WO2019178215A1 - Methods and compositions for treating, prognosing, and diagnosing esophageal cancer - Google Patents

Abstract

Methods and compositions are provided using the level of F. nucleatum in cancer patients. In certain embodiments, the levels of F. nucleatum are used to evaluate esophageal cancer patients and the risk of relapse.

Description

METHODS AND COMPOSITIONS FOR TREATING, PROGNOSING, AND

DIAGNOSING ESOPHAGEAL CANCER DESCRIPTION [0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No.62/642,392, filed March 13, 2018, hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION 1. Field of the Invention

[0002] The present invention relates generally to the fields of molecular biology and oncology. More particularly, it concerns methods and compositions involving biomarkers and cancer prognosis, diagnosis, and treatment.

2. Description of Related Art

[0003] Esophageal cancer is the sixth most common cause of cancer-related deaths worldwide. In spite of advances in multimodal therapies, including surgical removal of tumors, chemotherapy, radiotherapy and chemoradiotherapy, esophageal cancer remains a malignancy with high degree of fatality and overall 5-year survival rates of 15 to 20%. Globally, esophageal squamous cell cancer (ESCC) is the predominant histological subtype of esophageal cancer. In particular, ESCC cases make up to 80% of all esophageal cancers in developing countries. The standard treatment strategy for locally advanced esophageal cancer in western and Asian countries comprise of neoadjuvant chemoradiotherapy or chemotherapy (NAC), followed by surgery. Previous studies have shown that patients who respond well to NAC often exhibit improved overall survival. A combination of cisplatin and 5-fluorouracil (5-FU) is currently used as standard chemotherapeutic regimen for esophageal cancer patients; however, the reported response rates remain relatively poor. A recent study reported that addition of docetaxel to this regimen significantly improved the therapeutic response in patients with node- positive esophageal cancer. Nevertheless, most tumors acquire resistance to chemotherapeutic agents with subsequent treatment failure. Furthermore, there is currently no effective molecularly-targeted therapy available for esophageal cancer, and the efficacy of immunotherapy in these patients remains unclear. There is a need in the art for improved therapeutic methods to modify chemoresistance in patients. SUMMARY OF THE INVENTION

[0004] Methods and compositions are provided related to level of Fusobacterium nucleatum (F. nucleatum ) in cancer patients, particularly esophageal or colorectal cancer patients. Methods include one or more steps involving the level of F. nucleatum and compositions or kits.

[0005] Methods include but are not limited to the following: methods for measuring the level of F. nucleatum , methods for evaluating the level of F. nucleatum , methods for detecting the level of F. nucleatum , methods for quantifying the level of F. nucleatum , methods for comparing the level of F. nucleatum , methods for evaluating a esophageal cancer patient for recurrence of esophageal cancer, methods of identifying a patience at risk for recurrence of esophageal cancer, methods for measuring increased the level of F. nucleatum as compared to representative levels of F. nucleatum in patients who do not experience recurrence of esophageal cancer, methods for measuring levels of F. nucleatum that are representative of levels of F. nucleatum in patients who do not experience recurrence of esophageal cancer, methods for measuring decreased levels of F. nucleatum as compared to representative levels of F. nucleatum in patients who experience recurrence of esophageal cancer, methods of treating a patient for esophageal cancer recurrence, methods of treating a patient at risk for recurrence of esophageal cancer, methods of treating a patient for esophageal cancer after the patient was previously treated for esophageal cancer, and methods of administering a esophageal cancer treatment to a patient.

[0006] Steps for implementing any of the methods above or disclosed elsewhere in the application may include, but are not limited to, measuring the level of F. nucleatum , measuring the level of F. nucleatum , quantifying the level of F. nucleatum , determining the level of F. nucleatum, comparing the level of F. nucleatum to the level of F. nucleatum in a control, determining a risk score for recurrence, calculating a risk score for recurrence, evaluating the patient’s risk for recurrence, identifying the patient as at risk for esophageal cancer, diagnosing or prognosing the patient as having an increased risk for esophageal cancer recurrence, treating the patient for esophageal cancer, not treating the patient for esophageal cancer, and/or treating the patient as being at risk for esophageal cancer recurrence.

[0007] In some embodiments, there are methods comprising measuring the number of F. nucleatum in a biological sample from an esophageal or colorectal cancer patient. In some embodiments, the patient is treated after measuring the number of F. nucleatum in the biological sample. In some embodiments, a low number of F. nucleatum is measured as compared to a control level or sample. In some embodiments, a high number of F. nucleatum is measured as compared to a control level or sample.

[0008] In some embodiments, there are methods for predicting length of relapse free survival in an esophageal cancer patient comprising measuring the number of F. nucleatum in a biological sample from the patient, wherein a patient with a low number of F. nucleatum is predicted to have a longer time of relapse free survival than a patient with a high number of F. nucleatum. In some embodiments, methods further comprise administering chemotherapy to the patient after measuring the number of F. nucleatum in the biological sample. In some embodiments, methods further comprise administering one or more antibiotic(s) for F. nucleatum. In some embodiments, methods further comprise measuring the number of F. nucleatum in a sample from the patient after antibiotic(s) have been administered to the patient. In some embodiments, a low number of F. nucleatum is measured as compared to a control level or sample. In some embodiments, a high number of F. nucleatum is measured as compared to a control level or sample.

[0009] In some embodiments, there are methods for treating a patient with a gastrointestinal cancer comprising administering one or more antibiotics to the patient prior to administering chemotherapy to the patient, wherein a biological sample from the patient has been measured for F. nucleatum. In some embodiments, methods further comprise administering chemotherapy to the patient after measuring the number of F. nucleatum in the biological sample. In some embodiments, methods further comprise administering one or more antibiotic(s) for F. nucleatum. In some embodiments, methods further comprise measuring the number of F. nucleatum in a sample from the patient after antibiotic(s) have been administered to the patient. In some embodiments, a low number of F. nucleatum is measured as compared to a control level or sample. In some embodiments, a high number of F. nucleatum is measured as compared to a control level or sample.

[0010] In some embodiments, there are methods for increasing the time of relapse-free survival in an esophageal or colorectal cancer patient comprising measuring the number of F. nucleatum in a biological sample from the patient, administering an antibiotic to reduce the number of F. nucleatum, and administering chemotherapy to the patient, wherein the patient has increased length of time of relapse-free survival. In some embodiments, methods further comprise measuring the number of F. nucleatum in a sample from the patient after antibiotic(s) have been administered to the patient. In some embodiments, a low number of F. nucleatum [0011] In some embodiments, there are methods for treating a patient with a esophageal cancer comprising administering one or more antibiotics against F. nucleatum to the patient prior to administering chemotherapy to the patient, wherein a tumor sample from the patient has been measured for F. nucleatum. In some embodiments, methods further comprise administering chemotherapy to the patient after measuring the number of F. nucleatum in the biological sample. In some embodiments, methods further comprise administering one or more antibiotic(s) for F. nucleatum. In some embodiments, methods further comprise measuring the number of F. nucleatum in a sample from the patient after antibiotic(s) have been administered to the patient. In some embodiments, a low number of F. nucleatum is measured as compared to a control level or sample.

[0012] In some embodiments, a high number of F. nucleatum is measured as compared to a control level or sample.

[0013] Methods may also include a step of comparing a level of F. nucleatum to the level of F. nucleatum in a control sample or to a control level of F. nucleatum. In some embodiments, the control sample is representative of the number of F. nucleatum in a cohort of esophageal cancer patients with increased relapse-free survival or the control level of F. nucleatum or reference level of F. nucleatum is a range of F. nucleatum levels from a cohort of esophageal cancer patients with increased relapse-free survival. In some embodiments, the control sample is representative of the number of F. nucleatum in a cohort of esophageal cancer patients with decreased relapse-free survival or the control level of F. nucleatum or reference level of F. nucleatum is a range of F. nucleatum levels from a cohort of esophageal cancer patients with decreased relapse-free survival.

[0014] In some embodiments, methods concern measuring, quantifying, detecting, or determining the F. nucleatum level. In some embodiments, the F. nucleatum level is measured, quantified, detected, and/or determined. In some embodiments, a low number of F. nucleatum is measured as compared to a control level or sample. In some embodiments, a high number of F. nucleatum is measured as compared to a control level or sample.

[0015] It is specifically contemplated that a cohort comprises at least or at most 50, 100, 200, 300, 400, 500 or more patients (or any range derivable therein).

[0016] In some embodiments, the biological sample was a esophageal tissue sample, a blood sample, a stool sample, a saliva sample, a tumor sample, or other tissue sample. In some embodiments, the biological sample comprised tumor tissue. In some embodiments, the [0017] In further embodiments, the size of one or more esophageal tumors from the patient is known or considered. In some embodiments, the lymph node metastasis status of the patient is known or considered. Some methods comprise evaluating tumor size and/or evaluating lymph node metastasis status.

[0018] In some embodiments, methods include calculating a risk score of recurrence for the patient. The risk score is used to categorize the likelihood a patience will experience recurrence within 3, 6, 9, 12 months and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more of primary cancer treatment. The likelihood can be expressed in any way, such as a chance percentage of, for example, 10, 20, 30, 40, 50, 60, 70, 80, or 90% chance of detecting recurrence with a specified time period. In some embodiments, the risk score is determined based on comparing the level of F. nucleatum to a positive or negative control, wherein the positive control is a level of F. nucleatum representative of a cohort of esophageal cancer patients with decreased relapse-free survival and the negative control is a level of F. nucleatum representative of a cohort of esophageal cancer patients with increased relapse-free survival. In some embodiments, one or more F. nucleatum levels is compared to a control level or representative level indicative of increased or decreased relapse-free survival. The comparison may be done with the F. nucleatum level determined from a control sample that is similarly processed as the test sample or it may be done with a previously determined representative or threshold level of F. nucleatum that corresponds to increased or decreased relapse-free survival.

[0019] In some embodiments, methods include treating the patient for esophageal cancer after evaluating or measuring the level of F. nucleatum. This may or may not be done in conjunction with calculating a risk score for the patient. It may depend on a particular classifier that is used to evaluate the level of F. nucleatum and increased or decreased relapse-free survival.

[0020] In some embodiments, treating the patient for esophageal cancer comprises administering a platinum-based chemotherapeutic agent and/or a 5-fluorouracil-based chemotherapeutic agent. In some embodiments, the patient is treated with both a platinum- based chemotherapeutic agent and a 5-fluorouracil-based chemotherapeutic agent. In some embodiments, the patient is treated with 5-fluorouracil or a 5-FU-based compound and cisplatin or other platinum-based compound. In some embodiments, the patient is treated with docetaxel or other taxel-based compound. In some embodiments, treating the patient for esophageal cancer comprises administering an antibiotic for F. nucleatum. In some embodiments, the sulbactam, ampicillin, ertapenem, imipenem, metropenem, clindamycin, cefoxitin, salts thereof, and/or combinations thereof.

[0021] In some embodiments, the patient has previously undergone surgery to remove an esophageal tumor. In some embodiments, the patient has or had stage II or stage III esophageal cancer. In some embodiments, methods comprise calculating a risk score of recurrence for the patient based on the level of F. nucleatum, tumor size, and lymph node status. In some embodiments, the risk score identifies the patient as having a risk of recurrence that is or is at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or higher percentage of having recurrence within a certain time period, such as within 3, 6, 9, 12 months and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years. In some embodiments, methods comprise performing positron emission tomography on the patient to evaluate any tumor.

