WO2010088650A2 - Biomarker signature to predict cancer treatment response - Google Patents

Abstract

Currently cancer may be treated with chemotherapy, radiation therapy and surgery. The therapies are toxic and/or life altering. Previously there have been no methods to select therapy for a specific patient. Disclosed is a 3-biomarker signature that is highly associated with prediction and prognosis of patients. By assessing the signature in a patient's cancer specimen, the signature can identify patients who are likely to respond to chemotherapy and/or radiation treatment.

Description

BIOMARKER SIGNATURE TO PREDICT CANCER TREATMENT RESPONSE

[0001] This application claims the benefit of U.S. Provisional Application No. 61/149,204 filed February 2, 2009, the entirety of which is herein incorporated by reference.

TECHNICAL FIELD

[0002] The present invention relates generally to the fields of oncology, molecular biology, cell biology, and cancer. More particularly, it concerns the assessment of factors to predict the efficacy of cancer therapy.

BACKGROUND OF THE INVENTION

[0003] Cancer is a leading cause of death in most countries and the result of billions of dollars in healthcare expense around the world. Cancer is treated by a variety of methods including chemotherapy, radiation therapy, and surgery, depending on the type of cancer and the location of the cancer. These treatments are expensive and all have a wide variety of accompanying side effects that decrease the cancer patients quality of life.

[0004] It is presently unclear why some patients respond to certain cancer therapies while others do not. There is a need to identify specific patient subsets that will most benefit from certain treatment regiments. Currently, the ability to predict which patients respond well to which treatment regiments is limited. The ability to predict a patient' s response to therapy is a valuable asset in developing treatment strategies. For example, a patient with esophageal cancer may have a pathologic complete response (pathCR) to a treatment regiment of chemotherapy and radiation, and would therefore not need surgery. The ability to predict a favorable response to chemotherapy and/or radiation would then ameliorate the need for the patient to undergo surgery, and vice versa. Determining biomarkers that predict how an individual responds to therapy will help to individualize cancer therapy, thereby decreasing cost, increasing the effectiveness of cancer therapy and increasing the quality of life of cancer patients.

[0005] For example, patients with localized esophageal cancer are often treated with preoperative chemoradiation therapy despite equivocal results observed in several randomized trials. Approximately 25% of patients undergoing preoperative chemoradiation achieve a pathologic complete response (pathCR; Chirieac, 2005; Rohatgi, 2005; Berger, 2005). Patients who achieve a pathCR have a highly chemoradiation- sensitive cancer and tend to live significantly longer than those who do not achieve a pathCR (Chirieac, 2005; Rohatgi, 2005; Berger, 2005). The designation of pathCR can only be established by carefully evaluating the resected specimen (Chirieac, 2005; Wu, 2007). Previously there was no clinical or biomarker parameters that could predict a favorable response before therapy is initiated or even before surgery is performed. By determining if a patient will not need surgery after chemotherapy or radiation therapy, the esophagus of patients are spared. The other challenge is that the markers that predict sensitivity to or resistance to certain therapies should have an accuracy rate that is much higher than ordinary odds of -25%. One could argue that a 75% accuracy rate would be of considerable interest to pursue the strategy of preserving the esophagus in selected patients. One would be concerned about under treatment of patients; for example, it will not be desirable to spare esophagus (avoid surgery) in patients who have residual cancer after chemoradiation (leading to under- treatment). The under treatment can be avoided in many patients if the specificity is high (around >80%). In this scenario, high specificity is more valuable than high sensitivity because today nearly all operable patients with adenocarcinoma undergo chemoradiation and then surgery (esophagus is not spared in anyone) but if new tools, by accurately predicting pathCR in most such patients, can result in sparing of the esophagus in any pathCR patients, it would be an advantage.

[0006] There remains a need for improved methods and compositions for identifying how patients will respond to a certain anti-cancer therapy. Specifically, there is a need for a method of predicting a response to chemotherapy and/or radiation therapy for an individual having cancer.

BRIEF SUMMARY OF THE INVENTION

[0007] The present invention overcomes the deficiencies in the prior art by providing methods and compositions for identifying cancer cells that are either sensitive or resistant to a particular anti-cancer therapy. Accordingly, the present invention allows for more accurate diagnosis, prognosis, and/or monitoring of an individual's condition. Furthermore, the ability to assess an individual's resistance or sensitivity to a particular treatment regimen will permit more informed treatment decisions to be made at the onset of therapy.

[0008] In one general embodiment, there is a method of predicting a response to chemotherapy and/or radiation therapy for an individual having cancer comprising assessing the levels of deregulated SHH, GIi-I, and NF-κB proteins in cancer cells of the individual, wherein a higher level of deregulated SHH, GIi-I and NF-κB proteins in the cells, relative to a reference, is indicative that the individual's cancer is predisposed to be resistant to chemotherapy and/or radiation therapy, and an equal or lower level of deregulated SHH, GIi-I and NF-κB proteins relative to the reference is indicative that the individual's cancer is predisposed to be sensitive to chemotherapy and/or radiation therapy.

[0009] Any of the following embodiment/s of the invention maybe incorporated into this general embodiment, individually or in any combination.

[0010] In one embodiment, levels of deregulated SHH, GIi-I, and NF-κB are hallmarks of chemotherapy and/or radiation therapy resistant cells. In embodiments of the invention, levels of deregulated SHH, GIi-I, and/or NF-κB indicate a resistance of cancer. In specific embodiments, the levels of deregulated SHH, GIi-I, and/or NF- KB are considered elevated if they are over 0.1%, over 0.2%, over 0.3%, over 0.5%, over 0.6%, over 0.7%, over 0.8%, over 0.9%, over 1%, over 1.5%, over 2%, over 2.5%, over 3%, over 5%, over 4%, over 4.5%, over 5%, over 6%, over 7%, over 8%, over 9%, over 10%, over 12%, over 15%, over 17%, over 20%, over 25%, over 30%, over 35%, over 40%, over 45%, over 50%, over 55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, over 97%, over 98%, or over 99% than the relative expression in the reference. In embodiments of the invention, the level of deregulated SHH, GIi-I and NF-kB is the number of cells positive for deregulated SHH, GIi-I and NF-kB. In an embodiment of the invention, the higher the level of deregulated SHH, GIi-I, and/or NF-κB expression, the more resistant the cells are to chemotherapy and/or radiation therapy. In certain embodiments of the invention, a statistical method is used to determine if the degree of expression of deregulated SHH, GIi-I, and/or NF- KB indicates resistance or sensitivity. In specific embodiments of the invention, a statistical method is used to determine if the degree of sustained expression of deregulated SHH, GIi-I, and/or nuclear NF-κB indicates resistance or sensitivity to chemotherapy and/or radiation therapy. In specific embodiments of the invention, the statistical method determines the degree of sensitivity or resistance. In one embodiment of the statistical method that is utilized is the fitted multiple logistic regression model. In an embodiment of the invention, the resistance or sensitivity prediction is assessed by combining together and modeling the raw values of deregulated SHH, GIi-I and NF-κB proteins in cancer cells of the individual. In a further embodiment of the invention, the number of positive cells for each biomarker is combined into a prognostic score which predicts the response or resistance to cancer treatments. In a further embodiment of the invention, the score is a sliding scale that models degrees of partial resistance, full resistance, and full sensitivity to cancer treatments. In embodiments of the invention, the statistical model is used as the reference.

[0011] Deregulated SHH, GIi-I and/or NF-κB expression may be measured in any embodiment of the invention by determining the localization of each protein. In embodiments of the invention, NF-κB is considered deregulated if found in the nucleus, GIi-I is considered deregulated if found in the nucleus, and/or SHH is considered deregulated if found in the cytoplasm. In specific embodiments of the invention, nuclear factor kappa-B (NF-κB) is not considered deregulated if found in the cytoplasm, GIi-I is not considered deregulated if found in the cytoplasm, and SHH is not considered deregulated when found in the nucleus. In embodiments of the invention, the assessing step further comprises determining the cellular localization of SHH, GIi-I and NF-κB.

[0012] In embodiments of the invention, the reference is a control. A control is a collection of cells in which the expression of SHH, GIi-I and/or NF-κB is known. In particular embodiments of the invention, there are positive and/or negative controls. In specific embodiments of the invention, the control is an average of many individual controls + the standard deviation. In certain embodiments of the invention, a positive control is a sample that is known to be resistant to chemotherapy and/or radiation therapy. A non-limiting example of a positive control is a sample of cancerous cells from individuals that are known to be resistant to chemotherapy and/or radiation therapy. Another embodiment of a positive control is a cancerous cell line that is known to be resistant to chemotherapy and/or radiation therapy. In specific embodiments of the invention, exemplary positive control cell lines in the context of deregulated SHH, GIi-I and NF-κB are SKGT-4, SEG-I, 183A, MCF-7, A549, and SW480. In certain embodiments of the invention, positive controls comprise deregulated SHH, GIi-I, and NF-κB. In some embodiments of the invention, a negative control is a cell that is known to be sensitive to chemotherapy and/or radiation therapy. A non-limiting example of a negative control is cancerous cells taken from a patient prior to chemotherapy and/or radiation therapy, where the patient showed a complete pathologic response to the chemotherapy and/or radiation therapy. Another embodiment of a negative control are cancerous cell lines that are known to be sensitive to chemotherapy and/or radiation therapy. Negative controls may also be non-cancerous matched samples. For example, a matched sample may be taken from non-cancerous cells of the same tissue type, organ type, or localized region as the tumor. In some embodiments of the invention, the negative control is a sample of non-cancerous cells from the individual. In other embodiments of the invention, a negative control may be a sample that is known to express SHH, GIi-I, and NF-κB, but is treated with blocking agents or has the primary antibody removed from an immunohistochemistry assay. In some embodiments of the invention, positive or negative controls comprise particular cell lines. Exemplary negative control cell lines are LnCAP, MDA1386, UMSCClOB and Colo320. In the case of a statistical reference, the expression of SHH, GIi-I and NF-κB are compared to a reference to determine the probability the cancer will have a pathologic complete response, or be sensitive or resistant to chemotherapy and/or radiation therapy. In embodiments of the invention, the reference is a statistical reference such as a z-score.

[0013] The cancer may be any type of cancer. In certain embodiments of the invention the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, urogenital cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, an endocrine or neuroendocrine cancer or hematopoietic cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, renal cancer, biliary cancer, pheochromocytoma, pancreatic islet cell cancer, Li-Fraumeni tumors, thyroid cancer, parathyroid cancer, pituitary tumors, adrenal gland tumors, osteogenic sarcoma tumors, multiple neuroendrcine type I and type II tumors, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, or skin cancer. In specific embodiments of the invention, the cancer is inflammatory breast cancer, head and neck cancer, colorectal cancer, lung cancer, anal cancer or esophageal cancer. In further embodiments of the invention, the cancer is anal cancer or esophageal cancer. Any type of cancer cell listed or unlisted may be used in the invention in combination with any other embodiment of the invention.

[0014] Resistance or sensitivity to chemotherapy may be indicated for any type of chemotherapy in any embodiment of the invention. In specific embodiments of the invention, the cancer is inflammatory breast cancer, head and neck cancer, colorectal cancer, lung cancer, anal cancer or esophageal cancer. In further embodiments of the invention, the cancer is anal cancer or esophageal cancer. In specific embodiments the chemotherapy is 5-fluorouracil, mitomycin, cisplatin, taxane, camptothecin, irinotecan, and/or a platinum compound. Embodiments of the invention also include radiation and other types of cancer therapy. [0015] In any embodiment of the invention, the proteins may be detected by immunohistochemistry, an ELISA, an immunoassay, a radioimmunoassay (RIA), an immunoradiometric assay, a fluoroimmunoassay, a chemiluminescent assay, a bioluminescent assay, a gel electrophoresis, a Western blot analysis and/or an in situ hybridization assay. In a specific embodiment of the invention, the proteins are detected by immunohistochemistry. In an embodiment of the invention, mRNA levels are detected to assess the levels of deregulated and/or regulated SHH, GIi-I, NF-κB. In specific embodiments of the invention the mRNA levels are detected by in situ hybridization, Northern blotting, polymerase chain reaction, gene arrays, gene chips, and/or nuclease protection. Detection of proteins or mRNA may be manual, semi- automated, or fully automated.

[0016] Treatment regimens may be determined from an indication of sensitivity or resistance to a type of treatment, such as chemotherapy and radiation therapy. In certain embodiments of the invention, when the individual is determined to be predisposed to be resistant to chemotherapy and/or radiation therapy, the individual may be treated instead with surgery. In other embodiments of the invention, when the individual is determined to be predisposed to be sensitive to chemotherapy and/or radiation therapy, the individual may be treated with chemotherapy and/or radiation therapy. Depending on the degree of sensitivity or resistance determined, the individual may be treated with chemotherapy, radiation therapy, and surgery. In an embodiment of the invention, when the individual is determined to have a lesser degree of sensitivity to chemotherapy, the individual is treated with chemotherapy, radiation therapy, and surgery. In another embodiment of the invention, a high degree of sensitivity to chemotherapy and/or radiation therapy indicates the individual will have pathologic complete response to chemotherapy and/or radiation therapy and may not need surgery. In specific embodiments of the invention, the individual does not undergo surgery and instead follows an active surveillance program. In other embodiments of the invention, the individual does not undergo chemotherapy and radiation therapy and instead follows an active surveillance program.

[0017] A general embodiment of the invention is a kit comprising one or more suitably aliquoted antibodies to detect levels of SHH, GIi-I, and NF-κB protein, respectively, wherein said kit is housed in a suitable container. The kit may also comprise suitable references. In specific embodiments of the invention, the references are control samples. Additionally, the kit may also comprise software. Any method known to one of skill in the art may be utilized to procure a sample. In a specific embodiment of the invention, the sample is taken by biopsy. [0018] A general embodiment of the invention is a method of predicting a response to chemotherapy and/or radiation therapy for an individual having cancer comprising assessing the levels and localization of SHH, GIi-I, and NF-κB proteins in cancer cells of the individual, wherein when GIi-I and NF-κB protein are detected in the nucleus and SHH is detected in the cytoplasm of the cancer cells of the individual in sufficient levels, the individual is predisposed to be resistant to chemotherapy and/or radiation therapy, or wherein when GIi-I and NF-κB protein are not detected in the nucleus and SHH is not detected in the cytoplasm of the cancer cells of the individual in sufficient levels, the individual is predisposed to be sensitive to chemotherapy and/or radiation therapy. Any other embodiments of the invention may incorporated into this general embodiment, singly or in any combination.

[0019] Expression may be determined by any method known to those of skill in the art. In certain embodiments of the invention, expression is evaluated by assaying transcription levels. In other aspects of the invention, expression is evaluated by assaying protein levels.

[0020] In one embodiment of the invention, a favorable response to the therapy comprises reduction in tumor size or burden, blocking of tumor growth, reduction in tumor- associated pain, reduction in tumor associated pathology, reduction in tumor associated symptoms, tumor non-progression, increased disease free interval, increased time to progression, induction of remission, reduction of metastasis, increased patient survival, or pathCR.

[0021] In an embodiment of the invention, assessing the levels of SHH, GIi-I, and/or NF-κB comprises detecting mRNA levels of SHH, GIi-I, and/or NF-κB, respectively. In a specific embodiment of the invention, the respective mRNA sequences are capable of hybridizing under stringent conditions to one or more of the group consisting of SHH, GIi-I, and NF-KB. The SHH mRNA and protein sequences may be found under Genbank accession numbers NP_000184 (SEQ ID NO: 1) and NM_000193 (SEQ ID NO: 2). The GIi-I mRNA and protein sequences may be found under Genbank accession numbers NP_005260 (SEQ ID NO:3) and NM_005269 (SEQ ID NO: 4). The NF-KB mRNA and protein sequences may be found under Genbank accession numbers NP_068810 (SEQ ID NO:5), NM_021975 (SEQ ID NO: 6), NP_006500 (SEQ ID NO:7) and NM_006509 (SEQ ID NO: 8). In an embodiment of the invention, the respective mRNA sequences detected are at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% similar to one or more of the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and/or SEQ ID NO: 8. In an embodiment of the invention, mRNA levels are detected by in situ hybridization, Northern blotting, polymerase chain reaction assays, gene arrays, gene chips and/or nuclease protection. Embodiments may additionally comprise amplification of SHH, GIi- 1, and/or NF-κB nucleic acids by a method such as PCR.

[0022] The cancer cells may be a biological sample, e.g. a tissue sample comprising cancer cells obtained from the individual. In some embodiments, the cancer cells comprise tissue that is fixed, paraffin-embedded, or fresh or frozen. In particular embodiments, the tissue is from a biopsy. In specific embodiments, the cells are from a needle, core, or other type of biopsy. In other embodiments, the sample is from blood. In one embodiment, the sample is normalized. In a further embodiment, the sample is normalized by obtaining noncancerous cells from the individual, determining the levels of non- SHH, non- GIi-I, and non- NF-κB proteins, and normalizing the expression of SHH, GIi-I, and NF-κB in a cancerous sample from the same individual against the expression of the non-SHH, non-Gli-1, and non-NF- KB proteins in the non-cancerous cells.

