl, confirming the specificity of this antibody for GalNAc-T13 and that it binds to denatured forms of the protein. To determine which amino acid residues are crucial for mAb T13.5 binding we mapped the epitope using overlapping peptides covering the sequence RSLLPALRAVISRNQE. Peptide binding to immobilized antibody was assessed using BIAcore. The results obtained indicate that the epitope of mAb T13.5 could be mapped to residues LLPLAR. Considering that GalNAc-T13 expression was previously reported in neuroblastoma (Berois et ah, Clin Chem 2006; 52:1701-12), we performed an immunocytochemical analysis using MAb T13.5 on the IMR-32 cell line. We found a strong staining preponderantly detected in the perinuclear region, as expected for a glycosyltransferase localized in Golgi apparatus. The immunohistochemical evaluation in neuroblastoma tumors demonstrates that mAb T13.5 is able to detect GalNAc-T13 in paraffin embedded tissues used in the pathological routine diagnostic.

Example 3

Detection of GalNAc-Tl 3 in breast cancer

[0088] To evaluate the expression of GalNAc-T13 in breast cancer, we first analyzed its detection at mRNA level by RT-PCR. We found GalNAc-T13 in MCF-7 breast cancer cell line (Figure 1). We performed an immunohistochemical evaluation of breast tissues, using the anti-GalNAc-T13 monoclonal antibody T13.5. We found the expression of GalNAc-T13 in 10/15 primary tumors from breast cancer patients (Figure 2), and in 0/5 normal breast tissues.

Example 4

Detection of GalNAc-Tl 3 in colon cancer

[0089] We evaluated the expression of GalNAc-T13 in colon cancer. We found GalNac-T13 expression in human colon cancer cell lines, both using RT-PCR and indirect immunofluorescence using the MAb T13.5 (Figures 3A and 3B). We also observed that the SW620 cell line (high metastatic) strongly stained for GalNac-T13 compared to SW480 cell line (low metastatic, derived from the same patient as SW620), suggesting that GalNAc-T13 could be associated to a more aggressive phenotype. In the colon tumors evaluated by immunohistochemistry, we found GalNAc-T13 expression in 3/5 patients (Figure 3C) and not in the 2 samples or normal colon mucosa (Figure 3D). [0090] These results strongly suggest that GalNAc-T13 could be a new biomarker applicable to the evaluation of patients with breast and colon cancer.

Example 5

Immunohistochemical staining of tissue samples from patients with breast or colon cancer

[0091] For immunohistochemical staining 5 μιη thick histology sections were deparaffinized, hydrated, and heated in a steamer for 10 minutes with 10 mmol/liter of sodium citrate (pH 6.0) for antigen retrieval. Peroxide blocking was performed with 3% H2O2 in methanol at room temperature for 15 minutes, followed by 10% bovine serum albumin in TBS-tween for 30 minutes. The slides were incubated with primary antibody (T13.5 culture supernatant) for 65 minutes at room temperature, followed by 3 washes with phosphate-buffered saline, and incubation with biotin-labeled secondary antibody for 30 minutes. Finally, the samples were incubated with a 1 :40 solution of streptavidin-peroxidase for 30 minutes. The staining was then developed with 0.05% 3'3-diaminobenzidine tetrahydrochloride prepared in 0.05 mol/liter of Tris buffer at pH 7.6 containing 0.024% H2O2 and then counterstained with hematoxylin. For a negative control, the primary antibody was replaced with phosphate- buffered saline. Immunohistochemical expression was quantified blinded to the patient treatment and outcome, using a final score obtained by multiplying a 4-value intensity score (0-3 for negative, weak, moderate, and strong respectively), and the percentage of positive tumor cells. The score ranged from a minimum of 0 to a maximum of 300. The levels of GalNAc-T13 expression in tissue samples from patients with breast and colon cancer are shown in Tables 1, 2 and 3. For controls, GalNAc-T13 expression was evaluated in 5 normal breast tissue samples and in 5 normal colon tissue samples and expression of GalNAc-T13 was not detected.