[0022] In some embodiments, the kit further comprises one or more agents for measuring or detecting or quantifying the level of F. nucleatum or for detecting one or more controls. In some embodiments, the kit further comprises reagents for isolating nucleic acids from a biological sample. In some embodiments, the reagents are for isolating nucleic acids from a serum sample. In some embodiments, the reagents are for isolating nucleic acids from a sample described herein.

[0023] The term subject or patient may refer to an animal (for example a mammal), including but not limited to humans, non-human primates, rodents, dogs, or pigs. The methods of obtaining provided herein include methods of biopsy such as fine needle aspiration, core needle biopsy, vacuum assisted biopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy or skin biopsy.

[0024] In certain embodiments the sample is obtained from a biopsy from esophageal, stomach or the muscle tissue, mucosa or submucosa thereof. In other embodiments the sample may be obtained from any of the tissues provided herein that include but are not limited to gall bladder, skin, heart, lung, breast, pancreas, liver, muscle, kidney, smooth muscle, bladder, intestine, brain, prostate, or thyroid tissue.

[0025] Alternatively, the sample may include but not be limited to blood, serum, sweat, hair follicle, buccal tissue, tears, menses, urine, feces, or saliva. In particular embodiments, the sample may be a tissue sample, a whole blood sample, a urine sample, a saliva sample, a serum sample, a plasma sample or a fecal sample.

[0026] In certain aspects the sample is obtained from cystic fluid or fluid derived from a nurse or medical technician may obtain a biological sample for testing. In further aspects of the current methods, the patient or subject may obtain a biological sample for testing without the assistance of a medical professional, such as obtaining a whole blood sample, a urine sample, a fecal sample, a buccal sample, or a saliva sample.

[0027] In further embodiments, the sample may be a fresh, frozen or preserved sample or a fine needle aspirate. In particular embodiments, the sample is a formalin-fixed, paraffin- embedded (FFPE) sample. An acquired sample may be placed in short term or long term storage by placing in a suitable medium, excipient, solution, or container. In certain cases storage may require keeping the sample in a refrigerated, or frozen environment. The sample may be quickly frozen prior to storage in a frozen environment. In certain instances the frozen sample may be contacted with a suitable cryopreservation medium or compound. Examples of cryopreservation mediums or compounds include but are not limited to: glycerol, ethylene glycol, sucrose, or glucose.

[0028] Some embodiments further involve isolating nucleic acids such as ribonucleic or RNA from a biological sample or in a sample of the patient. Other steps may or may not include amplifying a nucleic acid in a sample and/or hybridizing one or more probes to an amplified or non-amplified nucleic acid. The methods may further comprise assaying nucleic acids in a sample. In certain embodiments, a microarray may be used to measure or assay the level of biomarker expression in a sample. The methods may further comprise recording the biomarker expression level in a tangible medium or reporting the expression level to the patient, a health care payer, a physician, an insurance agent, or an electronic system.

[0029] A difference between or among weighted coefficients ore expression levels or between or among the weighted comparisons may be, be at least or be at most about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0.19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000 times or–fold (or any range derivable therein).

[0030] In some embodiments, determination of calculation of a diagnostic, prognostic, or risk score is performed by applying classification algorithms based on the expression values of biomarkers with differential expression p values of about, between about, or at most about 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.020, 0.021, 0.022, 0.023, 0.024, 0.025, 0.026, 0.027, 0.028, 0.029, 0.03, 0.031, 0.032, 0.033, 0.034, 0.035, 0.036, 0.037, 0.038, 0.039, 0.040, 0.041, 0.042, 0.043, 0.044, 0.045, 0.046, 0.047, 0.048, 0.049, 0.050, 0.051, 0.052, 0.053, 0.054, 0.055, 0.056, 0.057, 0.058, 0.059, 0.060, 0.061, 0.062, 0.063, 0.064, 0.065, 0.066, 0.067, 0.068, 0.069, 0.070, 0.071, 0.072, 0.073, 0.074, 0.075, 0.076, 0.077, 0.078, 0.079, 0.080, 0.081, 0.082, 0.083, 0.084, 0.085, 0.086, 0.087, 0.088, 0.089, 0.090, 0.091, 0.092, 0.093, 0.094, 0.095, 0.096, 0.097, 0.098, 0.099, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or higher (or any range derivable therein). In certain embodiments, the prognosis score is calculated using one or more statistically significantly differentially expressed biomarkers (either individually or as difference pairs).

[0031] Any of the methods described herein may be implemented on tangible computer- readable medium comprising computer-readable code that, when executed by a computer, causes the computer to perform one or more operations. In some embodiments, there is a tangible computer-readable medium comprising computer-readable code that, when executed by a computer, causes the computer to perform operations comprising: a) receiving information corresponding to an expression level of a biomarkers in a sample from a patient; and b) determining a difference value in the expression levels using the information corresponding to the expression levels in the sample compared to a control or reference expression level for the gene.

[0032] In other aspects, tangible computer-readable medium further comprise computer- readable code that, when executed by a computer, causes the computer to perform one or more additional operations comprising making recommendations comprising: wherein the patient in the step a) is under or after a first treatment for cancer, administering the same treatment as the first treatment to the patient if the patient does not have increased expression level; administering a different treatment from the first treatment to the patient if the patient has [0033] In some embodiments, receiving information comprises receiving from a tangible data storage device information corresponding to the expression levels from a tangible storage device. In additional embodiments the medium further comprises computer-readable code that, when executed by a computer, causes the computer to perform one or more additional operations comprising: sending information corresponding to the difference value to a tangible data storage device, calculating a prognosis score for the patient, treating the patient with a traditional therapy if the patient does not have expression levels, and/or or treating the patient with an alternative esophageal therapy if the patient has increased expression levels.

[0034] The tangible, computer-readable medium further comprise computer-readable code that, when executed by a computer, causes the computer to perform one or more additional operations comprising calculating a prognosis score for the patient. The operations may further comprise making recommendations comprising: administering a treatment to a patient that is determined to have a decreased expression level.

[0035] As used herein, the terms“or” and“and/or” are utilized to describe multiple components in combination or exclusive of one another. For example,“x, y, and/or z” can refer to“x” alone,“y” alone,“z” alone,“x, y, and z,”“(x and y) or z,”“x or (y and z),” or“x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an

.

[0036] Throughout this application, the term“about” is used according to its plain and ordinary meaning in the area of cell biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0037] The term“comprising,” which is synonymous with“including,”“containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The phrase“consisting of” excludes any element, step, or ingredient not specified. The phrase“consisting essentially of” limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments described in the context of the term “comprising” may also be implemented in the context of the term“consisting of” or“consisting essentially of.”

[0038] It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the embodiments that may be implemented in the context of embodiments discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary of Invention, Detailed Description of the Embodiments, Claims, and description of Figure Legends. BRIEF DESCRIPTION OF THE DRAWINGS [0039] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0040] FIG. 1A-D. F. nucleatum expression in patients with ESCC. (A) The relative expression of F. nucleatum in 48 pairs of ESCC and adjacent normal tissue, and (B) in 344 ESCC tissue according to T factor in training cohort. (C) The relative amount of F. nucleatum in 45 pairs of ESCC and adjacent normal tissue, and (D) in 205 ESCC tissue according to T factor in validation cohort.

[0041] FIG.2A-D. High intratumoral F. nucleatum is associated with worse prognosis in ESCC. Kaplan-Meier analysis of RFS for ESCC patients with high (red) or low (blue) intratumoral F. nucleatum levels in (A) the training and (B) the validation cohort. Kaplan- Meier analysis of RFS for ESCC patients with low intratumoral F. nucleatum levels in T1 (blue) or T2-4 tumor (pink), or high intratumoral F. nucleatum levels in T1 (red) or T2-4 tumor (green) in (C) the training and (D) the validation cohort.

[0042] FIG.3A-F. Intratumoral F. nucleatum levels are associated with chemotherapeutic response. Chemotherapeutic response and the rate of responder comparing F. nucleatum high (red) and low (blue) patients using RECIST (A and B), PET-CT (C and D) and Tumor Regression Grade (E and F). * p<0.05; ** p<0.01, *** p<0.001

[0043] FIG.4. Overview of the study design

[0044] FIG. 5A-B. F. nucleatum is associated with ESCC recurrence. Comparison of F. nucleatum expression in patients with or without recurrence in (A) the training and (B) the validation cohort.

[0045] FIG.6A-B. T factors in ESCC is not associated with patient survival. Kaplan-Meier analysis of RFS for ESCC patients with T2 - 4 (red) or T1 (blue) factors in the training cohort (A) and the validation cohort (B). DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS [0046] Certain aspects of the invention provide a test that could assist physicians to select the optimal therapy for a patient from several alternative treatment options. A major clinical challenge in cancer treatment is to identify the subset of patients who will benefit from a therapeutic regimen, both in metastatic and adjuvant settings. The number of anti-cancer drugs and multi-drug combinations has increased substantially in the past decade, however, treatments continue to be applied empirically using a trial-and-error approach. Here methods and compositions are provided to diagnose patients to determine the optimal treatment option for cancer patients.

I. Definitions

[0047] The term“substantially the same”,“not significantly different”, or“within the range” refers to a level of expression that is not significantly different than what it is compared to. Alternatively, or in conjunction, the term substantially the same refers to a level of expression that is less than 2, 1.5, or 1.25 fold different than the expression level it is compared to or less than 20, 15, 10, or 5% difference in expression.

[0048] By“subject” or“patient” is meant any single subject for which therapy is desired, including humans, cattle, dogs, guinea pigs, rabbits, chickens, and so on. Also intended to be included as a subject are any subjects involved in clinical research trials not showing any clinical sign of disease, or subjects involved in epidemiological studies, or subjects used as controls.

[0049] 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.

[0050] As used herein,“increased expression” or“elevated expression” or“decreased expression” refers to an expression level of a biomarker in the subject’s sample as compared to a reference level representing the same biomarker or a different biomarker. In certain aspects, the reference level may be a reference level of expression from a non-cancerous tissue from the same subject. Alternatively, the reference level may be a reference level of expression from a different subject or group of subjects. For example, the reference level of expression may be of a subject or group of subjects with cancer. The reference level may be a single value or may be a range of values. The reference level of expression can be determined using any method known to those of ordinary skill in the art. In some embodiments, the reference level is an average level of expression determined from a cohort of subjects with cancer or without cancer. The reference level may also be depicted graphically as an area on a graph. In certain embodiments, a reference level is a normalized level, while in other embodiments, it may be a level that is not stable with respect to the tissue or biological sample being tested.

[0051] The term“determining” or“evaluating” as used herein may refer to measuring, quantitating, or quantifying (either qualitatively or quantitatively).

II. Esophageal Cancer Staging and Treatments

[0052] Methods and compositions may be provided for treating esophageal cancer with particular applications of microbial expression. Based on a biomarker or microbiome profile, different treatments may be prescribed or recommended for different cancer patients.

[0053] Esophageal cancer, also called esophagus cancer, begins in the cells that line the esophagus. Specifically, cancer of the esophagus begins in the inner layer of the esophageal wall and grows outward. If it spreads through the esophageal wall, it can travel to lymph nodes, which are the tiny, bean-shaped organs that help fight infection, as well as the blood vessels in the chest and other nearby organs. Esophageal cancer can also spread to the lungs, liver, stomach, and other parts of the body.