[0023] An embodiment of the invention is a computer program product predicting a response to chemotherapy and/or radiation therapy for an individual having cancer, the computer program product comprising: a computer usable medium having computer usable program code embodied therewith, the computer usable program code comprising: computer usable program code configured to instruct a device to carry out the steps of a) reading in levels of deregulated SHH, GIi-I, and NF-κB proteins in cancer cells of the individual; b) comparing the levels of deregulated SHH, GIi-I, and NF-κB to a model; c) outputting a prediction of response to chemotherapy and/or radiation therapy. In specific embodiments of the invention, the model is a z-score.

[0024] Any of the above embodiments of the invention may be used in concert with any other embodiments of the invention, in any combination.

[0025] The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] For a more complete understanding of the present invention, reference is now made to the following descriptions taken in conjunction with the accompanying drawings.

[0027] FIG. 1 illustrates the hedgehog signaling pathway. [0028] FIG. 2 illustrates the NF-κB signaling pathway.

[0029] FIG. 3 shows the expression of NF-κB pathway associated genes by transcription and differential expression of selected genes in relation to pathologic response.

[0030] FIG. 4 demonstrates SHH expression by transcriptional profiling.

[0031] FIG. 5 is the disease free survival (DFS) of patients with different degrees of pathologic response.

[0032] FIG. 6 is the predicative probability of pathCR vs. the linear Z score, based on the fitted multiple logistic regression model for pathCR for SHH, GIi-I and NF- KB.

[0033] FIG. 7 demonstrates the survival probably of pathCR and <pathCR patients. The top line is <pathCR, while the bottom line is pathCR.

[0034] FIG. 8 shows the pre-treatment NF-κB status and overall survival of patients.

[0035] FIG. 9 is detection of nuclear NF-κB expression in esophageal cancer.

[0036] FIG. 10 shows expression levels of SHH and GIi-I in pre-treatment cancer biopsies.

[0037] FIG. 11 demonstrates the results of IHC analyses showing increase of SHH and GIi-I expressions preceding increase in Ki67 (left axis) and tumor size (right axis) during tumor repopulation after chemoradiation. Each point is a mean of 3 independent experiments (Sims-Mourtada, 2006).

[0038] FIG. 12 is a second model of the predictive probability of exCRTR vs. the linear Z score, based on the fitted multiple logistic regression model for exCRTR based on SHH, GIi-I and NF-KB.

[0039] FIG. 13 is the ROC curve for predicting exCRTR using SHH and GIi-I only.

[0040] FIG. 14 is Kaplan-Meier estimates for DFS (n=30).

[0041] FIG. 15 shows the activation of the SHH embryonic pathway in a rat model of Barrett's Esophagus with progression to adenocarcinoma.

[0042] FIG. 16 shows the ROC curve for predicting pathCR using the 3 biomarkers.

DETAILED DESCRIPTION OF THE INVENTION

[0043] The ability to select the optimal anticancer therapy from several alternative treatment options would be an important clinical advance. Exemplary benefits of optimizing anti-cancer therapy includes minimizing cost and increasing patient comfort by only performing treatment protocols that are necessary. The ability to assess an individual's resistance or sensitivity to a particular treatment regiment will permit more informed treatment decision to be made prior to beginning therapy.

A. DEFINITIONS

[0044] In keeping with long-standing patent law convention, the words "a" and "an" when used in the present specification in concert with the word comprising, including the claims, denote "one or more." Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the invention. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

[0045] As used herein, the term "deregulated" refers to protein expression of SHH, GIi-I, and NF-κB that is no longer properly regulated. Under properly regulated conditions, SHH, GIi-I and NF-κB are not detectable in cells because any expression is very short. In embodiments of the invention, deregulated refers to upregulated and/or sustained expression, for example, proteins with a longer half life than normal cells. In embodiments of the invention deregulated SHH, GIi-I and NF-κB refers to elevated constitutive expression of SHH in the cytoplasm of cells, GIi-I in the nucleus of cells and NF-KB in the nucleus of cells. In specific embodiments of the invention, deregulated SHH, GIi-I and/or NF-κB protein refers to detectable levels of SHH protein found in the cytoplasm of cells, and/or detectable levels of GIi-I and NF- KB protein found in the nucleus of cells. In another embodiment of the invention, deregulated SSH, GIi-I and/or NF-κB protein refers to elevated levels of SHH protein found in the cytoplasm of cells, and/or detectable levels of GIi-I and NF-κB protein found in the nucleus of cells, when compared to a reference and/or control.

[0046] As used herein, an "individual" is an appropriate individual for the method of the present invention. An individual may be a mammal and in specific embodiments is any member of the higher vertebrate class Mammalia, including humans; characterized by live birth, body hair, and mammary glands in the female that secrete milk for feeding the young. Additionally, mammals are characterized by their ability to maintain a constant body temperature despite changing climatic conditions. Examples of mammals are humans, cats, dogs, cows, goats, mice, rats, horses, sheep, pigs and chimpanzees. Individuals may also be referred to as "patients," or "subjects."

[0047] The term "long-term" survival is used herein to refer to survival for a long period of time. This period of time may be least 2 year, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, or 20 years following treatment, depending on the type of cancer. The period of long term survival may be dependent on the type and location of the cancer.

[0048] Following treatment if no detectable evidence of residual cancer is found in a tissue sample, the response to treatment is considered a "pathologic complete response."

[0049] The term "prognosis" is used herein to refer to the prediction of the likelihood of cancer-attributable death or progression, including recurrence, metastatic spread, and drug resistance of a neoplastic disease, such as esophageal cancer or anal cancer, for example. The term "prediction" is used herein to refer to the likelihood that a patient will respond to a cancer treatment and the extent of the response. The predictive methods of the present invention can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular patient. The predictive methods of the present invention are valuable tools in predicting if a patient is likely to respond to a treatment regimen, such as chemotherapy and/or radiation therapy.

[0050] The term "therapy" or "treatment" refers to a process that is intended to produce a beneficial change in the condition of the patient. A beneficial change can, for example, include one or more of the following: restoration of function; reduction of symptoms; limitation or retardation of progress of a disease, disorder or condition; or prevention, limitation or retardation of a deterioration of a patient' s condition, disease or disorder. Such therapy can involve, for example, administration of chemotherapy, administration of radiation, surgery, or any such combination thereof.

[0051] The term "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.

[0052] "Patient response" can be assessed using any end point indicating a benefit to the patient, including without limitation, any inhibition of tumor growth, including slowing down and complete growth arrest; reduction in the number of tumor cells; reduction in tumor size; inhibition, i.e., reduction, slowing down or complete stopping, of tumor cell infiltration into adjacent peripheral organs and/or tissues; inhibition, i.e., reduction, slowing down or complete stopping of metastasis; enhancement of anti-tumor immune response, which may, but does not have to, result in the regression or rejection of the tumor; relief, to some extend of one or more symptoms associated with the tumor; increase in the length of survival following treatment; and/or decreased mortality at a given point of time following treatment.

[0053] The term "essentially equal" as used herein, refers to equal values or values within the standard of error for such values.

B. CANCER

[0054] The invention relates to predicting a response to chemotherapy and/or radiation therapy for an individual having cancer. Embodiments of cancer are further defined below, and are not limiting.

[0055] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Exemplary cancer for which treatment is contemplated in the present invention include the following: squamous cell carcinoma, basal cell carcinoma, adenoma, adenocarcinoma, linitis plastica, insulinoma, glucagonoma, gastrinoma, vipoma, cholangiocarcinoma, hepatocellular carcinoma, adenoid cystic carcinoma, carcinoid tumor, prolactinoma, oncocytoma, hurthle cell adenoma, renal cell carcinoma, endometrioid adenoma, cystadenoma, pseudomyxoma peritonei, Warthin's tumor, thymoma, thecoma, granulosa cell tumor, arrhenoblastoma, Sertoli-Leydig cell tumor, paraganglioma, pheochromocytoma, glomus tumor, melanoma, soft tissue sarcoma, desmoplastic small round cell tumor, fibroma, fibrosarcoma, myxoma, lipoma, liposarcoma, leiomyoma, leiomyosarcoma, myoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma, pleomorphic adenoma, nephroblastoma, brenner tumor, synovial sarcoma, mesothelioma, dysgerminoma, germ cell tumors, embryonal carcinoma, yolk sac tumor, teratomas, dermoid cysts, choriocarcinoma, mesonephromas, hemangioma, angioma, hemangiosarcoma, angiosarcoma, hemangioendothelioma, hemangioendothelioma, Kaposi's sarcoma, hemangiopericytoma, lymphangioma, cystic lymphangioma, osteoma, osteosarcoma, osteochondroma, cartilaginous exostosis, chondroma, chondrosarcoma, giant cell tumors, Ewing's sarcoma, odontogenic tumors, cementoblastoma, ameloblastoma, craniopharyngioma gliomas mixed oligoastrocytomas, ependymoma, astrocytomas, glioblastomas, oligodendrogliomas, neuroepitheliomatous neoplasms, neuroblastoma, retinoblastoma, meningiomas, neurofibroma, neurofibromatosis, schwannoma, neurinoma, neuromas, granular cell tumors, alveolar soft part sarcomas, lymphomas, non-Hodgkin's lymphoma, lymphosarcoma, Hodgkin's disease, small lymphocytic lymphoma, lymphoplasmacytic lymphoma, mantle cell lymphoma, primary effusion lymphoma, mediastinal (thymic) large cell lymphoma, diffuse large B-cell lymphoma, intravascular large B- cell lymphoma, Burkitt lymphoma, splenic marginal zone lymphoma, follicular lymphoma, extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT- lymphoma), nodal marginal zone B-cell lymphoma, mycosis fungoides, Sezary syndrome, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, subcutaneous panniculitis- like T-cell lymphoma, anaplastic large cell lymphoma, hepatosplenic T-cell lymphoma, enteropathy type T-cell lymphoma, lymphomatoid papulosis, primary cutaneous anaplastic large cell lymphoma, extranodal NK/T cell lymphoma, blastic NK cell lymphoma, plasmacytoma, multiple myeloma, mastocytoma, mast cell sarcoma, mastocytosis,mast cell leukemia, langerhans cell histiocytosis, histiocytic sarcoma, langerhans cell sarcoma dendritic cell sarcoma, follicular dendritic cell sarcoma, Waldenstrom macroglobulinemia, lymphomatoid granulomatosis, acute leukemia, lymphocytic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, adult T-cell leukemia/lymphoma, plasma cell leukemia, T-cell large granular lymphocytic leukemia, B-cell prolymphocytic leukemia, T- cell prolymphocytic leukemia, pecursor B lymphoblastic leukemia, precursor T lymphoblastic leukemia, acute erythroid leukemia, lymphosarcoma cell leukemia, myeloid leukemia, myelogenous leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, acute promyelocytic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, basophilic leukemia, eosinophilic leukemia, acute basophilic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, monocytic leukemia, acute monoblastic and monocytic leukemia, acute megakaryoblastic leukemia, acute myeloid leukemia and myelodysplastic syndrome, chloroma or myeloid sarcoma, acute panmyelosis with myelofibrosis, hairy cell leukemia, juvenile myelomonocytic leukemia, aggressive NK cell leukemia, polycythemia vera, myeloproliferative disease, chronic idiopathic myelofibrosis, essential thrombocytemia, chronic neutrophilic leukemia, chronic eosinophilic leukemia/ hypereosinophilic syndrome, post- transplant lymphoproliferative disorder, chronic myeloproliferative disease, myelodysplastic/myeloproliferative diseases, chronic myelomonocytic leukemia, myelodysplastic syndrome, inflammatory breast cancer, head and neck cancer, colorectal cancer, lung cancer, anal cancer and esophageal cancer.

[0056] The hyperproliferative disease or cancer may be treated after its initial diagnosis or subsequently by therapeutic nucleic acids or other therapies or combination of two or more therapies. A hyperproliferative disease or cancer recurrence may be defined as the reappearance or rediagnosis of a patent as having any hyperproliferative disease or cancer following any treatment including one or more of surgery, radiotherapy or chemotherapy. The patient with relapsed disease need not have been reported as disease free, but merely that the patient has exhibited renewed hyperproliferative disease or cancer growth following some degree of clinical response by the first therapy. The clinical response may be, but is not limited to, stable disease, tumor regression, tumor necrosis, absence of demonstrable cancer, reduction in tumor size or burden, blocking of tumor growth, reduction in tumor-associated pain, reduction in tumor associated pathology, reduction in tumor associated symptoms, tumor non-progression, increased disease free interval, increased time to progression, induction of remission, reduction of metastasis, or increased patient survival.

[0057] 1. Esophageal cancer

[0058] Carcinoma of the esophagus is a virulent disease. Localized esophageal adenocarcinoma (LEA) has increased dramatically in its incidence over the past 25 years (Pohl, 2005). Currently, adenocarcinoma is predominantly affecting Caucasian men. The incidence of adenocarcinoma has skyrocketed in the past 20 years with a rate ratio exceeding that of prostate cancer, brain cancer, and melanoma (Pohl, 2005). According to the American Cancer Society statistics, there has been a >40% increase in the incidence of esophageal cancer, mainly adenocarcinoma, since 1990 (Society, 2007). Moreover, the outcome of LEA treated with combined modality therapy has not improved despite a variety of cytotoxic combinations used concurrently with increasingly modern radiation therapy and improved surgical approaches (Kleinberg, 2007).

[0059] Approximately 600,000 new esophagus cancers are diagnosed each year in the world; including 15,500 in the United States (Jemal et al, 2006). For patients with the most common stage (II or III), the surgical 5-year cure rate is <20% (Devesa et al, 1998; Brenner et al , 2004) but often surgery is not the primary therapy. Despite equivocal data, these patients usually receive preoperative combined modality therapy (Urba, 2004; Iver et al., 2004; Tepper et al., 2006). Most patients' cancer (stage II or III) is treated empirically and by a similar approach. This is done because the clinical parameters (clinical stage, gender, location of the cancer within the thorax, histology, age, or baseline imaging) cannot predict response to chemoradiation (Chirieac et al, 2005; Berger et al, 2005; Rohatgi et al, 2005). However, the extent of pathologic response does determine patient's clinical outcome (Chirieac et al, 2005; Berger et al, 2005). Nearly all patients with stage II or III cancer are offered a similar strategy (chemoradiation followed by surgery) but their outcome (including pathCR or exCRTR) is heterogeneous and unpredictable. Moreover, preoperative chemoradiation and surgery have dire toxicity and quality of life consequences. A great deal of uncertainty exists in the current clinical decision-making and there are no tools to alter the current treatment paradigm. Meltzer et al. , have studied methylation abnormalities as a predictor of response to chemoradiation in 35 patients with esophageal cancer, however, they did not correlate it with pathCR or exCRTR.

[0060] Esophageal Cancer and Importance of Pathologic Response: Patients with localized carcinoma of the esophagus (most common stage is II or III) have poor 5-year survival rates (ref T). Currently, preoperative chemoradiation results in a 25% chance of achieving a pathCR or exCRTR. Identifying the subset of patients who are destined to have pathCR or exCRTR with a prediction accuracy of >70% and a high level of specificity (>80%) would be an important step towards therapy individualization. Individualization of therapy may be even more important in esophageal cancer than other cancers because chemoradiation and surgery have a high rate of morbidity. Patients can be divided in 3 distinct groups based on the pathologic findings in the resection specimen: (1) pathCR (2) >1% to <50% of residual cancer, and (3) >50% residual cancer or exCRTR. The median survival depends on the degree of residual cancer. Berger et al (Berger, 2005) and Rohatgi et al (Rohatgi et al 2005; Rohatgi et al, 2005b) among many others, have demonstrated that patients with a pathCR live significantly longer than those who have less than a pathCR. Approximately 25% of patients achieve a pathCR and 75% have <pathCR (-25% of all patients have exCRTR) (Rohatgi et al, 2005). A review of 18 published studies (>800 patients) using various cytotoxics and radiation doses, suggests that the class of cytotoxics or radiation utilized has little or no impact on the patterns of pathologic response (or overall survival (OS)). An analyses of 235 patients confirms this (Rohatgi et al, 2005; Rohatgi et al., 2005b). Clinical parameters do not predict therapeutic outcomes. There are no previous biomarkers that predict pathCR or exCRTR, therefore, the only way to determine the degree of pathologic response is to examine the resection specimen. The value of surgery in patients who have a pathCR is not known (one could argue that pathCR patients are being over-treated) and clearly the administration of chemoradiation for exCRTR patients should be questioned (another setting where patients are being over-treated). There is an opportunity to potentially avoid surgery (in pathCR patients) and chemoradiation (in exCRTR patients). However, it requires validated biomarkers that confer a high level of predictive accuracy with high specificity and reasonably high sensitivity. The normal odds for predicting a pathCR or exCRTR are only 25% with previous methods. A successful biomarker strategy would lead to organ preservation and better quality of life for some patients with a pathCR and reduction in morbidity/mortality of chemoradiation for most patients with exCRTR.

[0061] Imaging techniques do hold promise (Wieder et al., 2007; Lordick et al., 2007; Ott et al, 2006; Erasmus et al, 2006; Hong et al, 2005; Swisher et al, 2004; Swisher, 2004b; Flamene? al, 2002) but they have not been able to predict pathCR and considerably more sophistication is needed (Weissleder et al, 2008).