[0092] Table 1. Immunohistochemical analysis of GalNAc-T13 expression in tissue samples from patients with breast cancer. The numbers in high, middle and low columns indicate the number of patients in each group. For high GalNAc-T13 expression, the score is 200-300; for middle GalNAc-T13 expression, the score is 100-199; for low GalNAc-T13 expression, the score is below 100. <3a(NAc-T13 expression score

Patients >-; ~ 101 High Middle Low

Age in years 81 ,6 {31-87} .<> 5i- n ~ 3? n - 11

Tumor size in mm 3 .4 {1-130}

Tumor pTI 2? Q

stage pT2 8Θ 34 24

pT3 8 1 5

p = NS

Nodai o y 4

Status -v3 18 2

HZ 18 10 3 3

m 14 7 2

UiCC 9 6

stage !ia 29 8

ii 33 16

18

14

[0093] Table 2: Immunohistochemical analysis of GalNAc-T13expression in tissue samples from patients with breast cancer. The numbers in high, middle and low columns indicate the number of patients in each group. For high GalNAc-T13 expression, the score is 200-300; for middle GalNAc-T13 expression, the score is 100-199; for low GalNAc-T13 expression, the score is below 100.

Total Ga I Nac-T 3 «g|»e¾8jof> sc (¾¾

Population High Mid ite tow

81 37 19

HsstoSogicaitype Ductal

Lobular 19 8 8

Mixed 7 2 4

Cfibiform 4

Mucinous

Others 6

p■■ NS

His!oSogsca: 12

grade 39 16

50 23

ON 1 12

Nu fesr

GN2 62 33 28

a rade

G Z 14 6

p = NS

Mitotic 29 14 12

index M2 33 21 11

S.M3 39 18 14

p^■-^■· NS [0094] Table 3: Immunohistochemical analysis of GalNAc-T13 expression in tissue samples from patients with colon cancer.

Poor 10 (9,8} 1 2 (20)

[0095] The various methods and techniques described above provide a number of ways to carry out the application. Of course, it is to be understood that not necessarily all objectives or advantages described can be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein. A variety of alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular feature by inclusion of one, another, or several advantageous features.

[0096] Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments. [0097] Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the application extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

[0098] In some embodiments, the terms "a" and "an" and "the" and similar references used in the context of describing a particular embodiment of the application (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0099] Preferred embodiments of this application are described herein, including the best mode known to the inventors for carrying out the application. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context.

[0100] All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and/or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and/or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and/or the use of the term in the present document shall prevail.

[0101] It is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the application. Other modifications that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative configurations of the embodiments of the application can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.

Claims

CLAIMS What is claimed is:

1. An assay, comprising:

(i) obtaining a sample comprising a tumor cell from a patient with breast and/or colon cancer;

(ii) assaying the sample to determine the level of GalNac-T13; and

(iii) determining the subject has increased likelihood of breast and/or colon cancer if the level of GalNac-T13 is increased relative to a reference sample, or determining the subject has decreased likelihood of breast and/or colon cancer if the level of GalNac-T13 is the same as or decreased relative to the reference sample.

2. The assay of claim 1, wherein assaying the sample comprises detecting the level of nucleic acid encoding GalNac-T13, determining the level of GalNac-T13 protein, or a combination thereof.

3. The assay of claim 2, wherein detecting the level of nucleic acid encoding GalNAc-T13 comprises determining the amount of mR A, encoding GalNac-T13, present in the sample.

4. The assay of claim 2, wherein detecting the level of GalNAc-T13 protein comprises detecting the level of GalNac-T13 with an antibody specific to GalNac-T13.

5. The assay of claim 4, wherein the antibody is a monoclonal antibody.

6. The assay of claim 5, wherein the monoclonal antibody binds the epitope LLPALR of GalNAc-T13.

7. The assay of claim 5, wherein the antibody is T13.5 monoclonal antibody.

8. The assay of claim 1, wherein the sample is tissue, blood, plasma or a combination thereof.

9. The assay of claim 1, wherein the subject is human.

10. The assay of claim 1, wherein the reference value is the mean or median expression level of GalNAc-T13 in a population of subjects that do not have breast and/or colon cancer.

11. The assay of claim 1 , wherein the reference value is the expression level of GalNAc- T13 from the subject in a sample obtained from a different time point.

12. The assay of claim 1, further comprising prescribing a therapy to the subject if the subject has increased likelihood of breast or colon cancer.

13. The assay of claim 12, wherein the therapy is any one or more of surgery, radiation, chemotherapy, immunotherapy, vaccine, or a combination thereof.