[0054] There are two major types of esophageal cancer: esophagus squamous cell carcinoma (ESCC), which starts in squamous cells that line the esophagus, and usually develops in the upper and middle part of the esophagus, and esophagus adenocarcinoma (EAC). This type begins in the glandular tissue in the lower part of the esophagus where the esophagus and the stomach come together. In some embodiments, the patient has and/or has been diagnosed with ESCC. In some embodiments, the patient has or has been diagnosed with EAC.

A. Cancer staging

[0055] The esophageal cancer described herein may be an esophageal cancer of any of the following stages.

1. TNM staging system

[0056] One tool that doctors use to describe the stage is the TNM system. Tumor (T): How deeply has the primary tumor grown into the wall of the esophagus and the surrounding tissue? are 5 stages: stage 0 (zero) and stages I through IV (one through four). The following provides more information on the TNM staging system:

[0057] Using the TNM system, the "T" plus a letter or number (0 to 4) is used to describe the tumor, including whether the cancer has grown into the wall of the esophagus or nearby tissue, and if so, how deep. Some stages are also divided into smaller groups that help describe the tumor in even more detail. Specific tumor stage information is listed below.

[0058] In some embodiments, the patient has and/or has been diagnosed with Tx, T0, Tis, T1, T2, T3, or T4 esophageal cancer.

[0059] The“N” in the TNM staging system stands for lymph nodes. In esophageal cancer, lymph nodes near the esophagus and in the chest are called regional lymph nodes. Lymph nodes in other parts of the body are called distant lymph nodes.

[0060] In some embodiments, the patient has and/or has been diagnosed with NX, N0, N1, N2, or N3 esophageal cancer.

[0061] The "M" in the TNM system indicates whether the cancer has spread to other parts of the body.

[0062] In some embodiments, the patient has and/or has been diagnosed with MX, M0, or M1 esophageal cancer.

2. Grade (G)

[0063] Esophageal cancer can also be described by its grade (G), which describes how much cancer cells look like healthy cells when viewed under a microscope. The doctor compares the cancerous tissue with healthy tissue. Healthy tissue usually contains many different types of cells grouped together. If the cancer looks similar to healthy tissue and contains different cell groupings, it is called differentiated or a low-grade tumor. If the cancerous tissue looks very different from healthy tissue, it is called poorly differentiated or a high-grade tumor. The cancer’s grade may help the doctor predict how quickly the cancer will spread. In general, the lower the tumor’s grade, the better the prognosis.

[0064] In some embodiments, the patient has been diagnosed with and/or has been determined to have G1, G2, G3, or G4 cancer.

3. Cancer stage grouping

[0065] Doctors assign the stage of the cancer by combining the T, N, and M classifications. There are separate staging systems for the two most common types of esophageal cancer: squamous cell carcinoma and adenocarcinoma. The staging system for each is described below.

a. Staging of squamous cell carcinoma of the esophagus

[0066] In addition to the TNM classifications, for squamous cell carcinoma, the stages may be subdivided based on whether the tumor is located in the upper, middle, or lower section of the esophagus, as well as the grade (G) of the tumor cells.

[0067] In some embodiments, the patient has been diagnosed with and/or has been determined to have Stage 0, Stage IA, Stage IB, Stage IIA, Stage IIB, Stage IIIA, Stage IIIB, Stage IIIC or Stage IV squamous cell carcinoma of the esophagus b. Staging of adenocarcinoma of the esophagus

[0068] For adenocarcinoma, doctors use the T, N, and M classifications, as well as the grade (G).

[0069] In some embodiments, the patient has been diagnosed with and/or has been determined to have Stage 0, Stage IA, Stage IB, Stage IIA, Stage IIB, Stage IIIA, Stage IIIB, Stage IIIC, or Stage IV adenocarcinoma of the esophagus.

[0070] Recurrent cancer is cancer that has come back after treatment. It may come back in the esophagus or in another part of the body. If the cancer does return, there will be another round of tests to learn about the extent of the recurrence. These tests and scans are often similar to those done at the time of the original diagnosis. In some embodiments, the method is for treating recurrent cancer. In some embodiments, the patient has been determined to have recurrent cancer. In some embodiments, the method is for diagnosing or prognosing a patient’s risk of recurrence of a cancer.

B. Therapy

[0071] The following treatment steps/active ingredients are useful in the methods described herein. Embodiments of the disclosure may also exclude treatments and therapeutic regimens described herein. It is also contemplated that the following treatment steps/therapeutic agents may be specifically excluded in the embodiments described herein. For people with a tumor that has not spread beyond the esophagus and lymph nodes, it is often recommend combining different types of treatment: radiation therapy, chemotherapy, and surgery. The order of treatments varies, and several factors are considered, including the type of esophageal cancer.

[0072] Particularly for squamous cell cancer, chemotherapy and radiation therapy, a combination called chemoradiotherapy, are commonly recommended as the first treatment, with surgery afterwards depending how well chemoradiotherapy worked. Recent studies show using chemoradiotherapy before surgery is better than surgery alone.

[0073] For adenocarcinoma, the most common treatment in the United States is chemotherapy and radiation therapy followed by surgery. Surgery is almost always recommended after chemoradiotherapy, unless there are factors that increase the risks from surgery, such as a patient’s age or overall health.

[0074] For advanced esophageal cancer, treatment usually involves chemotherapy and radiation therapy.

[0075] Cancer and its treatment often cause side effects. In addition to treatment to slow, stop, or eliminate the cancer, an important part of cancer care is relieving a person’s symptoms and side effects. This approach is called palliative or supportive care, and it includes supporting the patient with his or her physical, emotional, and social needs.

[0076] Palliative care is any treatment that focuses on reducing symptoms, improving quality of life, and supporting patients and their families. Any person, regardless of age or type and stage of cancer, may receive palliative care. It works best when palliative care is started as early as needed in the cancer treatment process. People often receive treatment for the cancer and treatment to ease side effects at the same time. In fact, patients who receive both often have less severe symptoms, better quality of life, and report they are more satisfied with treatment.

[0077] Palliative treatments vary widely and often include medication, nutritional changes, relaxation techniques, emotional support, and other therapies. Palliative treatments may also include those similar to those meant to eliminate the cancer, such as chemotherapy, surgery, or radiation therapy.

1. Surgery

[0078] Surgery is the removal of the tumor and some surrounding healthy tissue during an operation. A surgical oncologist is a doctor who specializes in treating cancer using surgery. Surgery has traditionally been the most common treatment for esophageal cancer. However, currently, surgery is used as the main treatment only for patients with early-stage esophageal cancer.

[0079] For patients with locally-advanced esophageal cancer, a combination of chemotherapy and radiation therapy (see below) may be used before surgery to shrink the tumor. For people who cannot have surgery, the best treatment option is often a combination of chemotherapy and radiation therapy. [0080] The most common surgery to treat esophageal cancer is called an esophagectomy, where the doctor removes the affected part of the esophagus and then connects the remaining healthy part of the esophagus to the stomach so that the patient can swallow normally. The stomach or part of the intestine may sometimes be used to make the connection. The surgeon also removes lymph nodes around the esophagus.

[0081] In addition to surgery to treat the disease, surgery may be used to help patients eat and relieve symptoms caused by the cancer. This is called palliative surgery. To do this, surgeons and gastroenterologists can:

1.) put in a percutaneous gastrostomy or jejunostomy, also called a feeding tube, so that a person can receive nutrition directly into the stomach or intestine. This may be done before chemotherapy and radiation therapy is given to make sure that the patient can eat enough food to maintain his or her weight and strength during treatment; or 2.) create a bypass, or new pathway, to the stomach if a tumor blocks the esophagus but cannot be removed with surgery; this procedure is rarely used.

[0082] People who have had trouble eating and drinking may need intravenous (IV; into a vein) feedings and fluids for several days before and after surgery, as well as antibiotics to prevent or treat infections. Patients learn special coughing and breathing exercises to keep their lungs clear.

2. Endoscopic therapy

[0083] The following treatments use an endoscope (see Diagnosis) to treat esophageal cancer and to manage side effects caused by the tumor. Endoscopy and dilation is a procedure that expands the esophagus. It may have to be repeated if the tumor grows. Endoscopy with stent placement is a procedure that uses an endoscopy to insert a stent in the esophagus. An esophageal stent is a metal, mesh device that is expanded to keep the esophagus open.

[0084] Photodynamic therapy is a palliative or supportive care option used to make swallowing easier, especially for people who cannot or choose not to have surgery, radiation therapy, or chemotherapy. In photodynamic therapy, a light-sensitive substance is injected into the tumor and stays longer in cancer cells than in healthy cells. A light is then aimed at the tumor, destroying the cancer cells. Although photodynamic therapy may relieve swallowing problems for a short period of time, it does not cure esophageal cancer.

[0085] Electrocoagulation is a type of palliative treatment helps kill cancer cells by heating [0086] Cryotherapy is a type of palliative treatment that uses an endoscope with a probe attached that can freeze and remove tumor tissue. It can be used to reduce the size of a tumor to help a patient swallow better.

3. Radiation therapy

[0087] Radiation therapy is the use of high-energy x-rays or other particles to destroy cancer cells. A radiation therapy regimen (schedule) usually consists of a specific number of treatments given over a set period of time. The most common type of radiation treatment is called external-beam radiation therapy, which is radiation therapy given from a machine outside the body. When radiation treatment is given directly inside the body, it is called internal radiation therapy or brachytherapy. For esophageal cancer, this involves temporarily inserting a radioactive wire into the esophagus using an endoscope.

4. Chemotherapy

[0088] Chemotherapy and radiotherapy for esophageal cancer may be delivered preoperatively, postoperatively, or independent of surgery. Most chemotherapy that is currently used for the treatment of esophageal cancer include alkylating, antimetabolite, anthracycline, and antimicrotubular agents. Chemotherapy for squamous cell esophageal carcinoma, as with squamous cell carcinomas in general, may be based on cisplatin.

[0089] In some embodiments, chemoradiotherapy is administered, followed by surgery. In some embodiments, neoadjuvant therapy is used. In some embodiments, neoadjuvant therapy comprises a combination of radiotherapy and chemotherapy with a platinum compound and a DNA replication inhibitor. In some embodiments, the platinum compound is selected from cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenathriplatin, picoplatin, and satraplatin. In some embodiments, the platinum compound is cisplatin. In some embodiments, the DNA replication inhibitor is 5-fluorouracil.

[0090] In some embodiments, the chemotherapy comprises carboplatin, paclitaxel, cisplatin, 5-fluorouracil, epirubicin, docetaxel, cepecitabine, oxaliplatin, and combinations thereof. In some embodiments, the combination treatment comprises carboplatin and paclitaxel; cisplatin and 5-fluorouracil; epirubicin, cisplatin, and 5-fluorouracil; docetaxel, cisplatin, and 5-fluorouracil; cisplatin and cepacitabine; oxaliplatin and 5-fluorouracil; and oxaliplatin and capecitabine. 5. Targeted therapy

[0091] Targeted therapy is a treatment that targets the cancer’s specific genes, proteins, or the tissue environment that contributes to cancer growth and survival. This type of treatment blocks the growth and spread of cancer cells while limiting damage to healthy cells.

[0092] For esophageal cancer, the targeted therapy trastuzumab (Herceptin) may be used along with chemotherapy for patients with metastatic esophageal adenocarcinoma. Trastuzumab targets a protein called human epidermal growth receptor 2 (HER2). About 20% to 30% of esophageal adenocarcinomas make too much HER2.