2. Anal cancer

[0062] Anal carcinoma is an uncommon malignancy in the United States. Approximately 5,070 new cases of anal cancer were projected for the year 2008 out of >1.44 million new cancer diagnoses (Jemal et al, 2008). Anal carcinoma being highly sensitive to chemoradiation (Nigro et al, 1983), treatment with primary chemoradiation results in a disease free survival (DFS) rate of -65%. The DFS rates vary to some extent depending on known clinical factors such as gender, clinical nodal status, and tumor diameter (Sischy et al, 1989; Nilsson et al, 2005; Svensson et al, 1993; Ajani et al, 2008) but also treatment-related factors (Ajani et al, 2998). Ability to complete prescribed therapy may itself have an influence of DFS. In spite of these factors that are associated with poor disease-free survival (DFS), the outcome of patients in various prognostic categories still remains heterogeneous and unpredictable. Clinical biology of individual cancer is dictated by the genetic and epigenetic alterations that are commonly observed in cancers (Chin and Gray, 2008; Sawyers, 2008; van't Veer and Mernards, 2008; Vogelstein, 1990). Understanding the biology of anal carcinoma would explain the variations in clinical outcome with same or similar strategies. Research focusing on identification of biomarkers of DFS or other outcomes in anal carcinoma is limited because it is rare.

C. Cancer markers

[0063] A general embodiment of the invention is the method of predicting a response to chemotherapy and/or radiation therapy comprising assessing the levels of SHH, GIi- 1 and NF-κB in cancer cells of the individual. In embodiments of the invention, SHH, GIi-I and NF-κB are markers for resistance and/or sensitivity to chemotherapy and/or radiation therapy. Further description of SHH, GIi-I and NF-κB is found below, including description of pathway and activation.

[0064] Physiologically (i.e. in normal conditions), the protein expression of all three biomarkers, SHH, GIi-I and NF-κB, is strictly controlled by an "On-Off '-like switch mechanism. Upon stimulation or activation, these proteins are expressed and localized in specific cellular and extracellular compartments to exert their activity. The cellular half-life in the functional compartments is strictly regulated by degradation pathways, and is about <30 minutes. Genetic mutations and other molecular alteration lead to a continuous "On" state, which is characterized by a sustained expression in the functional compartments and defines deregulation.

1. Sonic Hedgehog and GIi-I

[0065] The Hedgehog signaling pathway. The Hedgehog (HH) signaling pathway (FIG. 1) is critical for growth and differentiation during embryonic development (Lum and Beachy, 2004). HH controls stem cell proliferation and survival throughout development and adulthood (Lum, 2004 #3282). Activation of the network, induced by one of the three hedgehog ligands (Sonic, Desert and Indian), leads to intracellular events that cause activation and nuclear translocation of the GIi family of transcription factors (GIi-I, -2, and -3) (Neumann, 2005). Secreted hedgehog molecules [Sonic (SHH), Desert (DHH) and Indian (IHH)] bind and inhibit the cell surface receptor patched (PTCH). This inhibition relieves the PTCH-mediated suppression of the transmembrane protein smoothened (SMO), leading to intracellular events that cause activation (i.e. proteolytic processing) and nuclear translocation of the GIi family of transcription factors (GIi-I, -2 and -3). The three GIi proteins vary considerably. While, both, Gli-2 and -3 have transcriptional activation and repression properties, GIi-I is a strong regulator of HH pathway targets and is itself a transcriptional target of the mammalian HH pathway. Transcriptional targets of the onco-protein GIi-I include genes implicated in cell cycle control, cell adhesion, signal transduction, vascularization, apoptosis, stem cell maintenance and PTCH itself. Vascular endothelial growth factor (VEGF) plays a role in tumor vascularization and normalization of oxygen and drug levels (Uasuda, 2008). Hypoxia plays a central role in radiation resistance through multiple mediators (Tannock, 2001; Fowler and Lindstrom, 1992).

[0066] The HH pathway is mostly switched off during adulthood and is restricted to specific areas in the skin, blood, prostate, nervous system and digestive tissues where it is involved in the maintenance of the stem cells and production of the progeny that differentiates in specialized cell lineages. The different HH ligand proteins, appear to control independent stem cell pools. In physiologic conditions, the HH pathway is silent in quiescent stem cells to become activated by other signaling, notably the Wnt/b-catenin axis during the regeneration processes. HH signaling is implicated in normal and pathogenic inflammation (including acute and chronic epithelial injury). Once activated, the HH pathway is responsible for sustained gradient- dependent proliferation and progeny differentiation. During adulthood, the HH pathway is involved in homeostatic organ maintenance through a tightly regulated signaling- and gradient- dependent regulatory network.

[0067] Hedgehog Pathway and Cancer. The initial observation of GIi-I involvement in glioma development suggested the importance of HH in tumorigenesis. Later, a definite link between the HH pathway and cancer was established by the identification of heterozygote germline mutations affecting the membrane receptor PTCH, and resulting in the abnormal activation of the HH pathway in basal cell carcinoma, rhabdomyosarcoma, and neural tumors. Several in vitro and in vivo studies suggest constitutive, ligand-dependent, activation of HH signaling in cancers of the digestive tract, including the biliary tract, stomach, and pancreas. Most gastrointestinal cancers lack mutations in the HH pathway genes, suggesting distinct pathway activation from other cancers. Studies in diverse cellular lineages have provided convincing evidence that HH signaling promotes cellular proliferation by opposing signals to physiologic growth arrest. Most of the HH pathway effects are transcriptionally mediated by the GIi family through the upregulation of cell cycle regulating genes, anti-apoptotic proteins, growth and pro-angiogenic factors. Aberrant activation or alteration of components of the HH pathway have been reported in several solid malignancies. The deregulation of HH pathway in cancer cells can contribute to cancer maintenance and progression by enhancing cell cycle alterations and production of cancer growth factors.

[0068] Genetic mutations in HH can lead to its constitutive activation of SHH in cancers including esophageal cancer (Kinzler et al, 2006; Berman et al, 2003; Lees et al, 2005). Studies of gastrointestinal malignancies, including esophagus, biliary track, pancreas, and stomach, have shown a constitutive activation of the HH network that are likely ligand- dependent. Previous observations also suggested a role of SHH in the mediation of chemotherapy- and radiation-resistance in esophageal cancer (Sims-Mourtada, 2006; Sims- Mourtada, 2007).

[0069] In one embodiment of the invention, nuclear GIi-I is a hallmark of a deregulated pathway. In a further embodiment of the invention, SHH found in the cytoplasm of cancer cells in combination with nuclear GIi-I marks cancer stem cells or cancer progenitor cells which are inherently more resistant to cancer treatment.

2. NF KB

[0070] NF-κB, a nuclear transcription factor, is a gatekeeper of survival processes and its constitutive activation has been reported in inflammatory diseases and with cancer progression (Karin et al, 2002; Aggarwal et al., 2004). NF-κB promotes survival of normal and cancer cells by preventing apoptosis in response to stress Karin et al, 2002). NF-κB is associated with chemotherapy and radiotherapy resistance (Nakanish and Toi, 2005; Bottero et al, 2001; Cusack et al, 2000; Bergstralh and Ting, 2006; Ashikawa et al, 2004; Russell and Tofilon, 2002; Brach et al, 1991).

[0071] NF-κB, Clinical Biology, and Therapy Outcome: NF KB, nuclear transcription factor, is involved in a wide array of physiologic processes, inflammatory diseases, and in cancer progression and maintenance(Karin et al, 2002; Aggarwal et al, 2004). Under normal conditions, NF-κB is present in the cytoplasm as an inactive heterotrimer consisting of p50, p65 and IKBOC subunits (FIG. 2). On activation and deregulation, IKBOC undergoes phosphorylation and ubiquitination-dependent degradation by the 26S proteosome, thus exposing nuclear localization signals on the p50-p65 heterodimer. This leads to nuclear translocation and binding to a consensus sequence in the promoter region of NF-κB -regulated genes and subsequent increase in expression of those genes. The phosphorylation of IKBOC occurs through the activation of IKK. The IKK complex consists of three proteins, IKKα, IKKβ, and IKKγ. The latter is also known as NF-κB essential modulator (NEMO). IKKα and IKKβ are capable of phosphorylating IKBOC, whereas IKKγ/NEMO is a scaffold protein that is critical for IKKα and IKKβ activity (Hayden and Ghosh, 2004). NF-κB can serve as a survival factor by preventing apoptosis in response to stress. NF-κB is associated with chemotherapy and radiotherapy resistance. NF-κB is activated in response to cytotoxics, including topoisomerases inhibitors, vinca alkaloids, platinols and taxanes (Nakanishi and Toi, 2005) . The mechanism of drug resistance is unclear but appears that different drug class initiates differing pathways that ultimately result in NF-κB activation (Nakanishi and Toi, 2005). For example, doxorubicin induces IKK-independent NF-κB activation while irinotecan induces mobilization and activation of the IKK complex without synthesis of intermediate protein products (Nakanishi and Toi, 2005; Bottero et al., 2001). Constitutively activated, or deregulated, NF-κB appears critical for chemotherapy resistance. Mabuchi et al. reported that low constitutive activity correlated with resistance to cisplatin in the Caov-3 ovarian cells (Mabuchi et al., 2004). Similarly, increased expression of NF-κB correlated with resistance to 5-FU in colorectal and breast cancer cells (Wang et al., 2004; Wang et al., 2005). NF-κB is also a potential key signaling molecule in radiation resistance since ionizing radiation up-regulates its expression and binding (Russell and Tofilon, 2002; Brach et al, 1991).

D. CANCER TREATMENTS

[0072] An "anti-cancer" or "cancer" treatment is capable of negatively affecting cancer in a subject, for example, by killing one or more cancer cells, inducing apoptosis in one or more cancer cells, reducing the growth rate of one or more cancer cells, reducing the incidence or number of metastases, reducing a tumor's size, inhibiting a tumor's growth, reducing the blood supply to a tumor or one or more cancer cells, promoting an immune response against one or more cancer cells or a tumor, preventing or inhibiting the progression of a cancer, or increasing the lifespan of a subject with a cancer. Anti-cancer agents include, for example, chemotherapy agents (chemotherapy), radiotherapy agents (radiotherapy), a surgical procedure (surgery), immune therapy agents (immunotherapy), genetic therapy agents (gene therapy), hormonal therapy, other biological agents (biotherapy) and/or alternative therapies. Further examples are given below and are exemplary in nature. 1. Anti-cancer agents

A. Chemotherapy

[0073] The term "chemotherapy" refers to the use of drugs to treat cancer. A

"chemotherapeutic agent" is used to connote a compound or composition that is administered in the treatment of cancer, as biochemotherapy involves the combination of a chemotherapy with a biological therapy.

[0074] Chemotherapeutic agents include, but are not limited to, 5-fluorouracil, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin (CDDP), cyclophosphamide, dactinomycin, daunorubicin, doxorubicin, estrogen receptor binding agents, etoposide (VP16), farnesyl-protein transferase inhibitors, gemcitabine, ifosfamide, mechlorethamine, melphalan, mitomycin, navelbine, nitrosurea, plicomycin, procarbazine, raloxifene, tamoxifen, taxol, temazolomide (an aqueous form of DTIC), transplatinum, vinblastine and methotrexate, vincristine, or any analog or derivative variant of the foregoing. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, corticosteroid hormones, mitotic inhibitors, and nitrosoureas, hormone agents, miscellaneous agents, and any analog or derivative variant thereof.

[0075] Chemotherapeutic agents and methods of administration, dosages, etc. are well known to those of skill in the art (see for example, the "Physicians Desk Reference", Goodman & Gilman's "The Pharmacological Basis of Therapeutics", "Remington's Pharmaceutical Sciences", "National Comprehensive Cancer Network (NCCN)" found online, "US Parmacopeia" and "The Merck Index, Eleventh Edition", incorporated herein by reference in relevant parts), and may be combined with the invention in light of the disclosures herein. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Examples of specific chemotherapeutic agents and dose regimes are also described herein. Of course, any dosages and agents described herein are exemplary rather than limiting, and other doses or agents may be used by a skilled artisan for a specific patient or application. Any dosage in-between these points, or range derivable therein is also expected to be of use in the invention.

2. Radiation

[0076] Radiotherapeutic agents include radiation and waves that induce DNA damage for example, γ-irradiation, X-rays, proton beam therapies (U.S. Patents 5,760,395 and 4,870,287), UV-irradiation, microwaves, electronic emissions, radioisotopes, and the like. Therapy may be achieved by irradiating the localized tumor site with the above described forms of radiations. It is most likely that all of these agents effect a broad range of damage DNA, on the precursors of DNA, the replication and repair of DNA, and the assembly and maintenance of chromosomes.

[0077] Radiotherapeutic agents and methods of administration, dosages, etc. are well known to those of skill in the art, and may be combined with the invention in light of the disclosures herein. For example, dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

[0078] Radiation therapy can be administered to the individual according to protocols commonly employed in the art and known to the skilled artisan. Such therapy may include cesium, iridium, iodine, or cobalt radiation. The radiation may be whole body irradiation, or may be directed locally to a specific site or tissue in or on the body, such as the lung, bladder, or esophagus. Radiation therapy may be administered in pulses over a period of time from about 1 to 2 weeks. The radiation therapy may also be administered over longer periods of time. The radiation therapy may be administered as a single dose or as multiple, sequential doses.

3. Surgery

[0079] Approximately 60% of persons with cancer will undergo surgery of some type, which includes, for example, preventative, diagnostic or staging, curative and palliative surgery. Surgery, and in particular a curative surgery, may be used in conjunction with other therapies, such as chemotherapy and/or radiation therapy.

[0080] Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised and/or destroyed. It is further contemplated that surgery may remove, excise or destroy superficial cancers, precancers, or incidental amounts of normal tissue. Treatment by surgery includes for example, tumor resection, laser surgery, cryosurgery, electro surgery, and miscopically controlled surgery (Mohs' surgery). Tumor resection refers to physical removal of at least part of a tumor. Upon excision of part of all of cancerous cells, tissue, or tumor, a cavity may be formed in the body.

[0081] Further treatment of the tumor or area of surgery may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer agent. Such treatment may be repeated, for example, about every 1, about every 2, about every 3, about every 4, about every 5, about every 6, or about every 7 days, or about every 1, about every 2, about every 3, about every 4, or about every 5 weeks or about every 1, about every 2, about every 3, about every 4, about every 5, about every 6, about every 7, about every 8, about every 9, about every 10, about every 11, or about every 12 months. These treatments may be of varying dosages as well.

4. Individualized cancer treatment

[0082] There are at least three groups of patients who may require different approaches based upon their response to chemoradiotherapy: 1) patients who are treated effectively with chemoradiotherapy and do not require immediate surgery (pathCR), 2) those who benefit from surgery and chemoradiotherapy (partial response with around 1% to 50% residual cancer), and 3) those who do not respond to chemoradiotherapy and can proceed directly to surgery (exCRTR). For the first group, it is important that a biomarker signature does not lead to under-treatment of many patients who are likely to benefit from surgery (those with a <pathCR). Therefore, signature should have a high specificity (around >80%) and at the same time, a moderate sensitivity (around >45%). A very high sensitivity is not always essential because preserving the tissue of any patient destined to have pathCR would be an advance over the current approach where all patients arbitrarily undergo surgery.

[0083] In some cases, the selection of a method of treatment, i.e., a therapeutic regimen, may incorporate selection of one or more from a plurality of medical therapies. Thus, the selection may be the selection of a method of methods which is/are more effective or less effective than certain other therapeutic regiments (with either having varying safety parameters). Likewise or in combination with the preceding selection, the selection may be the selection of a method or methods which is safer than certain other methods of treatment in the patient. Exemplary treatment therapies are surgery, chemotherapy, and/or radiation therapy. [0084] The selection of a therapy may involve either positive selection or negative selection or both, meaning that the selection can involve a choice that a particular therapy regimen would be an appropriate method to use and/or a choice that a particular therapy regimen would be an inappropriate method to use. Thus, in certain embodiments, an indication that the patient will respond and/or respond favorably to a certain method of treatment indicates that that treatment should be used to treat the patient. Stating that the treatment will be effective, means that the probability of beneficial therapeutic effect is greater than in a person not having the appropriate presence or absence of particular variances. In other embodiments, an indication that the patient will not respond to a certain methods of treatment indicates that the treatment should not be used to treat the patient. Stating that the treatment will not be effective means that the probability of non-beneficial therapeutic effect is greater than in a person that does not have an indication such as the 3-biomarker signature. For example, a treatment may be contra-indicated if the treatment results, or is more likely to result in undesirably side effects, or an excessive level of undesirable side effects. Determination of what constitutes excessive side effects will vary, for example, depending on the disease or condition being treated the availability of alternatives, the expected or experience efficacy of the treatment, and the tolerance of the patient. As for an effective treatment, this means that it is more likely that a desired effect will result from the treatment administration in a patient with a particular marker than in a patient who has a different marker signature. In some embodiments, the presence, localization, and/or levels of deregulated SHH, NF-κB, and GIi-I indicative that chemotherapy and/or radiation therapy will not be effective. In further embodiments, the presence, localization, and/or levels of deregulated SHH, NF-κB, and GIi-I indicate a the individual will be sensitive to, partially sensitive to, or resistant to radiation and/or chemotherapy.