14. An assay for determining an increased likelihood of breast and/or colon cancer in a subject in need thereof comprising:

(i) providing a biological sample from a subject having breast and/or colon cancer;

(ii) providing an antibody that specifically binds to GalNAc-T13;

(iii) contacting the biological sample with the antibody; and

(iv) detecting, using immunoassay, the level of antibody binding to GalNAc-T13, wherein the presence of binding in the biological sample from the subject relative to a reference sample is indicative of increased likelihood of breast and/or colon cancer in the subject.

15. An assay for selecting a therapy for a subject having breast and/or colon cancer, and optionally administering the therapy, the assay comprising:

(ii) providing an antibody that specifically binds to GalNAc-T13;

(iii) contacting the biological sample with the antibody;

(iv) detecting, using immunoassay, the level of antibody binding to GalNAc-T13, wherein an increase in binding in the biological sample from the subject relative to a reference sample is indicative of increased expression of GalNAc-T13 and increased likelihood of breast and/or colon cancer in the subject; and

(v) selecting a therapy comprising prescribing a therapy to the subject if the subject has an increased likelihood of breast and/or colon cancer.

16. The assay of claim 15, wherein the therapy is any is any one or more of surgery, radiation, chemotherapy, immunotherapy, vaccine, or a combination thereof.

17. The assay of claims 14 or 15, wherein detecting the level of antibodies reactive to GalNAc-T13 comprises:

(i) contacting the sample from the breast and/or colon cancer patient with an antibody or a fragment thereof that specifically binds GalNAc-T13;

(ii) forming an antibody-protein complex between the antibody and GalNAc-T13 present in the sample;

(iii) washing the sample to remove the unbound antibody;

(iv) adding a detection antibody that is labeled and is reactive to the antibody bound to GalNAc-T13 in the sample;

(v) washing to remove the unbound labeled detection antibody; and

(vi) converting the label to a detectable signal, wherein the detectable signal is indicative of the level of GalNAc-T13 in the sample from the patient.

18. The assay of claims 14 or 15, wherein the sample is tissue, blood, plasma or a combination thereof.

19. The assay of claims 14 or 15, wherein the antibody is a monoclonal antibody.

20. The assay of claim 19, wherein the antibody binds the epitope LLPALR of GalNAc- T13.

21. The assay of claim 19, wherein the antibody is the T13.5 monoclonal antibody.

22. The assay of claim 14 or 15, wherein the reference value is the mean or median GalNAc-T13 expression level from a population of subjects that do not have breast and/or colon cancer.

23. The assay of claim 14 or 15, wherein the reference value is the expression level of GalNAc-T13 from the subject in a sample obtained from a different time point.

24. The assay of claim 14 or 15, wherein the antibody is further conjugated to an effector component.

25. The assay of claim 24, wherein the effector component is a detectable moiety selected from the group consisting of a fluorescent label, a radioactive compound, an enzyme, a substrate, an epitope tag, electron-dense reagent, biotin, digonigenin, hapten and a combination thereof.

26. The assay of claim 17, wherein the detection antibody is labeled by covalently linking to an enzyme, labeled with a fluorescent compound or metal, labeled with a chemiluminescent compound.

27. The assay of claim 17, wherein the level of GalNAc-T13 is obtained by measuring a light scattering intensity resulting from the formation of an antibody-protein complex formed by a reaction of GalNAc-T13 in the sample with the antibody, wherein the light scattering intensity of at least 10% above a control light scattering intensity indicates the likelihood of cancer.

28. The assay of claim 14 or 15, wherein presence of binding is an increase in binding of the antibody to GalNAc-T13.

29. A method for treating a subject having breast and/or colon cancer, and optionally administering the therapy comprising:

(ii) providing an antibody that binds to GalNAc-T 13 ;

(iii) contacting the biological sample with the antibody;

(iv) detecting, using immunoassay, whether the antibody binds GalNAc-T13, wherein an increase in binding in the biological sample from the subject relative to a reference sample is indicative of increased expression of GalNAc-T13 and increased likelihood of breast and/or colon cancer in the subject; and

(v) prescribing a therapy to the subject if the subject has increased likelihood of breast and/or colon cancer.

30. The method of claim 29, wherein the therapy is any is any one or more of surgery, radiation, chemotherapy, immunotherapy, vaccine, or a combination thereof.