[0093] The targeted therapy ramucirumab (Cyramza) is also an option after first-line therapy, or the first treatments given, has not worked. It may be given by itself or with paclitaxel (Taxol), a type of chemotherapy.

C. Monitoring

[0094] In certain aspects, the biomarker-based method may be combined with one or more other esophageal cancer diagnosis or screening tests at increased frequency if the patient is determined to be at high risk for recurrence or have a poor prognosis.

[0095] The esophagus monitoring may include any methods known in the art. In particular, the monitoring include obtaining a sample and testing the sample for diagnosis. For example, the monitoring may include endoscopy of the esophagus and/or biopsy. Other monitoring test include imaging tests, barium swallow tests, CAT scan (computed tomography scan), magnetic resonance imaging (MRI) scan, positron emission tomography (PET) scan, endoscopy such as upper endoscopy, endoscopic ultrasound, bronchoscopy, thoracoscopy, laparoscopy, or combinations thereof.

[0096] In further aspects, the monitoring diagnosis may include lab tests such as HER2 testing of biopsy samples, a complete blood count (CBC) blood test to look for anemia, a check of a stool sample for occult blood, and/or blood tests to check for normal kidney or liver function.

III. Colorectal Cancer Staging and Treatments

[0097] Methods and compositions may be provided for treating, prognosing, and/or diagnosing colorectal cancer with particular applications of particular applications of microbial expression. Based on a biomarker or microbial profile, different treatments may be prescribed or recommended for different cancer patients. A. Cancer staging

[0098] Colorectal cancer, also known as colon cancer, rectal cancer, or bowel cancer, is a cancer from uncontrolled cell growth in the colon or rectum (parts of the large intestine), or in the appendix. Certain aspects of the methods are provided for patients that are stage I-IV colorectal cancer patients. In particular aspects, the patient is a stage II or III patient. In a further embodiment, the patient is a stage I or II patient. In a further embodiment, the patient is a stage I, II, or III patient. In some embodiments, the patient is diagnosed as having and/or determined to have Tis, N0, and/or M0; T1, N0, and/or M0; T2, N0, and/or M0; T3, N0, and/or M0; T4, N0, and/or M0; T1-2, N1, and/or M0; T3-4, N1, and/or M0; any T, N2, and/or M0; or any T, any N, and/or M1.

[0099] The most common staging system is the TNM (for tumors/nodes/metastases) system, from the American Joint Committee on Cancer (AJCC). The TNM system assigns a number based on three categories.“T” denotes the degree of invasion of the intestinal wall, “N” the degree of lymphatic node involvement, and“M” the degree of metastasis. The broader stage of a cancer is usually quoted as a number I, II, III, IV derived from the TNM value grouped by prognosis; a higher number indicates a more advanced cancer and likely a worse outcome. Details of this system are in the graph below:

AJCC TNM stage TNM stage criteria for colorectal cancer stage

Stage 0 Tis N0 M0 Tis: Tumor confined to mucosa; cancer-in-situ

Stage I T1 N0 M0 T1: Tumor invades submucosa

Stage I T2 N0 M0 T2: Tumor invades muscularis propria

Stage II-A T3 N0 M0 T3: Tumor invades subserosa or beyond (without other organs involved)

Stage II-B T4 N0 M0 T4: Tumor invades adjacent organs or perforates the visceral peritoneum

Stage III-A T1-2 N1 M0 N1: Metastasis to 1 to 3 regional lymph nodes. T1 or T2. Stage III-B T3-4 N1 M0 N1: Metastasis to 1 to 3 regional lymph nodes. T3 or T4. Stage III-C any T, N2 M0 N2: Metastasis to 4 or more regional lymph nodes. Any T. Stage IV any T, any N, M1: Distant metastases present. Any T, any N.

M1 B. Therapy

[00100] Methods of the disclosure may include a cancer therapy as described herein. In some embodiments, the cancer therapy comprises surgical removal of a tumor. This can either be neoadjuvant setting before surgery to shrink the cancer before attempting to remove it (neoadjuvant therapy). The two most common sites of recurrence of colorectal cancer is in the liver and lungs. In some embodiments, the treatment of early colorectal cancer excludes chemotherapy. In further embodiments, the treatment of early colorectal cancer includes neoadjuvant therapy (chemotherapy or radiotherapy before the surgical removal of the primary tumor), but excludes adjuvant therapy (chemotherapy and/or radiotherapy after surgical removal of the primary tumor.

[00101] In both cancer of the colon and rectum, chemotherapy may be used in addition to surgery in certain cases. In rectal cancer, chemotherapy may be used in the neoadjuvant setting.

[00102] In certain embodiments, there may be a decision regarding the therapeutic treatment based on microbiome profile. In some embodiments, the methods include the administration of a chemotherapeutic. In some embodiments, the chemotherapeutic comprises antimetabolites or thymidylate synthase inhibitors such as fluorouracil (5-FU). In some embodiments, the chemotherapeutic comprises cytotoxic drugs, such as irinotecan or oxaliplatin. In some embodiments, the chemotherapeutic comprises combinations such as irinotecan, fluorouracil, and Jeucovorin (FOLFIRI); and oxaliplatin, fluorouracil, and leucovorin (FOLFOX).

[00103] In some embodiments, the cancer therapy comprises an antibody. In some embodiments, the cancer therapy comprises Avastin® (bevacizumab) (Genentech Inc., South San Francisco CA) and/or epidermal growth factor receptor Erbitux® (cetuximab) (Imclone Inc. New York City). In some embodiments, the cancer therapy may include one or more of the chemical therapeutic agents including thymidylate synthase inhibitors or antimetabolites such as fluorouracil (5-FU), alone or in combination with other therapeutic agents. For example, in some embodiments, the first treatment to be tested for response therapy may be antimetabolites or thymidylate synthase inhibitors, prodrugs, or salts thereof. .

[00104] Antimetabolites can be used in cancer treatment, as they interfere with DNA production and therefore cell division and the growth of tumors. Because cancer cells spend more time dividing than other cells, inhibiting cell division harms tumor cells more than other cells. Anti-metabolites masquerade as a purine (azathioprine, mercaptopurine) or a pyrimidine, chemicals that become the building-blocks of DNA. They prevent these substances becoming incorporated in to DNA during the S phase (of the cell cycle), stopping normal development and division. They also affect RNA synthesis. However, because thymidine is used in DNA but not in RNA (where uracil is used instead), inhibition of thymidine synthesis via thymidylate In some embodiments, this treatment regimen is for advanced cancer. In some embodiments, this treatment regimen is excluded for early cancer.

[00105] Thymidylate synthase inhibitors are chemical agents which inhibit the enzyme thymidylate synthase and have potential as an anticancer chemotherapy. As an anti-cancer chemotherapy target, thymidylate synthetase can be inhibited by the thymidylate synthase inhibitors such as fluorinated pyrimidine fluorouracil, or certain folate analogues, the most notable one being raltitrexed (trade name Tomudex). Five agents were in clinical trials in 2002: raltitrexed, pemetrexed, nolatrexed, ZD9331, and GS7904L. Additional non-limiting examples include: Raltitrexed, used for colorectal cancer since 1998; Fluorouracil, used for colorectal cancer; BGC 945; OSI-7904L.

[00106] In further embodiments, there may be involved prodrugs that can be converted to thymidylate synthase inhibitors in the body, such as Capecitabine (INN), an orally- administered chemotherapeutic agent used in the treatment of numerous cancers. Capecitabine is a prodrug, that is enzymatically converted to 5-fluorouracil in the body. In some embodiments, this treatment regimen is for advanced cancer. In some embodiments, this treatment regimen is excluded for early cancer.

[00107] Further chemotherapeutic agents that may be used include capecitabine, fluorouracil, irinotecan, leucovorin, oxaliplatin and UFT. Another type of agent that is sometimes used are the epidermal growth factor receptor inhibitors.

[00108] In certain embodiments, alternative treatments may be prescribed or recommended based on the biomarker profile. In addition to traditional chemotherapy for colorectal cancer patients, cancer therapies also include a variety of combination therapies with both chemical and radiation based treatments. Combination chemotherapies include, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate, or any analog or derivative variant of the foregoing.

[00109] In people with incurable colorectal cancer, treatment options including palliative care can be considered for improving quality of life. Surgical options may include non-curative surgical removal of some of the cancer tissue, bypassing part of the intestines, or stent Non-operative methods of symptomatic treatment include radiation therapy to decrease tumor size as well as pain medications. In some embodiments, this treatment regimen is for advanced cancer. In some embodiments, this treatment regimen is excluded for early cancer.

[00110] Immunotherapies that are designed to boost the body’s natural defenses to fight the cancer may also be used. Immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells. Immune therapy methods are further described below:

2. Checkpoint Inhibitors and Combination Treatment

[00111] Embodiments of the disclosure may include administration of immune checkpoint inhibitors, which are further described below.

b. PD- 1, PDL1, and PDL2 inhibitors

[00112] PD-l can act in the tumor microenvironment where T cells encounter an infection or tumor. Activated T cells upregulate PD-l and continue to express it in the peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-l is to limit the activity of effector T cells in the periphery and prevent excessive damage to the tissues during an immune response. Inhibitors of the disclosure may block one or more functions of PD-l and/or PDL1 activity.

[00113] Alternative names for“PD-l” include CD279 and SLEB2. Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for“PDL2” include B7- DC, Btdc, and CD273. In some embodiments, PD-l, PDL1, and PDL2 are human PD-l, PDL1 and PDL2.

[00114] In some embodiments, the PD-l inhibitor is a molecule that inhibits the binding of PD-l to its ligand binding partners. In a specific aspect, the PD-l ligand binding partners are PDL1 and/or PDL2. In another embodiment, a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-l and/or B7-1. In another embodiment, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art such as described in U.S. Patent Application Nos. US2014/0294898, US2014/022021, and US2011/0008369, all incorporated herein by reference.

[00115] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD- 1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PDL1 inhibitor comprises AMP- 224. Nivolumab, also known as MDX-1106-04, MDX- 1106, ONO-4538, BMS-936558, and OPDIVO^®, is an anti-PD-1 antibody described in WO2006/121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA^®, and SCH-900475, is an anti-PD-1 antibody described in WO2009/114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in WO2009/101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010/027827 and WO2011/066342. Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[00116] In some embodiments, the immune checkpoint inhibitor is a PDL1 inhibitor such as Durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinations thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor such as rHIgM12B7.

[00117] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2 and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another embodiment, the antibody competes for binding with and/or binds to the same epitope on PD-1, PDL1, or PDL2 as the above- 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.

c. CTLA-4, B7-1, and B7-2

[00118] Another immune checkpoint that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an“off” switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA- 4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules. Inhibitors of the disclosure may block one or more functions of CTLA-4, B7-1, and/or B7-2 activity. In some embodiments, the inhibitor blocks the CTLA-4 and B7-1 interaction. In some embodiments, the inhibitor blocks the CTLA-4 and B7-2 interaction.

[00119] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[00120] Anti-human-CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti- CTLA-4 antibodies disclosed in: US 8,119,129, WO 01/14424, WO 98/42752; WO 00/37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No.6,207,156; Hurwitz et al., 1998; can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent Application No. WO2001/014424, WO2000/037504, and U.S. Patent No.8,017,114; all incorporated herein by reference. [00121] A further anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and Yervoy®) or antigen binding fragments and variants thereof (see, e.g., WO01/14424).