[0085] In reference to response to a treatment, the term "tolerance" refers to the ability of a patient to accept a treatment based, e.g. on deleterious effects and/or effects on lifestyle. Frequently, the term principally concerns the patients perceived magnitude of deleterious effects such as nausea, weakness, dizziness, and diarrhea, loss of tissue, among others. Such experienced effects, can, for example, be due to general or cell- specific toxicity, activity on non-target cells, cross reactivity on non-target cellular constituents (non-mechanism bases), and/or side-effects of activity on the target cellular substituent (mechanism based), or the cause of toxicity may not be understood. In any of these circumstances one may compare the extent of undesirable effects and the probability that an individual will respond to the treatment which results in the undesirable effects. In embodiments of the invention, the levels of deregulated SHH, NF-κB and GIi-I indicate the probability a patient will respond to a certain type of treatment.

[0086] An embodiment of the invention provides a method for selecting a treatment for a patient suffering from cancer by determining which treatments a patient will respond to, given levels of deregulated SHH, NF-κB and GIi-I.

[0087] Statistical methods used for determining individual response: In certain embodiments of the invention, a statistical model is calculated using expression levels of deregulated SHH, GIi- and NF-κB in cancer cells of individuals known to be sensitive, partially sensitive, or resistant to chemotherapy and/or radiation therapy. One of skill in the art knows of multiple methods to calculate the model, such as the classification and regression tree (CART) analyses, the fitted multiple logistic regression model, the linear Z score computation of multiple co-variants, and receiver-operator-characteristic (ROC) curves, for example. Expression levels of deregulated SHH, GIi-I and NF-κB protein from an individual with cancer prior to any treatment may then be compared to the model (reference) to determine how the individual will response to different types of therapy. For example, a linear Z score can be computed for each patient i, ie Z ,= β₀ + β_xli + β _Xl2 + β_xl3, , where xil, xi2 and xi3 are the expression level of SHH, GIi-I and NF-κB for the patient, plotted into the established model to identify the predicted probability of the patient i to be sensitive, partially sensitive or resistant to the chemotherapy and/or radiation In some embodiments of the invention, a training set with known outcomes is used. In specific embodiments, cross validation is performed with a test set and training set of patients with known outcomes.

E. Immunohistochemistry

[0088] Immunohistochemistry may be utilized in embodiments of the invention to determine the levels and/or localization of SHH, GIi-I and/or NF-κB protein.

1. Antibody preparation a. Polyclonal antibodies

[0089] Polyclonal antibodies to the trk receptor generally are raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the trk receptor and an adjuvant. It may be useful to conjugate the trk receptor or a fragment containing the target amino acid sequence to a protein that is immunogenic in the species to be immunized, e.g. keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glytaraldehyde, succinic anhydride, or SOCl₂.

[0090] Animals are immunized against the immunogenic conjugates or derivatives by combining 1 mg of 1 .mu.g of conjugate (for rabbits or mice, respectively) with 3 volumes of Freud's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with 1/5 to 1/10 the original amount of conjugate in Freud's complete adjuvant by subcutaneous injection at multiple sites. 7 to 14 days later the animals are bled and the serum is assayed for anti-trk receptor antibody titer. Animals are boosted until the titer plateaus. Preferably, the animal boosted with the conjugate of the same trk receptor, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are used to enhance the immune response. b. Monoclonal antibodies

[0091] Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0092] For example, the anti-trk receptor monoclonal antibodies of the invention may be made using the hybridoma method first described by Kohler & Milstein, Nature 256:495 (1975), or may be made by recombinant DNA methods (Cabilly, et al, U.S. Pat. No. 4,816,567).

[0093] In the hybridoma method, a mouse or other appropriate host animal, such as hamster is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, 1986).

[0094] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

[0095] Preferred myeloma cells are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-Il mouse tumors available from the SaIk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 cells available from the American Type Culture Collection, Rockville, Md. USA.

[0096] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against trk receptor. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).

[0097] The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson & Pollard, 1980.

[0098] After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods. (Goding, 1986). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium or RPMI- 1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.

[0099] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0100] DNA encoding the monoclonal antibodies of the invention is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (Morrison, et al, 1984), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In that manner, "chimeric" or "hybrid" antibodies are prepared that have the binding specificity of an anti-trk monoclonal antibody herein.

[0101] Typically such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody of the invention, or they are substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an trk receptor and another antigen-combining site having specificity for a different antigen.

[0102] Chimeric or hybrid antibodies also may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl- 4-mercaptobutyrimidate.

[0103] For diagnostic applications, the antibodies of the invention typically will be labeled with a detectable moiety. The detectable moiety can be any one which is capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as ³H, ¹⁴C, .³²P, ³⁵S, or ²⁵I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; biotin; radioactive isotopic labels, such as, e.g., ¹²⁵1, ³²P, ¹⁴C, or ³H, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.

[0104] Any method known in the art for separately conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter, et al, 1962; David, et al, 1974; Pain, et al, 1981; and Nygren, 1982.

[0105] The antibodies of the present invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, 1987). [0106] Competitive binding assays rely on the ability of a labeled standard (which may be an trk receptor or an immunologically reactive portion thereof) to compete with the test sample analyte (trk receptor) for binding with a limited amount of antibody. The amount of trk receptor in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies. To facilitate determining the amount of standard that becomes bound, the antibodies generally are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound.

[0107] Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three part complex (David & Greene, U.S. Pat. No. 4,376,110). The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an antiimmunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme. c. Bispedfic antibodies

[0108] Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for a trk receptor, the other one is for any other antigen, and preferably for another receptor or receptor subunit. For example, bispecific antibodies specifically binding a trk receptor and neurotrophic factor, or two different trk receptors are within the scope of the present invention.

[0109] Methods for making bispecific antibodies are known in the art.

[0110] Traditionally, the recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello, 1983). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in PCT application publication No. WO 93/08829 (published May 13, 1993), and in Traunecker et al, 1991.

[0111] According to a different and more preferred approach, antibody variable domains with the desired binding specificities (antibody- antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2 and CH3 regions. It is preferred to have the first heavy chain constant region (CHl) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are cotransfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance. In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in copending application Ser. No. 07/931,811 filed Aug. 17, 1992.

[0112] For further details of generating bispecific antibodies see, for example, Suresh et al, Methods in Enzymology 121, 210 (1986). f. Heteroconjugate antibodies

[0113] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (PCT application publication Nos. WO 91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

[0114] Any known antibody may be used in any embodiment of the present invention.

2. Immunodetection Methods

[0115] In still further embodiments, the present invention concerns immunodetection methods for binding, purifying, removing, quantifying and/or otherwise generally detecting biological components such as SHH, NF-κB, and/or GIi-I protein components. The SHH, NF-κB, and/or GIi-I antibodies prepared in accordance with the present invention may be employed to detect wild- type and/or mutant SHH, NF-κB, and/or GIi-I proteins, polypeptides and/or peptides. As described throughout the present application, the use of wild-type and/or mutant SHH, NF-κB, and/or GIi-I specific antibodies is contemplated. Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Doolittle MH and Ben-Zeev O, 1999; Gulbis B and Galand P, 1993; De Jager R et al, 1993; and Nakamura et al., 1987, each incorporated herein by reference.

[0116] In general, the immunobinding methods include obtaining a sample suspected of containing SHH, NF-κB, and/or GIi-I protein, polypeptide and/or peptide, and contacting the sample with a first anti- SHH, NF-κB, and/or GIi-I antibody in accordance with the present invention, as the case may be, under conditions effective to allow the formation of immunocomplexes.

[0117] These methods include methods for purifying wild- type and/or mutant SHH, NF- KB, and/or GIi-I proteins, polypeptides and/or peptides as may be employed in purifying wild- type and/or mutant SHH, NF-κB, and/or GIi-I proteins, polypeptides and/or peptides from patients' samples and/or for purifying recombinantly expressed wild-type or mutant SHH, NF-κB, and/or GIi-I proteins, polypeptides and/or peptides. In these instances, the antibody removes the antigenic wild-type and/or mutant SHH, NF-κB, and/or GIi-I protein, polypeptide and/or peptide component from a sample. The antibody will preferably be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the wild-type or mutant SHH, NF-κB, and/or GIi-I protein antigenic component will be applied to the immobilized antibody. The unwanted components will be washed from the column, leaving the antigen immunocomplexed to the immobilized antibody, which wild-type or mutant SHH, NF- KB, and/or GIi-I protein antigen is then collected by removing the wild-type or mutant SHH, NF-κB, and/or GIi-I protein and/or peptide from the column.

[0118] The immunobinding methods also include methods for detecting and quantifying the amount of a wild-type or mutant SHH, NF-κB, and/or GIi-I protein reactive component in a sample and the detection and quantification of any immune complexes formed during the binding process. Here, one would obtain a sample suspected of containing a wild- type or mutant SHH, NF-κB, and/or GIi-I protein and/or peptide, and contact the sample with an antibody against wild-type or mutant SHH, NF-κB, and/or GIi-I, and then detect and quantify the amount of immune complexes formed under the specific conditions.

[0119] In terms of antigen detection, the biological sample analyzed may be any sample that is suspected of containing a wild-type or mutant SHH, NF-κB, and/or GIi-I protein- specific antigen, such as esophageal or anal tissue section or specimen, a homogenized esophageal or anal tissue extract, a esophageal or anal tissue cell, separated and/or purified forms of any of the above wild-type or mutant SHH, NF- KB, and/or GIi-I protein-containing compositions, or even any biological fluid that comes into contact with the esophageal or anal tissue, including blood and/or serum, although tissue samples or extracts are preferred. Cancerous diseases that may be suspected of containing a wild- type or mutant SHH, NF- KB, and/or GIi-I protein-specific antigen include, but are not limited to, the collection of conditions classified as cancerous.

[0120] Contacting the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to, any SHH, NF-κB, and/or GIi-I protein antigens present. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or western blot, will generally be washed to remove any non- specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected. [0121] In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. U.S. Patents concerning the use of such labels include 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated herein by reference. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody and/or a biotin/avidin ligand binding arrangement, as is known in the art.

[0122] The SHH, NF-κB, and/or GIi-I antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

[0123] Further methods include the detection of primary immune complexes by a two step approach. A second binding ligand, such as an antibody, that has binding affinity for the antibody is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.

[0124] One method of immunodetection uses two different antibodies. A first step biotinylated, monoclonal or polyclonal antibody is used to detect the target antigen(s), and a second step antibody is then used to detect the biotin attached to the complexed biotin. In that method the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody/antigen complex. The antibody/antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody/antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin. This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence of the antibody/antigen complex by histoenzymology using a chromogen substrate. With suitable amplification, a conjugate can be produced which is macroscopically visible.

[0125] Another known method of immunodetection takes advantage of the immuno-PCR (Polymerase Chain Reaction) methodology. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA/biotin/streptavidin/antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls. At least in theory, the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.

[0126] The immunodetection methods of the present invention have evident utility in the diagnosis and prognosis of conditions such as various forms of cancer, such as esophageal or anal cancer. Here, a biological and/or clinical sample suspected of containing a wild-type or mutant SHH, NF-κB, and/or GIi-I protein, polypeptide, peptide and/or mutant is used. However, these embodiments also have applications to non-clinical samples, such as in the titering of antigen or antibody samples, for example in the selection of hybridomas.

[0127] In the clinical diagnosis and/or monitoring of patients with various forms of cancer, such as esophageal cancer or anal cancer, the detection of SHH, NF-κB, and/or GIi-I mutant, and/or an alteration in the levels of SHH, NF-κB, and/or GIi-I, in comparison to the levels in a corresponding biological sample from a normal subject is indicative of a patient with cancer, such as esophageal cancer or anal cancer. However, as is known to those of skill in the art, such a clinical diagnosis would not necessarily be made on the basis of this method in isolation. Those of skill in the art are very familiar with differentiating between significant differences in types and/or amounts of biomarkers, which represent a positive identification, and/or low level and/or background changes of biomarkers. Indeed, background expression levels are often used to form a "cut-off" above which increased detection will be scored as significant and/or positive.

a. ELISAs

[0128] As detailed above, immunoassays, in their most simple and/or direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and/or radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and/or western blotting, dot blotting, FACS analyses, and/or the like may also be used.

[0129] In one exemplary ELISA, the anti- SHH, NF-κB, and/or GIi-I antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the wild-type and/or mutant SHH, NF-κB, and/or GIi-I protein antigen, such as a clinical sample, is added to the wells. After binding and/or washing to remove non- specifically bound immune complexes, the bound wild- type and/or mutant SHH, NF-κB, and/or GIi-I protein antigen may be detected. Detection is generally achieved by the addition of another anti- SHH, NF-κB, and/or GIi-I antibody that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection may also be achieved by the addition of a second anti- SHH, NF-κB, and/or GIi-I antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

[0130] In another exemplary ELISA, the samples suspected of containing the wild-type and/or mutant SHH, NF- KB, and/or GIi-I protein antigen are immobilized onto the well surface and/or then contacted with the anti- SHH, NF-κB, and/or GIi-I antibodies of the invention. After binding and/or washing to remove non-specifically bound immune complexes, the bound anti- SHH, NF-κB, and/or GIi-I antibodies are detected. Where the initial anti- SHH, NF-κB, and/or GIi-I antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti- SHH, NF-κB, and/or GIi-I antibody, with the second antibody being linked to a detectable label.

[0131] Another ELISA in which the wild-type and/or mutant SHH, NF-κB, and/or GIi-I proteins, polypeptides and/or peptides are immobilized, involves the use of antibody competition in the detection. In this ELISA, labeled antibodies against wild-type or mutant SHH, NF-κB, and/or GIi-I protein are added to the wells, allowed to bind, and/or detected by means of their label. The amount of wild-type or mutant SHH, NF-κB, and/or GIi-I protein antigen in an unknown sample is then determined by mixing the sample with the labeled antibodies against wild- type and/or mutant SHH, NF- KB, and/or GIi-I before and/or during incubation with coated wells. The presence of wild- type and/or mutant SHH, NF-κB, and/or GIi- 1 protein in the sample acts to reduce the amount of antibody against wild-type or mutant SHH, NF- KB, and/or GIi-I protein available for binding to the well and thus reduces the ultimate signal. This is also appropriate for detecting antibodies against wild-type or mutant SHH, NF- KB, and/or GIi-I protein in an unknown sample, where the unlabeled antibodies bind to the antigen-coated wells and also reduces the amount of antigen available to bind the labeled antibodies.

[0132] Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non- specifically bound species, and detecting the bound immune complexes. These are described below.

[0133] In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

[0134] In ELISAs, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or a third binding ligand. [0135] "Under conditions effective to allow immune complex (antigen/antibody) formation" means that the conditions preferably include diluting the antigens and/or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.

[0136] The "suitable" conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25⁰C to 27⁰C, or may be overnight at about 4⁰C or so.

[0137] Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.

[0138] To provide a detecting means, the second or third antibody will have an associated label to allow detection. Preferably, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact or incubate the first and second immune complex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PB S -containing solution such as PBS- Tween).

[0139] After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl- benzthiazoline-6- sulfonic acid (ABTS), or H₂O₂, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generated, e.g., using a visible spectra spectrophotometer.

b. Immunohistochemistry

[0140] The antibodies of the present invention may also be used in conjunction with both fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks, or other method known by one of skill in the art, prepared for study by immunohistochemistry (IHC). The method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and/or is well known to those of skill in the art (Brown et al, 1990; Abbondanzo et al, 1990; Allred et al, 1990).

[0141] Briefly, frozen- sections may be prepared by rehydrating 50 ng of frozen "pulverized" tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and/or pelleting again by centrifugation; snap-freezing in -7O⁰C isopentane; cutting the plastic capsule and/or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and/or cutting 25-50 serial sections.

[0142] Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and/or embedding the block in paraffin; and/or cutting up to 50 serial permanent sections.

[0143] In some embodiments of the invention, the immunohistochemistry is automated. c. Immunoelectron Microscopy

[0144] The antibodies of the present invention may also be used in conjunction with electron microscopy to identify intracellular tissue components. Briefly, and electron-dense label is conjugated directly or indirectly to the anti- SHH, NF-κB, and/or GIi-I antibody. Examples of electron-dense labels according to the invention are ferritin and gold. The electron- dense label absorbs electrons and can be visualized by the electron microscope.

F. Expression systems

[0145] Numerous expression systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote- and/or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.

[0146] The insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Patents. 5,871,986, 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC^® 2.0 from INVITROGEN^® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH^®.

[0147] Other examples of expression systems include STRATAGENE^®'S COMPLETE CONTROL™ Inducible Mammalian Expression System, which involves a synthetic ecdysone- inducible receptor, or its pET Expression System, an E. coli expression system. Another example of an inducible expression system is available from INVITROGEN^®, which carries the T- REX™ (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter. INVITROGEN^® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.

[0148] It is contemplated a gene may be "overexpressed," i.e. expressed in increased levels relative to its natural expression in cells. Such overexpression may be assessed by a variety of methods, including radio-labeling and/or protein purification. However, simple and direct methods are contemplated, for example, those involving SDS/PAGE and protein staining or western blotting, followed by quantitative analyses, such as densitometric scanning of the resultant gel or blot. A specific increase in the level of the recombinant protein, polypeptide or peptide in comparison to the level in natural cells is indicative of overexpression, as is a relative abundance of the specific protein, polypeptides or peptides in relation to the other proteins produced by the host cell, e.g., visible on a gel.