31. The method of claim 29, wherein the antibody is a monoclonal antibody.

32. The assay of claim 31, wherein the antibody binds the epitope LLPALR of GalNAc- T13.

33. The assay of claim 31, wherein the antibody is the T13.5 monoclonal antibody.

34. An assay for determining an increased likelihood of breast or colon cancer in a subject in need thereof comprising:

(i) providing a biological sample from a subject having breast or colon cancer; (ii) providing an antibody that specifically binds to GalNAc-T13, wherein the antibody is the T13.5 monoclonal antibody that binds to LLPALR epitope on GalNAc-T13;

(iii) contacting the biological sample with the antibody; and

35. The assay of claim 34, wherein detecting the level of antibodies reactive to GalNAc- T13 comprises:

(iii) washing the sample to remove the unbound antibody;

(v) washing to remove the unbound labeled detection antibody; and

(vi) converting the label to a detectable signal, wherein the detectable signal is indicative of the level of GalNAc-T13 in the sample from the patient

36. The assay of claim 34, wherein the reference value is the mean or median GalNAc-T13 expression level from a population of subjects that do not have breast and/or colon cancer.

37. The assay of claim 34, wherein the reference value is the expression level of GalNAc- T13 from the subject in a sample obtained from a different time point.

38. The assay of claim 34, wherein the antibody is further conjugated to an effector component.

39. The assay of claim 38, wherein the effector component is a detectable moiety selected from the group consisting of a fluorescent label, a radioactive compound, an enzyme, a substrate, an epitope tag, electron-dense reagent, biotin, digonigenin, hapten and a combination thereof.

40. The assay of claim 35, wherein the detection antibody is labeled by covalently linking to an enzyme, labeled with a fluorescent compound or metal, labeled with a chemiluminescent compound.

41. The assay of claim 35, wherein the level of GalNAc-T13 is obtained by measuring a light scattering intensity resulting from the formation of an antibody-protein complex formed by a reaction of GalNAc-T13 in the sample with the antibody, wherein the light scattering intensity of at least 10% above a control light scattering intensity indicates the likelihood of cancer.

42. The assay of claim 34, wherein presence of binding is an increase in binding of the antibody to GalNAc-T13 relative to the reference sample.

43. A method for diagnosing breast and/or colon cancer in a subject in need thereof comprising:

(i) providing a biological sample from a subject having breast or colon cancer;

(ii) providing an antibody that specifically binds to GalNAc-T13, wherein the antibody is the T13.5 monoclonal antibody that binds to LLPALR epitope on GalNAc-T13;

(iii) contacting the biological sample with the antibody; and

44. The method of claim 43, wherein detecting the level of antibodies reactive to GalNAc- T13 comprises:

(i) contacting the sample from the cancer patient with an antibody or a fragment thereof that specifically binds GalNAc-T13;

(iii) washing the sample to remove the unbound antibody;

(v) washing to remove the unbound labeled detection antibody; and (vi) converting the label to a detectable signal, wherein the detectable signal is indicative of the level of GalNAc-T13 in the sample from the patient

45. The method of claim 43, wherein the reference value is the mean or median GalNAc- T13 expression level from a population of subjects that do not have breast and/or colon cancer.

46. The method of claim 43, wherein the reference value is the expression level of GalNAc- T13 from the subject in a sample obtained from a different time point.

47. The method of claim 43, wherein the antibody is further conjugated to an effector component.

48. The method of claim 47, wherein the effector component is a detectable moiety selected from the group consisting of a fluorescent label, a radioactive compound, an enzyme, a substrate, an epitope tag, electron-dense reagent, biotin, digonigenin, hapten and a combination thereof.

49. The method of claim 44, wherein the detection antibody is labeled by covalently linking to an enzyme, labeled with a fluorescent compound or metal, labeled with a chemiluminescent compound.

50. The method of claim 44, wherein the level of GalNAc-T13 is obtained by measuring a light scattering intensity resulting from the formation of an antibody-protein complex formed by a reaction of GalNAc-T13 in the sample with the antibody, wherein the light scattering intensity of at least 10% above a control light scattering intensity indicates the likelihood of cancer.

51. The method of claim 44, wherein presence of binding is an increase in binding of the antibody to GalNAc-T13 relative to the reference sample.