[00122] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another embodiment, the antibody competes for binding with and/or binds to the same epitope on PD-1, B7-1, or B7-2 as the above- mentioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.

3. Other immunotherapies

[00123] In some embodiments, the methods comprise administration of a cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer. Immunotherapies can be categorized as active, passive or hybrid (active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumour-associated antigens (TAAs); they are often proteins or other macromolecules (e.g. carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes and cytokines. Immumotherapies are known in the art, and some are described below.

a. Inhibition of co-stimulatory molecules

[00124] In some embodiments, the immunotherapy comprises an inhibitor of a co- stimulatory molecule. In some embodiments, the inhibitor comprises an inhibitor of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, OX40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Inhibitors include inhibitory antibodies, polypeptides, compounds, and nucleic acids.

b. Dendritic cell therapy

[00125] Dendritic cell therapy provokes anti-tumor responses by causing dendritic cells to immune system. In cancer treatment they aid cancer antigen targeting. One example of cellular cancer therapy based on dendritic cells is sipuleucel-T.

[00126] One method of inducing dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small parts of protein that correspond to the protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to increase the immune and anti-tumor responses. Other adjuvants include proteins or other chemicals that attract and/or activate dendritic cells, such as granulocyte macrophage colony-stimulating factor (GM-CSF).

[00127] Dendritic cells can also be activated in vivo by making tumor cells express GM- CSF. This can be achieved by either genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF.

[00128] Another strategy is to remove dendritic cells from the blood of a patient and activate them outside the body. The dendritic cells are activated in the presence of tumor antigens, which may be a single tumor-specific peptide/protein or a tumor cell lysate (a solution of broken down tumor cells). These cells (with optional adjuvants) are infused and provoke an immune response.

[00129] Dendritic cell therapies include the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibody and can induce the dendritic cells to mature and provide immunity to the tumor. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets.

c. CAR-T cell therapy

[00130] Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors) are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because they are fused of parts from different sources. CAR-T cell therapy refers to a treatment that uses such transformed cells for cancer therapy.

[00131] The basic principle of CAR-T cell design involves recombinant receptors that combine antigen-binding and T-cell activating functions. The general premise of CAR-T cells is to artificially generate T-cells targeted to markers found on cancer cells. Scientists can remove T-cells from a person, genetically alter them, and put them back into the patient for them to attack the cancer cells. Once the T cell has been engineered to become a CAR-T cell, domain to an intracellular signalling molecule which in turn activates T cells. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells are targeted, and not normal cells. The specificity of CAR-T cells is determined by the choice of molecule that is targeted.

[00132] Exemplary CAR-T therapies include Tisagenlecleucel (Kymriah) and Axicabtagene ciloleucel (Yescarta). In some embodiments, the CAR-T therapy targets CD19.

d. Cytokine therapy

[00133] Cytokines are proteins produced by many types of cells present within a tumor. They can modulate immune responses. The tumor often employs them to allow it to grow and reduce the immune response. These immune-modulating effects allow them to be used as drugs to provoke an immune response. Two commonly used cytokines are interferons and interleukins.

[00134] Interferons are produced by the immune system. They are usually involved in anti viral response, but also have use for cancer. They fall in three groups: type I (IFNa and IFNP), type II (IFNy) and type III (IFNk).

[00135] Interleukins have an array of immune system effects. IL-2 is an exemplary interleukin cytokine therapy.

e. Adoptive T-cell therapy

[00136] Adoptive T cell therapy is a form of passive immunization by the transfusion of T- cells (adoptive cell transfer). They are found in blood and tissue and usually activate when they find foreign pathogens. Specifically they activate when the T-cell's surface receptors encounter cells that display parts of foreign proteins on their surface antigens. These can be either infected cells, or antigen presenting cells (APCs). They are found in normal tissue and in tumor tissue, where they are known as tumor infiltrating lymphocytes (TILs). They are activated by the presence of APCs such as dendritic cells that present tumor antigens. Although these cells can attack the tumor, the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumour death. [60]

[00137] Multiple ways of producing and obtaining tumour targeted T-cells have been developed. T-cells specific to a tumor antigen can be removed from a tumor sample (TILs) or filtered from blood. Subsequent activation and culturing is performed ex vivo, with the results reinfused. Activation can take place through gene therapy, or by exposing the T cells to tumor antigens. [00138] It is contemplated that a cancer treatment may exclude any of the cancer treatments described herein. Furthermore, embodiments of the disclosure include patients that have been previously treated for a therapy described herein, are currently being treated for a therapy described herein, or have not been treated for a therapy described herein. In some embodiments, the patient is one that has been determined to be resistant to a therapy described herein. In some embodiments, the patient is one that has been determined to be sensitive to a therapy described herein.

C. Monitoring

[00139] In certain aspects, the methods of the disclosure may be combined with one or more other colon cancer diagnosis or screening tests at increased frequency if the patient is determined to be at high risk for recurrence or have a poor prognosis based on the biomarker described above.

[00140] The colon monitoring may include any methods known in the art. In particular, the monitoring include obtaining a sample and testing the sample for diagnosis. For example, the colon monitoring may include colonoscopy or coloscopy, which is the endoscopic examination of the large bowel and the distal part of the small bowel with a CCD camera or a fiber optic camera on a flexible tube passed through the anus. It can provide a visual diagnosis (e.g. ulceration, polyps) and grants the opportunity for biopsy or removal of suspected colorectal cancer lesions. Thus, colonoscopy or coloscopy can be used for treatment.

[00141] In further aspects, the monitoring diagnosis may include sigmoidoscopy, which is similar to colonoscopy—the difference being related to which parts of the colon each can examine. A colonoscopy allows an examination of the entire colon (1200–1500 mm in length). A sigmoidoscopy allows an examination of the distal portion (about 600 mm) of the colon, which may be sufficient because benefits to cancer survival of colonoscopy have been limited to the detection of lesions in the distal portion of the colon. A sigmoidoscopy is often used as a screening procedure for a full colonoscopy, often done in conjunction with a fecal occult blood test (FOBT). About 5% of these screened patients are referred to colonoscopy.

[00142] In additional aspects, the monitoring diagnosis may include virtual colonoscopy, which uses 2D and 3D imagery reconstructed from computed tomography (CT) scans or from nuclear magnetic resonance (MR) scans, as a totally non-invasive medical test.

[00143] The monitoring include the use of one or more screening tests for colon cancer including, but not limited to fecal occult blood testing, flexible sigmoidoscopy and 42% of malignancies are found. Virtual colonoscopy via a CT scan appears as good as standard colonoscopy for detecting cancers and large adenomas but is expensive, associated with radiation exposure, and cannot remove any detected abnormal growths like standard colonoscopy can. Fecal occult blood testing (FOBT) of the stool is typically recommended every two years and can be either guaiac based or immunochemical. Annual FOBT screening results in a 16% relative risk reduction in colorectal cancer mortality, but no difference in all- cause mortality. The M2-PK test identifies an enzyme in colorectal cancers and polyps rather than blood in the stool. It does not require any special preparation prior to testing. M2-PK is sensitive for colorectal cancer and polyps and is able to detect bleeding and non-bleeding colorectal cancer and polyps. In the event of a positive result people would be asked to undergo further examination e.g. colonoscopy.

IV. ROC analysis

[00144] In statistics, a receiver operating characteristic (ROC), or ROC curve, is a graphical plot that illustrates the performance of a binary classifier system as its discrimination threshold is varied. The curve is created by plotting the true positive rate against the false positive rate at various threshold settings. (The true-positive rate is also known as sensitivity in biomedical informatics, or recall in machine learning. The false-positive rate is also known as the fall-out and can be calculated as 1 - specificity). The ROC curve is thus the sensitivity as a function of fall-out. In general, if the probability distributions for both detection and false alarm are known, the ROC curve can be generated by plotting the cumulative distribution function (area under the probability distribution from–infinity to + infinity) of the detection probability in the y- axis versus the cumulative distribution function of the false-alarm probability in x-axis.

[00145] ROC analysis provides tools to select possibly optimal models and to discard suboptimal ones independently from (and prior to specifying) the cost context or the class distribution. ROC analysis is related in a direct and natural way to cost/benefit analysis of diagnostic decision making. ROC analysis provides a tool for creating cut-off values to partition patient populations into high expression and low expression of certain biomarkers.

[00146] The ROC is also known as a relative operating characteristic curve, because it is a comparison of two operating characteristics (TPR and FPR) as the criterion changes. ROC analysis curves are known in the art and described in Metz CE (1978) Basic principles of ROC analysis. Seminars in Nuclear Medicine 8:283-298; Youden WJ (1950) An index for rating diagnostic tests. Cancer 3:32-35; Zweig MH, Campbell G (1993) Receiver-operating Chemistry 39:561-577; and Greiner M, Pfeiffer D, Smith RD (2000) Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests. Preventive Veterinary Medicine 45:23-41, which are herein incorporated by reference in their entirety. V. Sample Preparation

[00147] In certain aspects, methods involve obtaining a sample from a subject. The methods of obtaining provided herein may include methods of biopsy such as fine needle aspiration, core needle biopsy, vacuum assisted biopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy or skin biopsy. In certain embodiments the sample is obtained from a biopsy from esophageal tissue by any of the biopsy methods previously mentioned. In other embodiments the sample may be obtained from any of the tissues provided herein that include but are not limited to non-cancerous or cancerous tissue and non-cancerous or cancerous tissue from the serum, gall bladder, mucosal, skin, heart, lung, breast, pancreas, blood, liver, muscle, kidney, smooth muscle, bladder, colon, intestine, brain, prostate, esophagus, or thyroid tissue. Alternatively, the sample may be obtained from any other source including but not limited to blood, sweat, hair follicle, buccal tissue, tears, menses, feces, or saliva. In certain aspects of the current methods, any medical professional such as a doctor, nurse or medical technician may obtain a biological sample for testing. Yet further, the biological sample can be obtained without the assistance of a medical professional.

[00148] A sample may include but is not limited to, tissue, cells, or biological material from cells or derived from cells of a subject. The biological sample may be a heterogeneous or homogeneous population of cells or tissues. The biological sample may be obtained using any method known to the art that can provide a sample suitable for the analytical methods described herein. The sample may be obtained by non-invasive methods including but not limited to: scraping of the skin or cervix, swabbing of the cheek, saliva collection, urine collection, feces collection, collection of menses, tears, or semen.

[00149] The sample may be obtained by methods known in the art. In certain embodiments the samples are obtained by biopsy. In other embodiments the sample is obtained by swabbing, endoscopy, scraping, phlebotomy, or any other methods known in the art. In some cases, the sample may be obtained, stored, or transported using components of a kit of the present methods. In some cases, multiple samples, such as multiple esophageal samples may be obtained for diagnosis by the methods described herein. In other cases, multiple samples, such as one or more samples from one tissue type (for example esophagus) and one or more samples some cases, multiple samples such as one or more samples from one tissue type (e.g. esophagus) and one or more samples from another specimen (e.g. serum) may be obtained at the same or different times. Samples may be obtained at different times are stored and/or analyzed by different methods. For example, a sample may be obtained and analyzed by routine staining methods or any other cytological analysis methods.