[0149] In some embodiments, the expressed proteinaceous sequence forms an inclusion body in the host cell, the host cells are lysed, for example, by disruption in a cell homogenizer, washed and/or centrifuged to separate the dense inclusion bodies and cell membranes from the soluble cell components. This centrifugation can be performed under conditions whereby the dense inclusion bodies are selectively enriched by incorporation of sugars, such as sucrose, into the buffer and centrifugation at a selective speed. Inclusion bodies may be solubilized in solutions containing high concentrations of urea (e.g., 8M) or chaotropic agents such as guanidine hydrochloride in the presence of reducing agents, such as β-mercaptoethanol or DTT (dithiothreitol), and refolded into a more desirable conformation, as would be known to one of ordinary skill in the art. [0150] The nucleotide and protein sequences for genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art. One such database is the National Center for Biotechnology Information's Genbank and GenPept databases. The coding regions for these known genes may be amplified and/or expressed using the techniques disclosed herein or by any technique that would be known to those of ordinary skill in the art. Additionally, peptide sequences may be synthesized by methods known to those of ordinary skill in the art, such as peptide synthesis using automated peptide synthesis machines, such as those available from Applied Biosystems (Foster City, CA).

G. Measuring mRNA expression

[0151] In an embodiment of the invention, mRNA levels are detected to assess the levels of deregulated and/or deregulated SHH, GIi-I, NF- KB. In specific embodiments of the invention the mRNA levels are detected by in situ hybridization, Northern blotting, sequences, gene arrays, gene chips, and/or nuclease protection. An exemplary description of mRNA detection is given below.

[0152] In addition to their use in directing the expression of SHH, GIi-I, and/or NF-κB proteins, polypeptides and/or peptides, the nucleic acid sequences disclosed herein have a variety of other uses. For example, they have utility as probes or primers for embodiments involving nucleic acid hybridization. a. Hybridization

[0153] The use of a probe or primer of between 13 and 100 nucleotides, preferably between 17 and 100 nucleotides in length, or in some aspects of the invention up to 1-2 kilobases or more in length, allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over contiguous stretches greater than 20 bases in length are generally preferred, to increase stability and/or selectivity of the hybrid molecules obtained. One will generally prefer to design nucleic acid molecules for hybridization having one or more complementary sequences of 20 to 30 nucleotides, or even longer where desired. Such fragments may be readily prepared, for example, by directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.

[0154] Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNAs and/or RNAs or to provide primers for amplification of DNA or RNA from samples. Depending on the application envisioned, one would desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of the probe or primers for the target sequence.

[0155] For applications requiring high selectivity, one will typically desire to employ relatively high stringency conditions to form the hybrids. For example, relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 5O⁰C to about 7O⁰C. Such high stringency conditions tolerate little, if any, mismatch between the probe or primers and the template or target strand and would be particularly suitable for isolating specific genes or for detecting specific mRNA transcripts. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.

[0156] For certain applications, for example, site-directed mutagenesis, it is appreciated that lower stringency conditions are preferred. Under these conditions, hybridization may occur even though the sequences of the hybridizing strands are not perfectly complementary, but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and/or decreasing temperature. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37⁰C to about 55⁰C, while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 2O⁰C to about 55⁰C. Hybridization conditions can be readily manipulated depending on the desired results.

[0157] In other embodiments, hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 1.0 mM dithiothreitol, at temperatures between approximately 2O⁰C to about 37⁰C. Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, at temperatures ranging from approximately 4O⁰C to about 72⁰C.

[0158] In certain embodiments, it will be advantageous to employ nucleic acids of defined sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of being detected. In preferred embodiments, one may desire to employ a fluorescent label or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known that can be employed to provide a detection means that is visibly or spectrophotometrically detectable, to identify specific hybridization with complementary nucleic acid containing samples.

[0159] In general, it is envisioned that the probes or primers described herein will be useful as reagents in solution hybridization, as in PCR™, for detection of expression of corresponding genes, as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single- stranded nucleic acid is then subjected to hybridization with selected probes under desired conditions. The conditions selected will depend on the particular circumstances (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Optimization of hybridization conditions for the particular application of interest is well known to those of skill in the art. After washing of the hybridized molecules to remove non- specifically bound probe molecules, hybridization is detected, and/or quantified, by determining the amount of bound label. Representative solid phase hybridization methods are disclosed in U.S. Patent Nos. 5,843,663, 5,900,481 and 5,919,626. Other methods of hybridization that may be used in the practice of the present invention are disclosed in U.S. Patent Nos. 5,849,481, 5,849,486 and 5,851,772. The relevant portions of these and other references identified in this section of the Specification are incorporated herein by reference. b. Amplification of Nucleic Acids

[0160] Nucleic acids used as a template for amplification may be isolated from cells, tissues or other samples according to standard methodologies (Sambrook et al, 1989). In certain embodiments, analysis is performed on whole cell or tissue homogenates or biological fluid samples without substantial purification of the template nucleic acid. The nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to first convert the RNA to a complementary DNA.

[0161] The term "primer," as used herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Typically, primers are oligonucleotides from ten to twenty and/or thirty base pairs in length, but longer sequences can be employed. Primers may be provided in double-stranded and/or single- stranded form, although the single- stranded form is preferred. [0162] Pairs of primers designed to selectively hybridize to nucleic acids corresponding to SHH, GIi-I and/or NF-κB are contacted with the template nucleic acid under conditions that permit selective hybridization. Depending upon the desired application, high stringency hybridization conditions may be selected that will only allow hybridization to sequences that are completely complementary to the primers. In other embodiments, hybridization may occur under reduced stringency to allow for amplification of nucleic acids contain one or more mismatches with the primer sequences. Once hybridized, the template- primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles," are conducted until a sufficient amount of amplification product is produced.

[0163] The amplification product may be detected or quantified. In certain applications, the detection may be performed by visual means. Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical and/or thermal impulse signals (Affymax technology; Bellus, 1994).

[0164] A number of template dependent processes are available to amplify the oligonucleotide sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (referred to as PCR™) which is described in detail in U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159, and in Innis et al, 1988, each of which is incorporated herein by reference in their entirety.

[0165] A reverse transcriptase PCR™ amplification procedure may be performed to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known (see Sambrook et al., 1989). Alternative methods for reverse transcription utilize thermostable DNA polymerases. These methods are described in WO 90/07641. Polymerase chain reaction methodologies are well known in the art. Representative methods of RT-PCR are described in U.S. Patent No. 5,882,864.

[0166] Another method for amplification is ligase chain reaction ("LCR"), disclosed in European Application No. 320 308, incorporated herein by reference in its entirety. U.S. Patent 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence. A method based on PCR™ and oligonucleotide ligase assy (OLA), disclosed in U.S. Patent 5,912,148, may also be used. [0167] Alternative methods for amplification of target nucleic acid sequences that may be used in the practice of the present invention are disclosed in U.S. Patent Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497, 5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905, 5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, GB Application No. 2 202 328, and in PCT Application No. PCT/US89/01025, each of which is incorporated herein by reference in its entirety.

[0168] Qbeta Replicase, described in PCT Application No. PCT/US 87/00880, may also be used as an amplification method in the present invention. In this method, a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence which may then be detected.

[0169] An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'- [alpha-thio] -triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention (Walker et al, 1992). Strand Displacement Amplification (SDA), disclosed in U.S. Patent No. 5,916,779, is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation.

[0170] Other nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al, 1989; Gingeras et al, PCT Application WO 88/10315, incorporated herein by reference in their entirety). European Application No. 329 822 disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double- stranded DNA (dsDNA), which may be used in accordance with the present invention.

[0171] PCT Application WO 89/06700 (incorporated herein by reference in its entirety) disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter region/primer sequence to a target single- stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts. Other amplification methods include "race" and "one-sided PCR" (Frohman, 1990; Ohara et al, 1989). c. Detection of Nucleic Acids

[0172] Following any amplification, it may be desirable to separate the amplification product from the template and/or the excess primer. In one embodiment, amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et al, 1989). Separated amplification products may be cut out and eluted from the gel for further manipulation. Using low melting point agarose gels, the separated band may be removed by heating the gel, followed by extraction of the nucleic acid.

[0173] Separation of nucleic acids may also be effected by chromatographic techniques known in art. There are many kinds of chromatography which may be used in the practice of the present invention, including adsorption, partition, ion-exchange, hydroxylapatite, molecular sieve, reverse-phase, column, paper, thin-layer, and gas chromatography as well as HPLC.

[0174] In certain embodiments, the amplification products are visualized. A typical visualization method involves staining of a gel with ethidium bromide and visualization of bands under UV light. Alternatively, if the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the separated amplification products can be exposed to x-ray film or visualized under the appropriate excitatory spectra.

[0175] In one embodiment, following separation of amplification products, a labeled nucleic acid probe is brought into contact with the amplified marker sequence. The probe preferably is conjugated to a chromophore but may be radiolabeled. In another embodiment, the probe is conjugated to a binding partner, such as an antibody or biotin, or another binding partner carrying a detectable moiety.

[0176] In particular embodiments, detection is by Southern blotting and hybridization with a labeled probe. The techniques involved in Southern blotting are well known to those of skill in the art (see Sambrook et al., 1989). One example of the foregoing is described in U.S. Patent No. 5,279,721, incorporated by reference herein, which discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids. The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention. [0177] Other methods of nucleic acid detection that may be used in the practice of the instant invention are disclosed in U.S. Patent Nos. 5,840,873, 5,843,640, 5,843,651, 5,846,708, 5,846,717, 5,846,726, 5,846,729, 5,849,487, 5,853,990, 5,853,992, 5,853,993, 5,856,092, 5,861,244, 5,863,732, 5,863,753, 5,866,331, 5,905,024, 5,910,407, 5,912,124, 5,912,145, 5,919,630, 5,925,517, 5,928,862, 5,928,869, 5,929,227, 5,932,413 and 5,935,791, each of which is incorporated herein by reference. d. Other Assays

[0178] Other methods for genetic screening may be used within the scope of the present invention, for example, to detect mutations in genomic DNA, cDNA and/or RNA samples. Methods used to detect point mutations include denaturing gradient gel electrophoresis ("DGGE"), restriction fragment length polymorphism analysis ("RFLP"), chemical or enzymatic cleavage methods, direct sequencing of target regions amplified by PCR™ (see above), single- strand conformation polymorphism analysis ("SSCP") and other methods well known in the art.

[0179] One method of screening for point mutations is based on RNase cleavage of base pair mismatches in RNA/DNA or RNA/RNA heteroduplexes. As used herein, the term "mismatch" is defined as a region of one or more unpaired or mispaired nucleotides in a double- stranded RNA/RNA, RNA/DNA or DNA/DNA molecule. This definition thus includes mismatches due to insertion/deletion mutations, as well as single or multiple base point mutations.

[0180] U.S. Patent No. 4,946,773 describes an RNase A mismatch cleavage assay that involves annealing single-stranded DNA or RNA test samples to an RNA probe, and subsequent treatment of the nucleic acid duplexes with RNase A. For the detection of mismatches, the single-stranded products of the RNase A treatment, electrophoretically separated according to size, are compared to similarly treated control duplexes. Samples containing smaller fragments (cleavage products) not seen in the control duplex are scored as positive.

[0181] Other investigators have described the use of RNase I in mismatch assays. The use of RNase I for mismatch detection is described in literature from Promega Biotech. Promega markets a kit containing RNase I that is reported to cleave three out of four known mismatches. Others have described using the Mu tS protein or other DNA-repair enzymes for detection of single-base mismatches. [0182] Alternative methods for detection of deletion, insertion or substitution mutations that may be used in the practice of the present invention are disclosed in U.S. Patent Nos. 5,849,483, 5,851,770, 5,866,337, 5,925,525 and 5,928,870, each of which is incorporated herein by reference in its entirety.

H. KITS

[0183] Any of the compositions described herein may be comprised in a kit. In a non-limiting example, antibodies to SHH, NF-κB, and GIi-I, and/or additional agent, may be comprised in a kit. The kits will thus comprise, in suitable container means, antibodies to SHH, NF-κB, and GIi-I, and/or an additional agent of the present invention.

[0184] The kits may comprise a suitably aliquoted antibody to SHH, NF-κB, and/or GIi-I, and/or additional agent compositions of the present invention, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay. The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the antibodies to SHH, NF- KB, and/or GIi-I, lipid, additional agent, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. Containers may also contain any agent that detects deregulated SHH, NF-κB, and/or GIi-I.

[0185] When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. The antibodies to SHH, NF-κB, and GIi-I compositions may also be formulated into a syringeable composition. In which case, the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit. [0186] However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

[0187] The container means will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the antibodies to or agents to detect deregulated SHH, NF-κB, and GIi-I protein formulation are placed, preferably, suitably allocated. The kits may also comprise a second container means for containing a sterile, acceptable buffer and/or other diluent.

[0188] The kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection and/or blow-molded plastic containers into which the desired vials are retained.

[0189] In still further embodiments, the present invention concerns immunodetection kits for use with the immunodetection methods described above. As the SHH, NF-κB, and/or GIi-I antibodies are generally used to detect wild- type and/or mutant SHH, NF- KB, and/or GIi-I proteins, polypeptides and/or peptides, the antibodies will preferably be included in the kit. However, kits including both such components may be provided. The immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to a wild- type and/or mutant SHH, NF-κB, and/or GIi-I protein, polypeptide and/or peptide, and/or optionally, an immunodetection reagent and/or further optionally, a wild-type and/or mutant SHH, NF-κB, and/or GIi-I protein, polypeptide and/or peptide.

[0190] In any embodiment of the invention, monoclonal antibodies may be used. In any embodiment, the first antibody that binds to the wild-type and/or mutant SHH, NF- KB, and/or GIi-I protein, polypeptide and/or peptide may be pre -bound to a solid support, such as a column matrix and/or well of a micro titre plate.

[0191] The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with and/or linked to the given antibody. Detectable labels that are associated with and/or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody. [0192] Further suitable immunodetection reagents for use in the present kits include the two-component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, a number of exemplary labels are known in the art and/or all such labels may be employed in connection with the present invention.

[0193] The kits may further comprise a suitably aliquoted composition of the wild- type and/or mutant SHH, NF-κB, and/or GIi-I protein, polypeptide and/or polypeptide, whether labeled and/or unlabeled, as may be used to prepare a standard curve for a detection assay. The kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, and/or as separate moieties to be conjugated by the user of the kit. The components of the kits may be packaged either in aqueous media and/or in lyophilized form.

[0194] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the antibody may be placed, and/or preferably, suitably aliquoted. Where wild- type and/or mutant SHH, NF-κB, and/or GIi-I protein, polypeptide and/or peptide, and/or a second and/or third binding ligand and/or additional component is provided, the kit will also generally contain a second, third and/or other additional container into which this ligand and/or component may be placed. The kits of the present invention will also typically include a means for containing the antibody, antigen, and/or any other reagent containers in close confinement for commercial sale. Such containers may include injection and/or blow-molded plastic containers into which the desired vials are retained.

[0195] All the essential materials and/or reagents required for detecting deregulated SHH, NF- KB and/or GIi-I nucleic acids in a sample may be assembled together in a kit. This generally may comprise a probe or primers designed to hybridize specifically to individual nucleic acids of interest in the practice of the present invention, including genes or mRNA to SHH, NF- KB, and/or GIi-I. Also included may be enzymes suitable for amplifying nucleic acids, including various polymerases (reverse transcriptase, Taq, etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification. Such kits may also include enzymes and other reagents suitable for detection of specific nucleic acids or amplification products. Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or enzyme as well as for each probe or primer pair. EXAMPLES

[0196] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1 EXPRESSION PROFILING

[0197] Sonic Hedgehog in Esophogeal adenocarinoma (EAC) In an expression profiling study intended to identify molecular signatures predictive of response to pre-operative chemoradiation (CTXRT) in EAC, a significantly increased expression of the SHH in pre- treatment EA tissue from patients resistant to CTXRT was found. Using IHC, pre-treatment tissue of 63 EA patients, undergoing CTXRT, for the levels of expression of SHH and its downstream effector GIi-I were examined. All the pre- CTXRT EAC (n=63) examined were found to have the SHH pathway activated (deregulated). The SHH labeling index (LI, number of positive cells) was significantly associated with the degree of pathologic response (p=<0.0001). The LI cut point for SHH at 0.145 by CART discriminated 15 of 16 patients with pathologic complete response and 38 of 47 patients with chemoradiation-resistance with a correct classification rate of 4%, sensitivity of 94%, and specificity of 81%. The SHH LI highly correlated with overall survival (p=0.0002) and the risk for death increased with higher proportion of residual cells expressing SHH (p=0.004) in the resected specimens n=47. In vitro studies conducted in EA cell lines, also showed different levels of SHH and GIi-I expression. Importantly, it was observed that the constitutive activation of SHH pathway was enhanced in cells harboring a more aggressive phenotype. Additionally, SHH inhibition, using siRNA, Forskolin and Cyclopamin, induced a Gl-S block, while exogenous SHH stimulation promoted cell proliferation. To establish a mechanistic link between SHH deregulation and CTXRT resistance in EA, a SEG-I (human cell line with SHH pathway deregulation) tumor xenografts model with CTXRT was treated and IHC studies overtime were performed. It was observed that an increase in SHH signaling preceded proliferation before tumor re-growth (Sims-Mourtada et al, 2006). Further, it was discovered that the SHH signaling regulates the transcription of two ABC drug transporters family members (MDRl and BCRP) (Sims-Mourtada et al, 2007). This data indicate that SHH deregulated activation is a common feature of EAC, and is closely associated with the burden of more aggressive cancer cells and patients clinical outcome. This evidence shows that SHH deregulation participates to the progression to EAC by enhancing cell adaptation and survival.

[0198] Transcriptional Profiling Correlates with NF-κB and SHH Pathways and Response to Chemoradiation. Pretreatment cancer biopsies were profiled from patients with esophageal cancers using the Affymetrix U133A platform and correlated their molecular signatures with pathologic response. Among 19 patients who received preoperative chemoradiation, 32% had a pathCR. Unsupervised hierarchical cluster analysis segregated cancers into two molecular subtypes (I and II, consisting of 10 and 9 specimens) (Luthra et al, 2006). Correlation with pathologic outcome revealed that 80% cancers with pathCR clustered in subtype I. Only one cancer with pathCR clustered in subtype II. Distinct expression profiles of the two categories in this study indicate that one biological entity is less likely to achieve pathCR compared to the other. Approximately 450 genes were differentially expressed between the two subtypes with an estimated false discovery rate of 5%. Greater than two fold differences in the expression levels were observed in 80 genes using t-test (p< 0.0001). Several genes, associated with apoptosis, calcium homeostasis, and stress response, were collectively down-regulated in subtype II in comparison with subtype I. Quantitative real-time PCR analysis of several randomly selected genes confirmed differential expression observed in the microarray data (Luthra et al, 2007). The gene expression analysis comparing the 19 cancers stratified per their pathological outcome (pathCR v <pathCR), also indicated that (FIG. 3) a number of genes either implicated in the upstream activation (i.e. TRAF2, FOXO3) or downstream targets (MMP9, PAFD, ICAM) of NF-κB were differentially expressed toward an NF-κB activated (deregulated) pathway in non-responders. Similarly, the expression levels of SHH ligand were significantly upregulated in the non-responder group (FIG. 4). These results indicate the use of the three biomarkers in a larger cohort of patients treated with chemoradiation. EXAMPLE 2

BIOMARKER SIGNATURE FOR PREDICTION OF RESPONSE IN ESOPHAGEAL

CANCER

Overview

[0199] An initial model using the 3 biomarker method (GIi-I, SHH, and NF-κB) was created from a ROC analysis on 60 patients, in which the specificity was 98% for pathCR, when the corresponding sensitivity was 45%; and with sensitivity up to 70%, >93% specificity could be maintained for pathCR. Based on ROC data, the specificity was 95% for exCRTR, when the corresponding sensitivity was 45%; and with sensitivity up to 70%, >83% specificity for exCRTR could be maintained. In this model, a few patients could end up without immediate surgery (surgery would be used as a salvage procedure) and only a few would not get chemoradiation when it is likely to be beneficial. However, the same model would correctly spare esophagectomy for -45% or more of pathCR patients (currently, 0% can avoid surgery) and avoid unnecessary chemoradiation for >80% of exCRTR patients (currently, 0% can avoid chemoradiation). Further specifics are given below.

[0200] As stated previously, approximately 25% of patients undergoing chemoradiation have >50% residual cancer in resected specimen. The median disease-free survival (DFS) of these patients is only 11 months and OS is 14 months (FIG. 5 panels C and D) (Rohatgi et al, 2005). It may be possible to identify patients with exCRTR to avoid ineffective chemoradiation therapy.

[0201] Methods: Pretreatment cancer specimens of esophageal adenocarcinoma patients who had preoperative chemoradiation and surgery were assessed for the expression of sonic hedgehog, GIi-I, and NF-κB. By combining the raw data as continuous variable for each biomarker, Z score was computed for each patient and the area under the curve (AUC) for receiver operating characteristics (ROC) curve were generated. Corrected AUCs were derived from two validation methods: 5-fold cross-validation (repeated 20 times) and bootstrap (re- sampled 200 times).

[0202] Results: There were 16 (27%) patients with pathCR among 60 patients studied. The rate of pathCR was not associated with the use of induction chemotherapy (p=0.96) or the class of cytotoxics (p=0.90). However, the ROC curve, from the fitted multiple logistic regression model, resulted in an estimated AUC of 0.953 (95% confidence interval: 0.906-1.000; FIG 16). The two validation methods, to control for the over- fitting of the data, resulted in the corrected AUC values of 0.943 and 0.940, respectively.

[0203] Conclusions: These data demonstrate that this pretreatment 3-biomarker signature is highly predictive of pathCR resulting in the high accuracy, sensitivity, and specificity rates.

[0204] Methods

[0205] Patient Selection and Evaluation Sixty operable patients with localized histologically confirmed adenocarcinoma of the thoracic esophagus and gastroesophageal juction were included in the study. Clinical evaluation included computed tomography (chest and abdomen), upper gastrointestinal barium radiographs, an esophago-gastroduodenoscopy with endoscopic ultrasonography (EUS), and when available, a positron emission tomography. Patients with T2-3 with any N, patients with MIa cancer (celiac nodes associated) and patients with TlNl were considered eligible. Patients with TlNO, or T4 lesions or with metastatic cancer were excluded. All patients were evaluated by multiple disciplines.

[0206] Patient Treatment Thirty-two patients were treated on clinical trials and 28 were treated off clinical trial. Induction chemotherapy was administered to 34 patients. 5- fluorouracil was administered to all patients. Twenty- seven also received a taxane, 25 received a camptothecin, and 7 received a platinum compound. The maximum duration of induction chemotherapy was 12 weeks. All 60 patients received approximately 50.4 Gy of radiation therapy in 28 fractions. During radiation, all patients received 5-fluorouracil and 43 received a taxane, 27 received platinum, and 45 received a camptothecin. Chemotherapy during radiation was given for 5 weeks. Five to 6 weeks after the completion of chemoradiation, a complete restaging was performed followed by surgery. Each resected esophageal specimen was examined in a systematic manner (Wu et al, , 2007; Chirieac, 2005) and was re-confirmed by one experienced gastrointestinal pathologist, without the knowledge of patient outcome. The pathologic response was assigned to one of two categories: pathCR (0% residual cancer) or <pathCR (>1% residual cancer) (Chirieac, 2005).

[0207] Patient Follow-up Each patient was assessed at 3, 6, 9, and 12 months, followed by every 6 months for an additional 2 years, and then every year or until death. Local- regional recurrence was defined as recurrence within the surgical field or mediastinal nodes. Metastatic recurrence was defined as evidence of cancer outside the regional area, or death from unknown causes within 3 years of study.

[0208] Tissue specimens for Immunohistochemical Studies All specimens were collected under an approved protocol. All tissue sections were matched to routine hematoxylin- eosin-stained slides used to evaluate for the presence of cancer by one pathologist.

[0209] Immunohistochemistry and protein expression Immunohistochemical staining for NF-κB, SHH and GIi-I protein expression was performed on 4-μm formalin-fixed, paraffin-embedded (FFPE) adjacent sections with the G96-337 monoclonal antibody (2 mg/mL; BD Pharmigen, Palo alto, CA), and the H- 160 (concentration 2 mg/mL) and H-330 (concentration 4 mg/mL) rabbit polyclonal antibodies (Santa Cruz Biotechnology, CA), respectively. The immunohistochemical procedure, including positive and negative controls were carried out as previously reported (Izzo et al, 2006; Izzo et al, 2007; Sims-Mourtada et al, 2006; Izzo et al, 2006). The positive control for deregulated hedgehog pathway was SEG-I cells, human esophageal adenocarcinoma cells. The hedgehog pathway negative control used was also SEG-I cells, which was pretreated with blocking peptide (Shh) and with the primary antibody (GIi-I) omitted from the assay. SKGT-4 esophageal cancer cells were used as a positive control for NF-κB. For scoring purposes only tumor cells were assessed for the presence of NF-κB and GIi-I nuclear immunoreactivity and of SHH cytoplasmic immunolabeling. The extent of NF-κB, GIi-I, and SHH positive cancer cells was expressed for each marker as the fraction of labeled cells, or labeling index (LI) in the cancers. To allow comparison between the three biomarkers on a region-by-region basis, digitized images of all cancer fields present in the tissue sections were captured with a high-resolution image analysis system (SimplePCI, Compix, Inc. Cranberry Town, PA) controlling a Nikon Optiphot microscope equipped with a motorized stage and a 3 CCD color video camera (Sony DXC-390), Sony, Inc. The digitized field-by-field montage images, representing the whole cancer tissue sections, were then used for scoring and comparison of spatial localization of the proteins. Three investigators, independently, in a blind fashion and without knowledge of the clinical data, analyzed protein expression. For each sample the final LI was determined by the average LI count of the three investigators. In the discrepant cases, a final opinion was made based on consensus by all three investigators, and if necessary a recount of the labeled cells was done.

[0210] Statistical Analysis The distribution of biomarker expression level were summarized by mean, standard deviation, median, and range. A multiple logistic regression model was used to fit for the endpoint of pathCR with GIi-I, SHH and NF-κB included as covariates. The goodness-of-fit was assessed by the method of Hosmer et al, (Hosmer, 1997) where high p values indicate a well calibrated model. The log-likelihood ratio test and AIC was used for model selection. Based on the final fitted model, a linear Z score was computed or each patient. For example, if the final model contains all 3 biomarkers, the linear Z score for patient i can be expressed as follows:

[0211] Z = β_c - &\,_: + β_ι \_tl - β₂ \,_ι .

[0212] where X₁₁, x_l2 and x_l3 are the expression level of GIi-I, SHH and NF-κB for the patient i, respectively, and i=l , 2, ... , 60. Also, the predicted probability of having pathCR for each patient i was computed based on the following formula:

A [ pathCR {∞ exCRTR) ] =

[0213] ⁱ ~ ^{€ r"'}

[0214] The functional relationship between the linear Z scores and the predicted probabilities of pathCR (or exCRTR) was plotted and the true pathCR (or exCRTR) patients were identified on this plot.

[0215] The area under the receiver operating characteristic (ROC) curve, ie, AUC, was used to assess the discriminating ability of the fitted model. The AUC is a threshold- independent measure and can take values between 0 and 1, where 0.5 indicates that the model has no predictive power (ie, not different from random); 1 signifies a perfect model; and values < 0.5 indicate a relationship worse than expected by chance. According to Swets (Swets, 1988), models providing values > 0.9 are considered highly accurate; those providing values in the range 0.7-0.9 useful; and those < 0.7 poorly accurate.

[0216] In this study, with a total sample size of 60 patients and assuming that 25% of these patients achieve pathCR, there is an 86% power to test the null hypothesis of HO: AUC= 0.65 (ie, poor discrimination) against the alternative hypothesis of Hl: AUC = 0.80 (ie, good discrimination), using the two-sided asymptotic Z-test and at 0.05 significance level. The powers were estimated using the %ROCPOWER macro in SAS (Zep, 1995). The functional relationship between the linear Z scores and the predicted probabilities of pathCR was plotted and the true pathCR patients was identified on this plot. The ROC curve was prepared for the fitted model, with the AUC estimated using the non-parametric method implemented in SAS. The 95% confidence interval for the AUC will also be estimated (%ROC macro in SAS). Two resampling techniques were used to validate the estimated AUC. First, 5-fold cross-validation was used and in order to obtain more accurate estimate for the corrected AUC, the cross-validation process will be repeated 20 times. The average AUC from these 20 repeated cross validations were obtained. Secondly, a bootstrap with 200 resampling runs was performed. Through resampling with replacement, the bootstrap allowing estimation of the optimism in AUC of predictive accuracy, and then subtraction of the estimate of optimism from the initial estimation to obtain a corrected estimate. All statistical analyses were carried out in Splus (Venables, 1999).

[0217] Results

[0218] Patient characteristics Pretreatment cancer biopsies from 60 patients with esophageal adenocarcinoma was analyzed. Patient characteristics are shown in Table 1. The median age was 59 (range, 35-76); males predominated (98.4%) and all had adenocarcinoma histology. Clinical stage included: stage HA in 40%, HB in 5%, III in 50%, and IVA in 5%.

[0219] PathCR was observed in 16 (26.7%) patients; the remaining 44 (73.3%) patients had <pathCR. The median follow-up time was 44+ months (range, 5.8-104). The median OS time was 44.4 months (95% confidence interval [CI]; 23.2 - 65.7) with the 3- and 5-year survival rates were 57.2% (95% CI: 50.4 - 63.9%) and 43.3% (95% CI: 35.0 - 51.6%), respectively.

[0220] On univariate analysis, the type of pathologic response assessed in the resected specimen was associated with OS. Patients who achieved a pathCR survived significantly longer (median not reached) compared to patients who achieved <pathCR (median of 34 months; P=0.005, log-rank test; FIG. 6).

TABLE 1: PATIENT CHARACTERISTICS

[0221] Influence of Class Cytotoxics and Induction Chemotherapy: The use of induction chemotherapy had no influence on OS (p=1.0 by the Fisher's exact test) and similarly, the class of cytotoxics used had no influence of OS (p=0.51 by the Fisher's exact test).

[0222] Expression of Three Biomarkers: The summary statistics for the LI for each biomarker are summarized in Table 2. The fitted multiple logistic regression model for pathCR is shown in Table 3.

TABLE 2: SUMMARY STATISTICS FOR SHH, GLI-I AND NF-KB

TABLE 3: FITTED MULTIPLE LOGISTIC REGRESSION MODEL FOR PATHCR (N=60) [

[0223] Z Score for Each Patient FIG. 6 shows the curve for Z score. All pathCR patients had a similar score and only a few non-pathCR patients had a positive score. This indicates that scoring model is highly accurate for pathCR prediction.

[0224] ROC Curves The ROC curve for the fitted multiple logistic regression model, which had an estimated AUC of 0.953 (95% CI: 0.906 - 1.000; FIG. 16). To control for the overfitting of the data, two validation methods were used: 5- fold cross-validation and bootstrap. The 5-fold cross validation was repeated 20 times and the average corrected AUC was calculated. A bootstrap with 200 resampling runs was additionally performed. The corrected AUC values after cross-validation and bootstrapping were 0.943 and 0.940, respectively.

[0225] Discussion

[0226] The data on 60 patients with 3 biomarkers demonstrate that the 3- biomarker signature has high accuracy, specificity, and sensitivity rates based on the corrected AUCs generated by the ROC curves. On the Z score curve, patients with pathCR congregated with a positive (FIG. 6). In addition to biomarkers, sophisticated imaging techniques complements the strategy of individualized therapy for LEA, in specific embodiments of the invention. In conclusion, the data demonstrate that a 3-biomarker signature for SHH, GIi-I, and NF- KB is highly predictive of pathCR in patients with adenocarcinoma of the esophagus who undergo chemoradiation and surgery.

EXAMPLE 3

BIOMARKER SIGNATURE FOR PREDICTION OF RESPONSE IN ESOPHAGEAL

CANCER ADDITIONAL IHC DATA

[0227] For all IHC studies described below (total data available for 80 patients), also studied were NF-κB in 80 patients, NF-κB plus GIi-I in 75 patients, and NF-κB, GIi-I, and SHH in 60 patients (patients from Example 2, used to create another model).

[0228] NF-κB and Chemoradiation-resistance: In a previous study, tissue from 43 patients was examined for NF- KB and its expression status and it correlated with histologic features, pathologic response, metastatic potential, and overall survival (OS). Twenty one (72%) of 29 patients having nonpathCR had an NF-κB - positive cancer but only one (7%) of 14 patients achieving pathCR had an NF-κB -positive cancer (P<0.001). Activated (deregulated) NF-κB was associated with aggressive pathologic features (P=0.0004) and 10 (48%) of 21 patients with NF-κB positive cancer had died compared to only one (5%) of 22 patients with NF- KB negative cancer (P=O.0013). In a multivariate model NF-κB activation was the only independent predictor of OS (P=O.015). This cohort was expanded to 75 patients and correlated with pathologic response to chemoradiation and OS (FIG. 8). FIG. 9A illustrates examples of immunohistochemical (IHC) expression of NF-κB. Upper panel: NF-κB positive biopsies. Lower panel: NF-κB negative biopsies. Activated (deregulated) NF-κB prior to any therapy was associated with <pathCR; P=0.006 (FIG. 9B). Forty-five (78%) of 58 patients achieving <pathCR had an activated (deregulated) NF-κB vs. 2 (9%) of 22 patients with pathCR (P=OOOl). Of 46 (61%) patients with negative NF-κB cancer, 43% had a pathCR and of 29 positive NFkB cancers, only 7% had a pathCR. 25 (53%) of 47 patients with activated (deregulated) NF-κB cancer had died versus 3 (9%) of 33 patients with negative NF-κB cancer.