[00150] In some embodiments the biological sample may be obtained by a physician, nurse, or other medical professional such as a medical technician, endocrinologist, cytologist, phlebotomist, radiologist, or a pulmonologist. The medical professional may indicate the appropriate test or assay to perform on the sample. In certain aspects a molecular profiling business may consult on which assays or tests are most appropriately indicated. In further aspects of the current methods, the patient or subject may obtain a biological sample for testing without the assistance of a medical professional, such as obtaining a whole blood sample, a urine sample, a fecal sample, a buccal sample, or a saliva sample.

[00151] In other cases, the sample is obtained by an invasive procedure including but not limited to: biopsy, needle aspiration, endoscopy, or phlebotomy. The method of needle aspiration may further include fine needle aspiration, core needle biopsy, vacuum assisted biopsy, or large core biopsy. In some embodiments, multiple samples may be obtained by the methods herein to ensure a sufficient amount of biological material.

[00152] General methods for obtaining biological samples are also known in the art. Publications such as Ramzy, Ibrahim Clinical Cytopathology and Aspiration Biopsy 2001, which is herein incorporated by reference in its entirety, describes general methods for biopsy and cytological methods. In one embodiment, the sample is a fine needle aspirate of a esophageal or a suspected esophageal tumor or neoplasm. In some cases, the fine needle aspirate sampling procedure may be guided by the use of an ultrasound, X-ray, or other imaging device.

[00153] In some embodiments of the present methods, the molecular profiling business may obtain the biological sample from a subject directly, from a medical professional, from a third party, or from a kit provided by a molecular profiling business or a third party. In some cases, the biological sample may be obtained by the molecular profiling business after the subject, a medical professional, or a third party acquires and sends the biological sample to the molecular profiling business. In some cases, the molecular profiling business may provide suitable containers, and excipients for storage and transport of the biological sample to the molecular [00154] In some embodiments of the methods described herein, a medical professional need not be involved in the initial diagnosis or sample acquisition. An individual may alternatively obtain a sample through the use of an over the counter (OTC) kit. An OTC kit may contain a means for obtaining said sample as described herein, a means for storing said sample for inspection, and instructions for proper use of the kit. In some cases, molecular profiling services are included in the price for purchase of the kit. In other cases, the molecular profiling services are billed separately. A sample suitable for use by the molecular profiling business may be any material containing tissues, cells, nucleic acids, genes, gene fragments, expression products, gene expression products, or gene expression product fragments of an individual to be tested. Methods for determining sample suitability and/or adequacy are provided.

[00155] In some embodiments, the subject may be referred to a specialist such as an oncologist, surgeon, or endocrinologist. The specialist may likewise obtain a biological sample for testing or refer the individual to a testing center or laboratory for submission of the biological sample. In some cases the medical professional may refer the subject to a testing center or laboratory for submission of the biological sample. In other cases, the subject may provide the sample. In some cases, a molecular profiling business may obtain the sample. VI. Evaluating Levels of Biomarkers

[00156] In certain aspects a meta-analysis of expression or activity can be performed. In statistics, a meta-analysis combines the results of several studies that address a set of related research hypotheses. This is normally done by identification of a common measure of effect size, which is modeled using a form of meta-regression. Generally, three types of models can be distinguished in the literature on meta-analysis: simple regression, fixed effects meta- regression and random effects meta-regression. Resulting overall averages when controlling for study characteristics can be considered meta-effect sizes, which are more powerful estimates of the true effect size than those derived in a single study under a given single set of assumptions and conditions. A meta-gene expression value, in this context, is to be understood as being the median of the normalized expression of a biomarker gene or activity. Normalization of the expression of a biomarker gene is preferably achieved by dividing the expression level of the individual marker gene to be normalized by the respective individual median expression of this marker genes, wherein said median expression is preferably calculated from multiple measurements of the respective gene in a sufficiently large cohort of test individuals. The test cohort preferably comprises at least 3, 10, 100, 200, 1000 individuals minimized allowing multiple datasets to be combined for meta-analyses (See Sims et al. BMC Medical Genomics (1:42), 1-14, 2008, which is incorporated herein by reference in its entirety).

[00157] The calculation of a meta-gene expression value is performed by: (i) determining the gene expression value of at least two, preferably more genes (ii) "normalizing" the gene expression value of each individual gene by dividing the expression value with a coefficient which is approximately the median expression value of the respective gene in a representative breast cancer cohort (iii) calculating the median of the group of normalized gene expression values.

[00158] A gene shall be understood to be specifically expressed in a certain cell type if the expression level of the gene in the cell type is at least about 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, or 10000-fold higher (or any range derivable therein) than in a reference cell type, or in a mixture of reference cell types. Reference cell types include non-cancerous tissue cells or a heterogenous population of cancers.

[00159] In certain algorithms a suitable threshold level is first determined for a marker gene. The suitable threshold level can be determined from measurements of the marker gene expression in multiple individuals from a test cohort. The median expression of the marker gene in said multiple expression measurements is taken as the suitable threshold value.

[00160] Comparison of multiple marker genes with a threshold level can be performed as follows: 1. The individual marker genes are compared to their respective threshold levels. 2. The number of marker genes, the expression level of which is above their respective threshold level, is determined. 3. If a marker genes is expressed above its respective threshold level, then the expression level of the marker gene is taken to be "above the threshold level".

[00161] Some embodiments include determining that a measured expression level is higher than, lower than, increased relative to, decreased relative to, equal to, or within a predetermined amount of a reference expression level. In some embodiments, a higher, lower, increased, or decreased expression level is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 50, 100, 150, 200, 250, 500, or 1000 fold (or any derivable range therein) or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900% different than the reference level, or any derivable range therein. These values may represent a predetermined threshold level, and some embodiments include determining that the measured expression level is higher by a predetermined amount or lower by a predetermined amount than a reference level. In some embodiments, a level of expression may be qualified as“low” or samples meeting particular criteria. The level or range of levels in multiple control samples is an example of this. In some embodiments, that certain level or a predetermined threshold value is at, below, or above 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percentile, or any range derivable therein. Moreover, a threshold level may be derived from a cohort of individuals meeting a particular criteria. The number in the cohort may be, be at least, or be at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or more (or any range derivable therein). A measured expression level can be considered equal to a reference expression level if it is within a certain amount of the reference expression level, and such amount may be an amount that is predetermined. This can be the case, for example, when a classifier is used to identify the molecular subtype of a metastasis. The predetermined amount may be within 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50% of the reference level, or any range derivable therein.

[00162] For any comparison of gene or miRNA expression levels to a mean expression levels or a reference expression levels, the comparison is to be made on a gene-by-gene and miRNA- by-miRNA basis. For example, if the expression levels of gene A, gene B, and miRNA X in a patient’s cancerous sample are measured, a comparison to mean expression levels in cancerous samples of a cohort of patients would involve: comparing the expression level of gene A in the patient’s cancerous sample with the mean expression level of gene A in cancerous samples of the cohort of patients, comparing the expression level of gene B in the patient’s sample with the mean expression level of gene B in samples of the cohort of patients, and comparing the expression level of miRNA X in the patient’s metastasis with the mean expression level of miRNA X in cancerous samples of the cohort of patients. Comparisons that involve determining whether the expression level measured in a patient’s sample is within a predetermined amount of a mean expression level or reference expression level are similarly done on a gene-by-gene and miRNA-by-miRNA basis, as applicable. VII. Nucleic Acid Assays

[00163] Aspects of the methods include assaying nucleic acids to determine expression levels. Arrays can be used to detect differences between two samples. Specifically contemplated applications include identifying and/or quantifying differences between bacterial populations from a sample that is normal and from a sample that is not normal, between a cancerous condition and a non-cancerous condition, or between two differently treated samples. Also, microbiome profiles may be compared between a sample believed to be susceptible to a particular disease or condition and one believed to be not susceptible or resistant to that disease or condition. A sample that is not normal is one exhibiting phenotypic trait(s) of a disease or condition or one believed to be not normal with respect to that disease or condition. It may be compared to a cell that is normal with respect to that disease or condition. Phenotypic traits include symptoms of, or susceptibility to, a disease or condition of which a component is or may or may not be genetic or caused by a hyperproliferative or neoplastic cell or cells.

[00164] An array comprises a solid support with nucleic acid probes attached to the support. Arrays typically comprise a plurality of different nucleic acid probes that are coupled to a surface of a substrate in different, known locations. These arrays, also described as "microarrays" or colloquially "chips" have been generally described in the art, for example, U.S. Pat. Nos.5,143,854, 5,445,934, 5,744,305, 5,677,195, 6,040,193, 5,424,186 and Fodor et al., 1991), each of which is incorporated by reference in its entirety for all purposes. Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. No.5,384,261, incorporated herein by reference in its entirety for all purposes. Although a planar array surface is used in certain aspects, the array may be fabricated on a surface of virtually any shape or even a multiplicity of surfaces. Arrays may be nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate, see U.S. Pat. Nos.5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992, which are hereby incorporated in their entirety for all purposes.

[00165] In addition to the use of arrays and microarrays, it is contemplated that a number of difference assays could be employed to analyze nucleic acids, their activities, and their effects. Such assays include, but are not limited to, nucleic amplification, polymerase chain reaction, quantitative PCR, RT-PCR, in situ hybridization, Northern hybridization, hybridization protection assay (HPA)(GenProbe), branched DNA (bDNA) assay (Chiron), rolling circle amplification (RCA), single molecule hybridization detection (US Genomics), Invader assay (ThirdWave Technologies), and/or Bridge Litigation Assay (Genaco).

VIII. Methods of Determining Microbiome Composition

[00166] In some embodiments, the methods relate to obtaining a microbiome profile. In some embodiments, obtaining a microbiome profile comprises the steps of or the ordered steps of: i) obtaining a sample obtained from a subject (e.g., a human subject), ii) isolating one or more bacterial species from the sample, iii) isolating one or more nucleic acids from at least one bacterial species, iv) sequencing the isolated nucleic acids, and v) comparing the sequenced nucleic acids to a reference nucleic acid sequence. When performing the methods necessitating genotyping, any genotyping assay can be used. For example, this can be done by sequencing the 16S or the 23S ribosomal subunit or by metagenomics shotgun DNA sequencing associated with metatranscriptomics.

[00167] Methods for determining microbiome composition may include one or more microbiology methods such as sequencing, next generation sequencing, wester blotting, comparative genomic hybridization, PCR, ELISA, etc.

IX. Administration of Therapeutic Compositions

[00168] The therapy provided herein may comprise administration of a combination of therapeutic agents, such as antibiotics and cancer therapies, such as chemotherapy or a therapy described herein. The therapies may be administered in any suitable manner known in the art. For example, the antibiotic and the cancer treatment may be administered sequentially (at different times) or concurrently (at the same time). In some embodiments, the compositions are administered in a separate composition. In some embodiments, antibiotic and cancer therapy or second therapeutic agent are in the same composition.