[0229] Activated (deregulated) SHH Signaling and Chemoradiation Resistance: To determine the role of SHH signaling in chemoradiation-resistance, expression levels of cytoplasmic SHH and nuclear GIi-I were examined in pre- and post-treatment esophageal cancer specimens from 59 patients who received preoperative chemoradiation. All pre-treatment cancer biopsies showed the presence of SHH expression (FIG. 10). Strikingly, a lower labeling index of, both, SHH and GIi-I, was observed in cancers with pathCR than those with <pathCR [median; (range)]: Gli-1-pathCR = 0.08 (0.005-0.5) vs Gli-l-<pathCR = 0.55 (0.01-0.90); SHH-pathCR = 0.10 (0.01-0.5) vs SHH-<pathCR = 0.30 (0.01-0.8); P = 0.009 and P = 0.062, respectively; FIG. 10). Importantly, activation of the SHH pathway (defined by the presence of nuclear GIi-I expression in > 30% of tumor cells) was significantly correlated with the lack of pathCR. 36 (87.3%) of the 43 resistant cancers had a sustained activation of the SHH pathway. The spatial localization of SHH and GIi-I IHC within a tumor showed that SHH was usually clustered in small patches surrounded by larger areas of GIi-I, indicating a regional activation of the HH pathway. Substantial activation of the SHH pathway with <pathCR indicating secretion of SHH and subsequent activation of HH signaling (with the expression of SHH and GIi-I) in proliferation and repopulation after treatment. FIG. 11 shows that, in a xenograft model after chemoradiation, before the tumor volume increases there is rapid rise in the labeling indices (LIs) of SHH and GIi-I (even before Ki67). This substantiates the role SHH/Gli-1 play in repopulation of the tumor bed after chemoradiation injury. [0230] Data on NF-κB, SHH, and GH-I in 60 patients: Next, the three biomarkers (NF-κB, SHH and GIi-I) in 60 patients was analyzed again to create a second statistical model (patients are a subset of the above described cohort), for which biomarkers data on cancer tissue were available (Table 2).

[0231] Statistical methods: Summary statistics are provided for the 3 biomarkers, NF- KB, SHH and GIi-I. A multiple logistic regression model was fit for the binary endpoints of pathCR or exCRTR, using NF-κB, SHH and GIi-I as predictors. The goodness-of-fit was assessed by the method of Hosmer et al (Hosmer, 1997), where high p values indicate a well- calibrated model. The predictive ability was assessed using a threshold-independent measure of the area under the receiver operating characteristic (ROC) curve. The AUC can take values between 0 and 1, where 0.5 indicates that the model has no predictive power (ie, no different from random); 1 signifies a perfect model; and values < 0.5 indicate a relationship worse than expected by chance. According to Swets (1988), models providing values > 0.9 are considered highly accurate; those providing values in the range 0.7-0.9 useful; and those < 0.7 poorly accurate. Two resampling techniques were used to validate the AUC for the final fitted model. First, 5-fold cross-validation was used and in order to obtain more accurate estimate for the corrected AUC, the cross validation process was repeated 20 times. The average AUC from these 20 repeated cross-validations is reported. Secondly, a bootstrap with 200 re-sampling runs was performed. Through resampling with replacement, the bootstrap allows one to estimate the optimism in any measure, such as AUC, of predictive accuracy, and then subtract the estimate of optimism from the initial apparent measure to obtain a corrected estimate (Efron & Tibshirani, 1993). Both methods were implemented using the Design library (Harrell, 2001) in Splus

[0232] Calculations were performed for exCRTR as described above. Table 4 shows a regression model for exCRTR with SHH, GIi-I, and NF-κB and FIG. 12 shows the z- score vs. predicted probability of exCRTR. Statistical calculations were also performed for just SHH and GIi-I, as described above (FIG. 13; Table 5).

TABLE 4: FITTED MULTIPLE LOGISTIC REGRESSION MODEL FOR exCRTR (FULL

MODEL)

TABLE 5: FITTED MULTIPLE LOGISTIC REGRESSION MODEL FOR exCRTR (FINAL

MODEL)

[0233] Conclusion

[0234] The data on 60 patients with 3 biomarkers demonstrate that the 3- biomarker signature has high accuracy, specificity, and sensitivity rates based on the corrected AUCs generated by the ROC curves. On the Z score curve, patients with pathCR congregated with a positive score. This demonstrates the efficacy of systematic individualization of therapy for patients with esophageal carcinoma, in certain embodiments of the invention.

TABLE 6: PREDICTION OF PATHCR BY INDIVIDUAL MARKERS WITH 40 PATIENTS

[0235] The combination of all three biomarkers demonstrates both a higher degree of sensitivity and a higher degree of specificity than then each biomarker on its own in a much larger sample of patients (FIGS. 13 and 16, Table 6). In conclusion, the data demonstrate that a 3-biomarker signature for SHH, GIi-I, and NF-κB is highly predictive of pathCR and exCRTR in patients with adenocarcinoma of the esophagus who undergo chemoradiation and surgery.

EXAMPLE 4

BIOMARKERS CORRELATE WITH DISEASE-FREE SURVIVAL IN PATIENTS WITH ANAL CANCER TREATED WITH CHEMORADIATION

[0236] Overview [0237] Clinical and biomarker data in 30 patients with anal carcinoma who had chemoradiation were analyzed. Patient selection was based on the availability of untreated cancer for biomarkers, completion of prescribed chemoradiation, and patient outcomes (-50% disease-free) non-representative of published cohorts but conducive to biomarker discovery. Ten biomarkers, Ki67, hTERT, EGFR, p53, pl6, Bcl-2, VEGF, NF-κB, Shh, and GIi-I, were studied. Raw data as continuous variable (only EGFR was trichotomized) were analyzed. Univariate and multivariate Cox models were utilized to assess relationship between DFS and biomarkers.

[0238] Twenty-three of 30 patients were women, tumor diameter was >5cm in 30%, and 37% had clinically positive nodes. Fourteen (47%) patients had a DFS event after chemoradiation. In univariate analysis, NF-κB (p=0.01), SHH (p=0.02), GIi-I (p=0.02), and tumor diameter (0.03) were significantly associated with DFS and Ki67 (p=0.07) was marginally significant. In multivariate analysis, tumor diameter (p=0.003), Ki67 (p=0.005), NF-κB (0.002), SHH (0.02), and GIi-I (0.02) were significantly associated with DFS. The data indicate that several biomarkers (Ki67, NF-κB, SHH, and GIi-I) are associated with DFS.

[0239] Methods and Patients

[0240] Patients with histologically documented localized squamous cell carcinoma of the anal canal cancer were eligible. The clinical and biomarker studies were conducted under a approved protocol.

[0241] Patient selection: Through an established database patients were identified who met the following conditions: (1) availability of sufficient untreated tissue to perform a 10- biomarker analysis, (2) completed prescribed chemoradiation to reduce confounding DFS factors such as: undue treatment interruptions or discontinuation, and (3) selected approximately 50% of patients had a DFS event (persistent or recurrent cancer or death) after chemoradiation. This was done to assure that there were patients in two risk categories for biomarkers to show any difference if it existed, recognizing that the cohort of 30 patients would not be representative of the anal canal carcinoma population in the literature but this approach may be conducive to biomarker discovery. Patients with certain tumor diameter or those who had clinically positive nodes were not selected, however, it turns out that these two parameters match closely with the population of anal canal carcinoma patients at large.

[0242] Pretreatment evaluations: Pretreatment evaluation included complete history, physical examination, proctoscopy, chest radiograph, computerized tomography of abdomen and pelvis, serum chemistry, and complete blood count. Clinical nodal involvement and tumor diameters were documented prior the initiation of chemoradiation.

[0243] Therapy: All patients were treated with concurrent chemoradiation. The median dose of radiation was 55 Gy administered in approximately 6 weeks. There was no provision for a planned treatment break. The details of radiation therapy are provided in a previous report (Das et al., 2007). All patients received concurrent 5-fluorouracil as an infusion and cisplatin (only one patient received induction therapy) as previously described (Das et al, 2007).

[0244] Follow-up: After completion of chemoradiation, patients were examined at least every 2 months until there was assurance that a complete clinical response had occurred or in its absence persistent cancer could be documented. After this phase, patients were followed approximately every 4 months for 2 years and then every 6 months for additional 3 years. Proctoscopy and computerized tomography was performed routinely during these visits. DFS was documented in each patient by reviewing all the records.

[0245] Tissue collection: Residual unstained untreated tissue was procured from 30 patients for this biomarker study.

[0246] Immunihistochemistry: Each tissue block was resectioned to obtain ten 4 μ unstained charged slides and one hematoxylin-and-eosin-stained slide to confirm the presence of tumor. Immunohistochemistry was performed using monoclonal antibodies for EGFR (clone 31G7, Zymed, San Francisco, CA, 1:50 dilution), VEGF (SC-152, Santa Cruz Biotechnology, Santa Cruz, CA, 1:10 dilution), Bcl-2 (100, Biogenex Laboratories Inc, San Ramon, CA, 1:200 dilution), pl6 (16P07, Lab Vision/Neomarkers, Freemont, CA, 1:40 dilution), p53 (DO-7, DAKO, Carpinteria, CA, 1:100 dilution), Ki-67 (MIB-I, DAKO, 1:200 dilution) and nucleolin (nucleolin, Novacastra Vision Biosystems Inc, Norwell, MA, 1:25 dilution). The slides were deparaffinized in xylene, and rehydrated in 100% to 70% ethyl alcohol. The slides were then subjected to heat-induced antigen retrieval in 0.01 mol/L citrate buffer, pH 6.0, preheated to 90 ⁰C and heated in an electric steamer for 45 minutes and cooled to room temperature for 20 minutes. The slides for EGFR immunohistochemistry instead were treated with p24 protease for 1 minute in 1 M Tris-HCl, pH 7.4 with 0.2% Tween 20. The endogenous peroxidase activity was blocked by 3% hydrogen peroxide in absolute methanol for 5 minutes. The secondary antibody was visualized by avidin-biotin system (LS AB2 peroxidase kit, DAKO) using 3-3'- diaminobenzedine as the chromogen, and the slides were counterstained with Mayer hematoxylin. All slides were reviewed by the one pathologist. Staining was graded as percent of tumor cells staining for the marker. For EGFR membranous staining of tumor cells was evaluated and scored in a four-tier system (no, faint, intermediate and intense staining of tumor cells). For VEGF, Bcl-2, and pl6 cytoplasmic staining of tumor cells was evaluated, for Ki-67 and p53 nuclear staining was evaluated, and for nucleolin nucleolus staining was assessed.

[0247] Immunohistochemical staining for NF-κB, SHH, GIi-I protein expression was performed on 4-mm formalin-fixed, paraffin-embedded (FFPE) adjacent sections with the G96-337 (2 mg/ml, BDPharMigen, Palo Alto, CA) H- 160(2 mg/ml, Santa Cruz Biotechnology, CA) and H-330 (4 mg/ml, Santa Cruz Biotechnology, CA) antibodies, respectively. The immunohistochemical procedure, including positive and negative controls were carried out as previously reported (Sims-Mourtada et al., 2006; Sims-Mourtada et al., 2007; Izzo et al., 2006). Only cytoplasm SHH and nuclear NF-κB, and GIi-I immunoreactivity in tumor cells were considered positive for scoring purposes. Staining intensity for SHH was defined as undetectable or detectable. Staining for NF-κB and GIi-I protein was evaluated on a three-point semiquantitative scale as follow: 0, no staining; 1, weak to moderate and 2, strong nuclear staining, whereas cells with staining >1 were considered positive. The extent of positive cancer cells with positive SHH and Glil was then expressed as the fraction of labeled (e.g. staining levels 1 and 2) cells, or labeling index (LI) in the cancers.

[0248] Two investigators independently, in a blind fashion and without knowledge of the clinical data, analyzed protein expression. In the discrepant cases, a final opinion was made based on consensus by all three investigators, and if necessary a recount of the labeled cells was done.

[0249] Statistical methods: Patient characteristics were summarized using median (range) for continuous variable and frequency (percentage) for categorical variables. Univariate and multivariate logistic regression models were fit to assess the association between patient characteristics and biomarkers and response. Overall survival (OS) and Disease-free survival (DFS) were estimated using the method of Kaplan and Meier (Kaplan, 1958), where a DFS event was defined as evidence of persistent or recurrent anal cancer or death. Univariate and multivariate Cox proportional hazards models (Cox, 1972) were used to assess the ability of patient characteristics and biomarkers to predict DFS, with goodness of fit assessed by the Grambsch-Therneau test, Schoenfeld residual plots, and martingale residual plots (Therneau, 2000). Due to the strong associations among several biomarkers, in both the logistic and Cox regression analyses several alterative multivariate models were fit to avoid the co-linearity among biomarkers. All statistical analyses were carried out in Splus (Venables, 1999).

[0250] Results

[0251] Patient characteristics are shown in Table 7. There were 22 women and 8 men. Thirty percent had the diameter of the primary cancer >5 cm and 37% clinical nodal involvement. Table 8 shows the distribution of biomarkers. Except for EGFR, the data represented are in % of staining cells. The % of cell staining was used for a particular biomarker as continuous variable without a pre-specified cut point. The only exception was EGFR, which was trichotamized in the standard manner.

TABLE 7. PATIENT CHARACTERISTICS (N=30)

TABLE 8: DISTRIBUTION OF 10 BIOMARKERS IN 30 PATIENTS WITH ANAL CANCER

[0252] Outcome of the entire population: Complete clinical response was noted in 22 (73%) of patients in the immediate observation period after the completion of chemoradiation. These 22 patients were designated as responders and 8 patients as non-responders. A total of 14 (47%) had a disease relapse or died. All 10 (33%) deaths in this cohort were related to anal carcinoma. The median survival of 30 patients was 10.9 years (95% confidence interval: 7.0 years to not reached). The median DFS time was 7 years (95% confidence interval: 3.7 years to not reached). FIG. 14 shows the DFS curve of the entire population.

[0253] Biomarkers of Response Table 9 shows the univariate logistic regression model for response and Ki67 (p=0.05), VEGF (p=0.05), and T-stage [T3-4 vs. Tl -2] (p=0.02) were significantly correlated and tumor diameter (as a continuous variable) had a marginally significant trend (p=0.06).

TABLE 9: UNIVARIATE LOGISTIC REGRESSION MODEL FOR RESPONSE (N=30).

[0254] Table 10 shows two alternative multivariate logistic regression models for response, due to the correlation between VEGF and Ki67. VEGF (p=0.05) and tumor diameter (P=OM) (Table 10a), or Ki67 (p=0.03) and tumor diameter (p=0.05) (Table 10b) are independent predictors of response. The fitted models suggest that higher VEGF or Ki67 level is associated with a greater probability of response; while larger tumor size is associated with a lower probability of response.

TABLE 10: TWO ALTERNATIVE MULTIVARIATE LOGISTIC REGRESSION MODELS

FOR RESPONSE (N=30).

[0255] Biomarkers of DFS: Table 11 demonstrates the univariate Cox proportional hazards models for DFS. NF-κB (p=0.01), SHH (p=0.02), GIi-I (p=0.02) and tumor size (p=0.03) were significantly associated with DFS. Ki67 demonstrated a marginally significant trend (p=0.07), but p53, hTERT, pl6, Bcl-2, VEGF, EGFR, clinical stage, and gender were not significantly associated with DFS.

TABLE 11: UNIVARIATE COX PROPORTIONAL HAZARDS MODELS FOR DFS (N=30).

[0256] Table 12 shows the multivariate analyses. Again, due to the correlations among the three biomarkers, NF-κB, SHH and GIi-I, three alternative models were fit. These models suggest that tumor size, Ki67, together with NF-κB (0.002), SHH (p=0.02) or GIi-I (p=0.02) were independent predictors of DFS. Patients with large tumor size, high % of cells with NF-KB, SHH or GIi-I tend to have a shorter DFS; while patients with high level of Ki67 tend to have a longer DFS.

TABLE 12: THREE ALTERNATIVE MULTIVARIATE COX PROPORTIONAL HAZARDS

MODELS FOR DFS (N=30).

[0257] Discussion

[0258] The limited number of reports defining the molecular biology of anal carcinoma range from attempts to characterize the process of carcinogenesis to defining prognostic markers. Methylation of several genes (Zhang et al., 2005), human papilloma virus (HPV), p53, and AKT have been implicated in carcinogenesis of anal carcinoma (Patel et al, 2007). Nilsson et al. (2006) have reported the largest attempt to discover the prognostic factors for survival by studying the immunohistochemical expression of p53, p21, Cyclin A and CD 31 in 215 patients with anal carcinoma. The tumor- specific survival (p=0.009) and local regional failure (p=<0.05) was favorably correlated with high Cyclin A expression. In a multivariate Cox analysis, Cyclin A was the only biomarker correlating with tumor- specific survival. In a gene expression profiling study of 55 patients with HPV-positive anal carcinoma by Bruland et al. (2008) found that MCM7 protein (MCM7 gene is induced by HPV) was associated with relapse- free survival (p=0.02) and cancer-specific survival (p=0.03). pl6 (CDKN2A) was not associated with survival. In addition, expression of EGFR (Alvarez et al., 2006), 18q along with other biomarkers have beed described (Gervaz et al, 2004) but have not been correlated with response or outcome.

[0259] Two major challenges face patients with localized anal carcinoma: uniformly toxic effects of chemoradiation with rate of grade 3 or 4 events often reaching above 80% (Ajani et al, 2008) and fear of colostomy. Many patients do have a complete clinical response after primary chemoradiation, however, approximately 20% who have persistent cancer after chemoradiation, could obviously be better off without chemoradiation and all the unpleasant side effects. The prognostic factors that are associated with poor DFS and overall survival have been described but no previous predictive factors for response to therapy existed and there were no known biomarkers associated with DFS. The results indicate that relevant biomarkers predict for response and are prognostic for DFS. Biomarkers were chosen that are associated with response to either to chemotherapy, radiotherapy, or both. In specific embodiments, tumor size is an independent prognostic factor.