[00169] Embodiments of the disclosure relate to compositions and methods comprising therapeutic compositions. The different therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of the agents may be employed, for example, an antibiotic is“A” and a cancer therapeutic is“B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A [00170] In some embodiments, the antibiotic is administered prior to the cancer therapy. In some embodiments, the antibiotic is administered at least, at most, or exactly 1, 2, 3, 5, 6, 12, 24 hours or 2, 3, 4, 6, 8, 10, days or 2, 3, 4, 5, 6, 7, or 8 weeks (or any derivable range therein) prior to the cancer therapy. In some embodiments, at least 1, 2, 3, 4, 5, 6, or 7 doses (or any derivable range therein) of the antibiotic is administered at least, at most, or exactly 1, 2, 3, 5, 6, 12, 24 hours or 2, 3, 4, 6, 8, 10, days or 2, 3, 4, 5, 6, 7, or 8 weeks (or any derivable range therein) prior to the cancer therapy. In some embodiments, the antibiotic is administered after the cancer therapy. In some embodiments, the antibiotic is administered at least, at most, or exactly 1, 2, 3, 5, 6, 12, 24 hours or 2, 3, 4, 6, 8, 10, days or 2, 3, 4, 5, 6, 7, or 8 weeks (or any derivable range therein) after the cancer therapy or after at least one of the cancer therapies or after at least 2 of the therapies. In some embodiments, at least 1, 2, 3, 4, 5, 6, or 7 doses (or any derivable range therein) of the antibiotic is administered at least, at most, or exactly 1, 2, 3, 5, 6, 12, 24 hours or 2, 3, 4, 6, 8, 10, days or 2, 3, 4, 5, 6, 7, or 8 weeks (or any derivable range therein) after the cancer therapy or after at least one of the cancer therapies or after at least 2 of the cancer therapies.

[00171] In some embodiments, the antibiotic composition is formulated for oral administration. The skilled artisan knows a variety of formulas which can encompass living or killed microorganisms and which can present as food supplements (e.g., pills, tablets and the like) or as functional food such as drinks or fermented yogurts.

[00172] The agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some embodiments, the cancer therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[00173] The treatments may include various“unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and infusion over a set period of time. In some embodiments, a unit dose comprises a single administrable dose.

[00174] The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain embodiments, it is contemplated that doses in the range from 10 mg/kg to 200 mg/kg can affect the protective capability of these agents. Thus, it is contemplated that doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 µg/kg, mg/kg, µg/day, or mg/day or any range derivable therein. Furthermore, such doses can be administered at multiple times during a day, and/or on multiple days, weeks, or months.

[00175] In certain embodiments, the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 µM to 150 µM. In another embodiment, the effective dose provides a blood level of about 4 µM to 100 µM.; or about 1 µM to 100 µM; or about 1 µM to 50 µM; or about 1 µM to 40 µM; or about 1 µM to 30 µM; or about 1 µM to 20 µM; or about 1 µM to 10 µM; or about 10 µM to 150 µM; or about 10 µM to 100 µM; or about 10 µM to 50 µM; or about 25 µM to 150 µM; or about 25 µM to 100 µM; or about 25 µM to 50 µM; or about 50 µM to 150 µM; or about 50 µM to 100 µM (or any range derivable therein). In other embodiments, the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 mM or any range derivable therein. In certain embodiments, the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent. Alternatively, to the extent the therapeutic agent is not metabolized by a subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[00176] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual Factors affecting dose include physical and (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[00177] It will be understood by those skilled in the art and made aware that dosage units of µg/kg or mg/kg of body weight can be converted and expressed in comparable concentration units of µg/ml or mM (blood levels), such as 4 µM to 100 µM. It is also understood that uptake is species and organ/tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.

X. Kits

[00178] Certain aspects of the present invention also concern kits containing compositions of the invention or compositions to implement methods of the invention. In some embodiments, kits can be used to evaluate one or more microbial isolations or patient sample. In certain embodiments, a kit contains, contains at least or contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more probes, synthetic molecules or inhibitors, or any value or range and combination derivable therein. In some embodiments, there are kits for evaluating biomarker activity in a cell.

[00179] Kits may comprise components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means.

[00180] Individual components may also be provided in a kit in concentrated amounts; in some embodiments, a component is provided individually in the same concentration as it would be in a solution with other components. Concentrations of components may be provided as 1x, 2x, 5x, 10x, or 20x or more.

[00181] Kits for using probes, synthetic nucleic acids, nonsynthetic nucleic acids, and/or inhibitors of the disclosure for prognostic or diagnostic applications are included as part of the disclosure. Specifically contemplated are any such molecules corresponding to any biomarker identified herein.

[00182] In certain aspects, negative and/or positive control nucleic acids, probes, and inhibitors are included in some kit embodiments. The control molecules can be used to verify transfection efficiency and/or control for transfection-induced changes in cells [00183] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined. The claims originally filed are contemplated to cover claims that are multiply dependent on any filed claim or combination of filed claims.

[00184] Any embodiment of the invention involving specific biomarker by name is contemplated also to cover embodiments involving biomarkers whose sequences are at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to the mature sequence of the specified nucleic acid.

[00185] Embodiments of the disclosure include kits for analysis of a pathological sample by assessing biomarker profile for a sample comprising, in suitable container means, two or more biomarker probes, wherein the biomarker probes detect one or more of the biomarkers identified herein. The kit can further comprise reagents for labeling nucleic acids in the sample. The kit may also include labeling reagents, including at least one of amine-modified nucleotide, poly(A) polymerase, and poly(A) polymerase buffer. Labeling reagents can include an amine- reactive dye.

XI. Examples

[00186] 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 which 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 - Intratumoral Fusobacterium nucleatum levels predict therapeutic response to neoadjuvant chemotherapy in esophageal squamous cell carcinoma

[00187] Emerging evidence indicates that gut microbiome plays a crucial role in the cancer pathogenesis. Although Fusobacterium nucleatum (F. nucleatum) is associated with poor prognosis in multiple cancers, its clinical significance in predicting response to chemotherapy in patients with esophageal squamous cell carcinoma (ESCC) remains unclear.

[00188] Methods: The F. nucleatum levels were quantified by qPCR assays in tumor tissues from 551 ESCC patients from two independent cohorts, including 101 patients who received neoadjuvant chemotherapy prior to curative resection. Associations between F. nucleatum evaluated using response evaluation criteria in solid tumors (RECIST), primary tumor metabolic response defined by PET-CT changes in maximum standardized uptake value (SUVmax), and pathological tumor regression grade (TRG).

[00189] Results: High burden of F. nucleatum in ESCC patients associated with poor RFS in both training (log-rank p=0.003; Hazard Ratio [HR]=1.96; p=0.004) and validation cohorts (log-rank p=0.02; HR=1.61; p=0.03). Importantly, ESCC patients with high levels of F. nucleatum displayed poor chemotherapeutic response for all three evaluation methods: RECIST (p=0.04), PET-CT SUVmax (p=0.0004), and TRG (p=0.003).

[00190] Conclusions: The inventors provide first evidence that high intratumoral F. nucleatum levels are prognostic for poor RFS in ESCC. More importantly, the inventors provide a novel evidence that high F. nucleatum in ESCC patient are predictive of poor therapeutic response to neoadjuvant chemotherapy, suggesting the possibility that a simple and inexpensive antibiotic intervention against this bacterium may significantly improve therapeutic response in ESCC patients.

A. INTRODUCTION

[00191] In order to improve treatment response in esophageal cancer patients, it is of paramount importance to elucidate the underlying mechanism(s) that confer chemotherapeutic resistance in these patients. Accordingly, in the present study, the inventors undertook this first effort, and provide novel evidence that increased levels of intratumoral F. nucleatum associate with advanced tumor stage and poor survival. The inventors also observed that higher burden of this microorganism in ESCC tissues predicted relapse-free survival (RFS), as well as associated with poor response to NAC in patients with ESCC.

B. MATERIALS AND METHODS

1. Patients and sample collection procedures

[00192] This study analyzed a total of 551 cases with ESCC, which consisted of two independent clinical cohorts. The first patient cohort (training) comprised of 344 ESCC patients who underwent surgical resections, including 316 with radical surgeries, at the Kumamoto University Hospital, Kumamoto, Japan, between 2005 and 2016. Furthermore, this cohort included 187 patients that experienced surgery alone, 41 who received chemoradiation therapy and 116 patients with neoadjuvant chemotherapy (NAC). Among these 116 patients in the NAC group, 101 patients were treated with two cycles of docetaxel, cisplatin and 5-FU (DCF) and October 2015. The study workflow is summarized in FIG.4. Tumor depth (clinical T1–3) and regional lymph node involvement without distant metastases (N1) were used as the selection criteria for selecting patients for NAC treatment. Relapse-free survival (RFS) was defined as the time period between the date of surgery to the time of tumor recurrence or death. Cancer-specific survival (CSS) was defined as the time duration between the date of surgery and the date of death attributable specifically to esophageal cancer. A written informed consent was obtained from each patient, and the institutional review boards of all participating institutions approved this study. The patient characteristics are summarized in Table 1. The median follow-up duration for all cases after surgery was 43.5 months in the training cohort and 20.4 months in the validation cohort. The pathological diagnosis of all ESCC tumor tissue specimens was confirmed histologically, and the tumor node-metastasis (TNM) staging was determined according to the American Joint Committee on Cancer staging handbook (7th edition) [24], prior to and after surgery.

2. Patient treatments

[00193] The NAC regimen consisted of 2 h intravenous administration of 60 mg/m2 docetaxel beginning on day 1, a 24 h continuous intravenous infusion of 350 mg/m25-FU from days 1 thru 5, and 1 h intravenous administration of 6 mg/m2 cisplatin from days 1 thru 5. Two scheduled courses of NAC regimen were administered 3 weeks apart prior to esophagectomy. Surgery was carried out within 4 to 6 weeks following the final treatment day of preoperative chemotherapy, when curative resection was considered feasible.

3. DNA extraction and quantitative PCR assays

[00194] Genomic DNA from formalin-fixed paraffin-embedded (FFPE) tissues in the training cohort was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). Likewise, genomic DNA from frozen tissue specimens in the validation cohort was extracted using AllPrep DNA/RNA/miRNA Universal Kit (Qiagen). The amount of F. nucleatum DNA was quantified by use of a quantitative PCR (qPCR) assay. The nus G gene of F. nucleatum and the reference human gene SLCO2A1 were amplified using custom TaqMan primer/probe sets (Applied Biosystems, Carlsbad, CA, USA) in 384-well optical PCR plates, as described previously [22].

4. Evaluation of response to chemotherapy using RECIST

[00195] The response to chemotherapy was assessed using response evaluation criteria in all clinical and radiological evidence of the tumor; partial response (PR), as decrease of 30% or more in the sum of longest diameters of all target measurable lesions; progressive disease (PD), as increase of more than 20% of the sum of longest diameters of all target measurable lesions or the appearance of new lesions; and stable disease (SD), as all other indications. Patients with CR or PR were defined as responders, while PD and SD were classified as non- responders.

5. PET/CT imaging

[00196] A total of 86 out of 101 patients who received NAC also underwent positron emission computed tomography (PET/CT) using a hybrid PET/CT imager, consisting of a dedicated GSO full-ring PET scanner and a 16-slice helical CT scanner (Gemini GXL16, Philips Medical Solutions, Amsterdam, Netherlands). All patients fasted for a minimum of 5 h prior to the examination. Emission scans were acquired in a 3D mode, with a 144×144 matrix, 60 min after intravenous injection of 185–300 MBq 18F-fluoro-deoxy-glucose (FDG), immediately after urination. PET/CT transmission data were acquired for the area defined from the base of the skull to the proximal thighs. Standardized uptake value (SUVmax) response was classified as follows [26]: complete metabolic response (CMR), as complete resolution of FDG uptake within the measurable target lesion, with the appearance of no new lesion; partial metabolic response (PMR), with at least 30% reduction in SUVmax of FDG uptake; progressive metabolic disease (PMD), with more than 30% increase in the SUVmax of the FDG uptake or appearance of FDG avid new lesion/s that is/are morphologically typical of cancer; stable metabolic disease (SMD), as disease which did not qualify for CMR, PMR, or PMD. Patients with CMR or PMR were defined as responders.