[0260] In conclusion, the data on 30 patients with anal carcinoma indicates that SHH, GIi-I, and NF-κB are biomarkers that are predictive variables for DFS. EXAMPLE 5 SHH, GLI-I AND NF-KB ARE MARKERS FOR MULTIPLE TYPES OF CANCER

[0261] To investigate the expression levels of SHH, GIi-I and NF-KB in solid malignancies, a variety of exemplary human cancer cell lines and an animal model of inflammation-induced esophageal adenocarcinoma was studied.

[0262] Experimental Design

[0263] Immunohistochemistry was used to examine the protein expression of SHH, GIi-I and NF-κB in a rat model of Barrett's esophagus with progression to esophageal adenocarcinoma. This is a non-carcinogenic surgery-based animal model, which depends on the entero-esophageal reflux of bile salts into the lower end of esophagus. This animal model closely resembles gastro-esophageal reflux disease dependent development of Barrett's esophagus in human (Buttar et al., 2002).

[0264] The protein expression of key components of the ligand Sonic Hedgehog (SHH), and its transcriptional effector GIi-I were examined by Western Blot on whole cell lysates and supernatants (for SHH only). The proteins were screened for since the mRNA of both SHH and GIi-I can be found in several cancer cell type, however it is not always translated in to proteins. Additionally, SHH is not always processed (from its pro-form of 42 KDa to the secreted form of 19 KDa) and secreted by cells. The basal expression of these proteins was investigated without exogenous stimulation (i.e. without cytotoxic stress). The same lysates were also screened for the presence of NF-KB protein at baseline. A panel of exemplary human cell lines was investigated and included: 10 breast adenocarcinoma, 2 inflammatory breast adenocarcinoma, 15 head and neck squamous cell carcinoma, 5 colo-rectal adenocarcinoma, 8 lung cancer lines, and 2 prostate adenocarcinoma cell lines.

[0265] Results

[0266] Rat model of esophageal adenocarcinoma. SHH ligand expression was detected in 2/7 (28.5%) esophagitis lesions, the activation of the network (defined by the nuclear localization of GIi-I) was seen in 4/5 (80%) of the BE and 7/7 (100%) of the invasive esophageal adenocarcinoma. FIG. 15 contains examples of SHH and GIi-I expression by IHC in this animal model. Thus, aberrant expression NF-κB can be found in esophageal adenocarcinoma developed in an experimental rat animal model of Barrett's esophagus induced by Bile salt reflux.

TABLE 13: HUMAN CANCER CELL LINES

[0267] In the table above: 1, IBC, is Inflammatory Breast Cancer; 2, HNSCC is Head&Neck Squamous Cell Carcinoma; 3, CRC, is ColoRectal Cancer; 4, NSCLC, is Non small Cell Lung Cancer; 5, SHH intra, is intracellular and SHH, Seer, is secreted in the media; 6, Concomitant expression, is the presence of expression of all 3 proteins in the same whole cell lysate.

[0268] Western blots were duplicated for all protein expression. Actin protein expression was used as internal positive control for each cell line; the SEGl cell line, expressing constitutive SHH and GIi-I positive was used as positive control in the blots; LNCap and PC3, prostate cancer cell lines, were used as negative controls for SHH and GIi-I, because at baseline they do not express detectable amount of neither proteins.

TABLE 14: SUMMARY OF RESULTS

[0269] The numbers in parenthesis above are percentages. 1, IBC, is Inflammatory Breast Cancer; 2, HNSCC, is Head&Neck Squamous Cell Carcinoma; 3, CRC, is ColoRectal Cancer; and 4, NSCLC, is Non small Cell Lung Cancer.

[0270] The screening indicates that the SHH signaling pathway is expressed in a wide variety of human cell lines derived from solid malignancies. In this study, the presence of SHH and GIi-I protein expressions in baseline cell growth conditions was investigated.

[0271] Further studies characterize a specific embodiment of the invention wherein the pathway is constitutively active (deregulated), in addition to characterizing exposure of the cells to cytotoxic conditions induces sustained activation also in cells that have not shown baseline expression. Additional studies to assess the activation status of NF-κB, defined by its binding to DNA, with regard to SHH expression and activation are also performed.

[0272] In conclusion, the three biomarker signature is predictive of sensitivity or resistance to chemotherapy and/or radiation therapy in a variety of different types of cancer. EXAMPLE 6 AUTOMATION [0273] Overview

[0274] The first critical step in developing biomarkers with potential widespread utilization is the establishment of robust standardized staining assays in a CLIA environment. Thus, the primary goal of this study is to transfer optimized and reproducible IHC assays from the research laboratory into a CLIA certified. The second aspect is to develop automated scoring procedures to establish a precise, objective, and reproducible methods of interpretation.

[0275] IHC Standardization

[0276] General Approach: IHC assays achieving >80% technical reproducibility is transferred to an institutional CLIA certified IHC laboratory for standardization (i.e. IHC technique and scoring) and routine testing. After standardization, the CLIA laboratory proceeds with markers immunostaining in 90 (>50%) tissue specimens randomly chosen from 175 tumor specimens currently available. The IHC assays for all three NF-κB, SHH and Glil biomarkers are transferred for standardization.

[0277] Methods: Transfer of IHC and Standardization of Immunostaining Conditions: The research, RHL and CLIA laboratories closely collaborate to optimize the conditions for standardization. Standard assay format optimization is done by modifying reagents and conditions. For each IHC biomarker the stepwise procedures is as follows: 1. Identification of FFPE tissues that can serve as positive and negative controls. In previous experiments, human cancer cell lines served as positive and negative, tissue may also be used. 2. Immunolabeling of the identified FFPE tissue controls using the previously optimized IHC conditions (Sims- Mourtada et al, 2006; Sims-Mourtada et al, 2007; Izzo et al, 2006. A slide of the positive/negative control cell lines is also be included in the IHC batch. CLIA laboratory proceeds using a fully automated staining system (either the DAKO Autostainer Universal Staining System or the BioGenex Automated System). Quality is assessed and efficiency of staining and of which 25% is independently and blindly reviewed of randomly chosen slides for further assessments. Under guidance, the CLIA laboratory proceeds to further optimize and standardize the procedure. This may include: (a) changes in the antigen retrieval step using different reagents (up to 4) and times of retrieval; (b) assessment of the efficiency of the retrieval procedure on tissues fixed in different chronology and with different fixatives, (b) titration of the primary antibody (up to 10 dilutions); and (c) changes in the visualization step. Once the IHC conditions are standardized, the CLIA laboratory stains 90 untreated cancer specimens, randomly selected from the 175 samples studied in previous experiments. Positive and negative tissue controls are either placed on the same slide of the target tissue or a slide of each is included with each batch of staining. For cross-comparison, 45 FFPE specimens, randomly chosen, are run in parallel in the research laboratory using as controls the newly established tissue controls. The CCC is used to evaluate the reproducibility between the two laboratories and the reproducibility is deemed as excellent if the one-sided 95% lower confidence limit is at least about 0.8.

[0278] Assessment of Immunolabeling and Scoring Procedures: Quality control procedures for assessing "scorability" of each immunolabeled cancer specimen is performed as described in previous experiments. Each slide is scored in an independent and blinded fashion by two different people of ordinary skill in the art and reading are compared. Blind reviews of 25% of randomly chosen slides are also performed. The CCC is used to assess scoring agreement and IHC reproducibility following a similar procedure as described in SAl.

[0279] Establishment of automated scoring procedures

[0280] General Approach: Along with standardization of immunostaining techniques, establishment of automated scoring procedures is performed allowing minimization of inter-observer variability and to develop objective, consistent evaluation of the predictive/prognostic markers. Recent advances in imaging technology and automation have provided high-resolution investigative tools permitting objective, and reliable view of specimens amenable to the generation of quantifiable results. Some of the newly developed imaging systems, such as the Ariol SL-50, generate images and data that can be integrated seamlessly, through system networking, into existing third-party laboratory, thus allowing multiple independent case review without huddling around the microscope. This example review routine techniques used to develop and establish new automated scoring procedures which is not a substitute for the standardized manual scoring protocols described previously. The high- resolution Applied Imaging Ariol SL-50 system is used for IHC automated scoring. The Ariol SL-50 is a high throughput automated image analysis system approved by the FDA for in vitro diagnostic use of Her-2/neu, ER and PR IHC in breast cancer. The system is designed to carry out frame-by-frame image capture of the tissue slides using a high-resolution charge-coupled- devise camera, optimized for low-light-sensitive imaging across the spectra, and which works with a high-performance Olympus upright BX-61 microscope (Olympus America, Inc.). This fully automated system allows a user to: scan slides and obtain a seamless stitch of the images for virtual view of the whole tissue at high resolution; (2) measure color, intensity, and morphology in nuclear, cytoplasmic, and membranous structures; (3) link sequential tissue slices for simultaneous analysis and automatic selection of analysis regions across linked slides; (4) share digitized images and review of the samples.

[0281] Methods

[0282] An automated scoring protocol is developed for all the IHC biomarkers transferred to the CLIA laboratory. For all biomarkers, the procedure includes:

[0283] (1) scanning and digitization of all reference H&E slides;

[0284] (2) selection of 60 specimens for SHH, NF-κB, and GIi-I each from the pool of specimens stained in previous examples.

[0285] The general procedure takes advantage of two specialized functions performed by the Ariol system, (a) the trainable classifier for automated size and shape recognition allowing selection of specified nuclear, cytoplasmic, or membranous morphology, and (b) the quantification of color intensity. Thus, the system is first "trained" to recognize brown staining only in cells with the morphology of tumor cells. Then, three consecutive scan- pass are performed, at low-resolution to recognize the location of the tissue, at 5x magnification to obtain the overview image which is utilized to select the cancer regions of interest manually by a pathologist, and at 2Ox magnification to obtain a high-resolution image, which is used by the system to calculate the percentage of positive cells based on intensity of staining and nuclear/cytoplasmic or membranous morphology. In addition, the reproducibility between automated scoring based on the selection of the regions of interest by a pathologist and programming the Ariol system is assessed to automatically select the regions. The CCC is used to assess scoring agreement between manual and automatic scoring, or "guided" and "programmed" automatic scoring. The scoring agreement is accepted if the lower bound of onesided 95% lower confidence limit is at least 0.8 (i.e. CCC of 0.87 for a total of 60 cases).

EXAMPLE 7 COMPUTER-DRIVEN IMAGE ANALYSIS

[0286] Biomarker studies in human specimens are done using computer-driven image analysis. Over the past decade, a number of diverse computer-driven image analysis systems have been used to spatially visualize, and measure biomarkers in formalin-fixed tissue sections. In an earlier study, the spatial distribution of cyclin Dl (CDl) protein expression (IHC) and gene copy numbers (FISH) and chromosome 9 numbers (FISH) was analyzed in upper- aerodigestive tract (UADT) epithelia adjacent to cancer, using the Magiscan Image Analysis System attached to a Nikon microscope with a controller-driven stage. Using a light pen, each scored cells was first recorded for positive or negative staining or number of copies and its relative spatial x and y coordinates in the epithelium. Subsequently, using a "in house software" a topological map of CDl expression, gene and chromosome copy numbers was constructed (from independently captured fields) displaying the results as a two-dimensional color map where the degree of expression, the gene copy number or the chromosome copy numbers can be represented by different colors (Izzo et al, 1998; Izzo et al, 1998b; Izzo et al, 1999; Izzo et al, 2001; Izzo et al, 2003). Illustrations may be made using this type of spatial analysis, where dots represent the location of each cell and the color represent its characteristics (i.e. normal (cyan) or altered (red) cyclin Dl expression). Using this unique approach, it was reported that CDl dysregulation is an early event in UADT tumorigenesis, as it occurs already in premalignant lesions, associated with increased chromosome instability and subsequent gene amplification (Izzo et al., 1998; Izzo et al., 1998b). Next a nearest neighbor average analysis was carried out on the epithelia using S-Plus graphical statistics software and a new genetic map of the epithelium scribing the spatial pattern of average chromosome copy numbers was derived. If a group of cells showed one-half the normal chromosome copies per cell, the cell would be labeled part of a monosomic clone. Conversely, if a group of cells showed three-halves or twice the normal chromosome copies per cells (e.g. three or four instead of two), the cell was labeled part of a trisomic or tetrasomic clone, respectively. Using this approach, it was shown that the increase in copy number in those lesions with both increased CDl expression and 9p21 loss was associated with outgrowth of tetraploid clone. Recent studies measured relative expression of cytoplasmic (cortactin, β-catenin) and membranous (E-cadherin) using the SimplePCI (Compix, Inc., Cranberry Town, PA) system controlling a Nikon Optiphot microscope equipped with a motorized stage and a 3 CCD color video camera (den Hollander et al, 2001). After capture, the digitized field-by-field seamless montage images representing the whole tissue were used to measure relative protein expression using the MetaMorph software (Universal Imaging Corporation, Downington, PA). Regions of interest were circled and further characterized for their total integrated optical intensity, area (in pixels) and the relative coordinates of their center of gravity, each region of interest measured was characterized for both staining intensity and its relative position in the tissue, we were able to construct topological maps of protein expression in the tissue wherein the levels of Cortactin, β- catenin and E-cadherin were displayed on a relative pseudocolor scale.

[0287] These data demonstrated the ability of those of ordinary skill in the art to perform biomarkers studies in fixed tissues using a diverse image analysis systems. Studies using the 3-biomarker signature on cells are done to create a reliable computational method to determine the levels and/or localization of SHH, GIi-I and/or NF-κB.

[0288] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

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Claims

CLAIMSWhat is claimed is:

1. A in vitro method of predicting a response to chemotherapy and/or radiation therapy for an individual having cancer comprising assessing the levels of deregulated SHH, GIi-I, and NF-κB proteins in cancer cells of the individual, wherein a higher level of deregulated SHH, GIi-I and NF-κB proteins in said cells, relative to a reference, is indicative that the individual's cancer is predisposed to be resistant to chemotherapy and/or radiation therapy, and lower or essentially equal levels of deregulated SHH, GIi-I and NF-κB proteins relative to said reference is indicative that the individual's cancer is predisposed to be sensitive to chemotherapy and/or radiation therapy.

2. The method of claim 1, wherein assessing the levels the of the deregulated of SHH, GIi-I, and NF-κB proteins comprises determining the levels of SHH in the cytoplasm and determining the levels of GIi-I and NF-κB proteins in the nucleus.

3. The method of claim 1, wherein the reference comprises positive and/or negative controls.

4. The method of claim 3, wherein the positive control comprises cells that are known to be resistant to chemotherapy and/or radiation therapy.

5. The method of claim 4, wherein the negative control comprises cells that are known to sensitive to chemotherapy and/or radiation therapy.

6. The method of claim 5, wherein the negative control comprises non-cancerous cells of the individual with cancer.

7. The method of claim 3, wherein the control comprises cells that are not from the individual with cancer.

8. The method of claim 1, wherein the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, urogenital cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, an endocrine or neuroendocrine cancer or hematopoietic cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, renal cancer, biliary cancer, pheochromocytoma, pancreatic islet cell cancer, Li- Fraumeni tumors, thyroid cancer, parathyroid cancer, pituitary tumors, adrenal gland tumors, osteogenic sarcoma tumors, multiple neuroendrcine type I and type II tumors, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, or skin cancer.

9. The method of claim 8, wherein the cancer is inflammatory breast cancer, head and neck cancer, colorectal cancer, lung cancer, anal cancer or esophageal cancer.

10. The method of claim 9, wherein the cancer is anal cancer or esophageal cancer.

11. The method of claim 1 , wherein the chemotherapy is 5- fluorouracil, mitomycin, cisplatin, taxane, camptothecin, irinotecan, and/or a platinum compound.

12. The method of claim 1, wherein the levels of deregulated of SHH, GIi-I, and NF-κB proteins are assessed by immunohistochemistry.

13. The method of claim 1, wherein when the individual is predisposed to be resistant to chemotherapy and/or radiation therapy, the individual is treated with surgery.

14. The method of claim 1, wherein when the individual is predisposed to be sensitive to chemotherapy and/or radiation therapy, the individual is treated with chemotherapy and/or radiation therapy.

15. The method of claim 1, wherein said cancer cells are paraffin- embedded.

16. The method of claim 1, wherein said reference is a z score.

17. The method of claim 1, wherein the z- score was determined from a set of individuals with known outcomes.

18. A kit comprising suitably aliquoted antibodies to detect levels of SHH, GIi-I, and NF-κB protein, respectively, wherein said kit is housed in a suitable container.

19. The kit of claim 16, wherein at least one of the antibodies is a monoclonal antibody.

20. The kit of claim 16, wherein at least one of the antibodies is a polyclonal antibody.

21. The kit of claim 16, wherein any of the antibodies are chimeric, bispecific, trispecific, recombinant or engineered.

22. The kit of claim 16, wherein the kit is configured to be used to assess the levels of deregulated of SHH, GIi-I, and NF-κB in cells.