6. Pathological tumor regression grading criteria

[00197] The histopathological response to NAC was classified into four categories according to the following criteria [27]: grade 1, as no evidence of viable tumor cells; grade 2, with less than 10% viable tumor cells; grade 3, with 11–50% viable tumor cells; and grade 4, with more than 50% viable tumor cells. Subsequently, grade 1-3 tumors were combined as the group of patients with response (TRG 1, 2, 3), while grade 4 tumors were classified as non-responders (TRG 4).

7. Statistical analysis

[00198] All statistical analyses were carried out using JMP, version 10 (SAS Institute, Cary, were calculated using a two-sided test, and a p-value of <0.05 was considered statistically significant. For time-to-event analyses, survival estimates were calculated using the Kaplan- Meier analysis, and the survival differences between groups were compared using the log-rank test. Associations between RFS and clinicopathological features was evaluated by univariate Cox proportional hazards regression analysis. Parameters determined to be significant by univariate analysis were included in multivariate Cox proportional hazards regression analysis. Receiver operating characteristic (ROC) curve values were used to evaluate the performance of F. nucleatum status in tumor tissues for chemotherapeutic response.

C. RESULTS

1. The levels of F. nucleatum are significantly higher in ESCC patients

[00199] The inventors first assessed the burden of F. nucleatum in ESCC tissues by a qPCR assay in two independent patient cohorts, where matched cancer and normal tissues were available. The inventors observed that F. nucleatum DNA levels were significantly higher in cancer tissues compared to the paired adjacent normal tissues in both cohorts (cohort-1; n = 48, P <0.0001, cohort-2; n=45, P=0.006; FIG.1A and 1B, respectively).

[00200] The inventors next analyzed the abundance of F. nucleatum in the training (n=344) and validation (n=207) cohorts, based upon all tumor stages. Interestingly, the inventors observed a marked enrichment of F. nucleatum in ESCC patients with advanced (T2-T4) vs. those with an earlier stage disease (T1), in both cohorts (P <0.05; FIG.1C and 1D).

2. Higher levels of F. nucleatum associated with advanced stage disease in ESCC

[00201] Next, the inventors determined the associations between F. nucleatum burden and various clinicopathological features in two independent ESCC patient cohorts (training cohort; n=344 and validation cohort; n=207). The cut-off thresholds to categorize tumors into the high and low groups were determined using ROC analysis and Youden’s index, based on the level of F. nucleatum that provided the highest sensitivity and specificity to predict ESCC recurrence in the training cohort. The same cut-off values were then applied to the patient in the validation cohort to evaluate survival. The inventors observed that there was no effect of age (p=0.82), gender (p>0.99), tumor location (p=0.42), N stage (p=0.12), or type of surgery (radical or not radical; p=0.24) on the expression of expression of F. nucleatum in the cancer tissues within the training cohort. Similar results were noted in the validation cohort for age (p=0.26), gender (p=062) location (029) and N stage (p>099) However high levels of intratumoral F <0.0001), higher tumor stage (p = 0.035), and in patients who underwent preoperative NAC treatment (p = 0.01) in the training cohort (Table 1). Similarly, in the validation cohort, high intratumoral F. nucleatum levels were associated with higher T factor (p = 0.03) and in patients who received preoperative NAC (p = 0.03). Collectively, the inventors’ results indicate that high levels of F. nucleatum associate with a higher tumor stage and are predict responsiveness to NAC in ESCC patients.

3. Increased burden of F. nucleatum associated with higher tumor recurrence, poor RFS, and serve as a prognostic indicator for early stage ESCC patients

[00202] Considering that presence of F. nucleatum is cancer cells is associated with advanced disease, the inventors were curious to interrogate its relationship with tumor recurrence in ESCC patients. Therefore, the inventors determined the relationship between the F. nucleatum levels and cancer recurrence in 316 ESCC patients in the training cohort, which included 91 patients without recurrence and 225 with recurrence. The inventors noted that the overall levels of F. nucleatum were significantly higher in neoplastic tissues in ESCC patients with recurrence vs. those without (P <0.05; FIG. 5A). Likewise, even within the validation cohort of 207 patients (87 patients with recurrence and 120 without recurrence), the inventors observed a significant association for higher F. nucleatum levels in patients with recurrence (P <0.05; FIG. 5B).

[00203] In order to determine whether intratumoral F. nucleatum burden in ESCC patients is associated with RFS, the inventors performed Kaplan-Meier analysis in both cohorts. Interestingly, patients in the training cohort with high vs. low intratumoral F. nucleatum levels exhibited a significantly poor RFS (log-rank p = 0.003, FIG. 2A); a finding which was also true when interrogated in the independent validation cohort (log-rank p = 0.02, FIG. 2B).

[00204] Since the inventors observed a higher burden of F. nucleatum in advanced ESCC patients (T2-4 vs. Tl), they investigated whether the presence of this bacterium has any effect on patient survival, even in early ESCC. While no significant differences were observed in RFS between the two groups when stratified by the T-factor alone (T2-4 vs. Tl; FIG. 6A and 6B) in both patient cohorts. Importantly however, when the T-factor was combined together with the F. nucleatum levels, the inventors observed that even early stage Tl ESCC patients with high levels of this bacterium exhibited a worse RFS, which was similar to the one noted for patients with advanced disease, in both cohorts (FIG. 2C and 2D). These findings highlight that presence of high levels of F. nucleatum indicate an important prognostic biomarker potential for this bacteria in ESCC patients. 4. High levels of F. nucleatum serve as an independent risk factor for RFS in ESCC patients

[00205] Next, the inventors were curious to investigate the clinical significance of F. nucleatum levels in term of patient survival in the context of other clinicopathological features, using univariate and multivariate analysis, in both patient cohorts. In the training cohort, univariate and Cox regression analysis revealed that patients who received pre-operative NAC (p = 0.02), and those with higher TNM stages (III/IV vs. I/II; p <0.0001), and those with high levels of F. nucleatum were associated with poor RFS (HR=1.96; 95% CI, 1.23–3.04; p = 0.004; Table 2). These findings were further evaluated in a multivariate Cox model adjusted for various clinicopathological features, which were in agreement with the inventors’ univariate analysis and demonstrated that F. nucleatum levels were significantly associated with poor RFS (HR=1.70; 95% CI, 1.06–2.65; p = 0.027), suggesting that this bacterium was indeed an independent risk factor for predicting poor RFS in the patients within the training cohort.

[00206] The inventors subsequently confirmed their findings in an independent validation cohort, wherein, once again they observed that higher burden of F. nucleatum was significantly associated with worse RFS in both, univariate (HR = 1.61; 95% CI, 1.06–2.52; p = 0.03) and multivariate analysis (HR = 1.72; 95% CI, 1.12–270; p = 0.01). Collectively, these data demonstrate that high levels of intratumoral F. nucleatum are an independent risk factor for poor RFS in ESCC patients.

5. Intratumoral F. nucleatum burden correlates with worse chemotherapeutic response in ESCC patients

[00207] The inventors examined whether higher burden of intratumoral F. nucleatum have any correlation with response to NAC in ESCC patients. The inventors first investigated this association in the context of imaging data available to us from the CT scans. Of the 101 patients who underwent NAC treatment, the F. nucleatum-high group had a significantly lower number of responders (i.e. patients with CR or PR; 42.9% (12/28) vs. 67.1% (49/73) in the F. nucleatum-low group; p = 0.04; FIG.3A and 3B).

[00208] Next, the inventors interrogated this correlation as determined by the metabolic response rates determined by SUVmax values obtained from PET-CT imaging. Reassuringly, these analyses also revealed that patients with higher burden of F. nucleatum had a significantly fewer responders (i.e. patients with CMR or PMR; 47.6% (10/21) vs.87.7% (57/65) in the low [00209] Finally, the inventors performed the pathological assessment of all patients based upon tumor regression grade (TRG) analysis. In these analysis, the inventors noted that F. nucleatum levels were significantly higher in ESCC patients with a low vs. high pathological response (TRG 4 vs. TRG 1, 2 and 3; p = 0.003; FIG.3E and 3F). Taken together, these results illustrate that patients with high intratumoral levels of F. nucleatum appear to have greater resistance to NAC treatment.

6. High levels of F. nucleatum serve as an independent risk factor for predicting response to neoadjuvant chemotherapy in ESCC patients

[00210] Next, the inventors analyzed the results of CT (RECIST), PET-CT and TRG in univariate and multivariate settings to determine the clinical significance of F. nucleatum as a potential biomarker of chemotherapeutic response in ESCC patients. The univariate logistic regression analysis revealed that higher levels of F. nucleatum associated with an overall poor chemotherapeutic response to NAC in all three approaches (RECIST: Odds Ratio [OR], 2.72; 95% CI 1.12–6.78, p = 0.027; PET-CT: OR, 7.84; 95% CI 2.58–25.4, p = 0.0003; and TRG: OR, 11.6; 95% CI 2.25–214, p = 0.0013; Table 3).

[00211] Likewise, multivariate analysis also revealed that high levels of intratumoral F. nucleatum burden was an independent risk factor for poor response to NAC in all three criteria (RECIST: OR, 2.97; 95% CI 1.19–7.73, p = 0.02; PET-CT: OR, 7.66, 95% CI 2.37–26.8, p = 0.0006; and TRG: OR, 10.3; 95% CI 1.96–190, p = 0.003). Collectively, these results illustrate that F. nucleatum is an important independent risk factor and a potential biomarker for predicting response to NAC in ESCC patients.

D. DISCUSSION

[00212] In this present study, the inventors for the first time, interrogated the clinical significance of F. nucleatum as a potential prognostic and predictive biomarker of response to neoadjuvant chemotherapy in large, multiple, independent cohort of ESCC patients. In this study, the inventors make several novel observations. First, the inventors demonstrate that F. nucleatum is significantly overexpressed in cancer vs. normal tissues, and that its burden is significantly higher in ESCC patients with advanced disease stage. Second, the inventors describe that higher levels of this bacterium are present in patients with recurrence, and are an independent risk factor for predicting poor RFS in ESCC. Third, the inventors illustrate that using RECIST, PET-CT and TRG analysis, higher burden of F. nucleatum predicts poor response to neoadjuvant chemotherapy (NAC) in ESCC patients; collectively highlighting the possibility of using an antibiotic intervention to target this bacterium for improving the therapeutic response rates to chemotherapy in ESCC patients.

[00213] To the best of the inventors’ knowledge, no previous studies have thus far evaluated the clinical significance of F. nucleatum in the context of responsiveness to chemotherapeutic treatment in cancer patients. Herein, the inventors fill this important gap in knowledge, and evaluated therapeutic response using three commonly used and well-established approaches for drug resistance in ESCC patients. In spite of the use of RECIST as one of the most widely used tumor response metric [25], it has several limitations due to its dependence on morphologic changes [37]. RECIST criteria can often select lymph nodes as target lesions in ESCC patients. In contrast, 18F-FDG PET is considered as a superior method which overcomes the limitations of RECIST. Since metabolic changes are thought to be more closely related to malignant potential of tumors [38], PET-CT is emerging as a more accurate non-invasive imaging modality for initial staging and response assessment in ESCC patients [39]. Based on these findings, PET response criteria in solid tumors (PERCIST), which is RECIST using 18F- FDG PET, has recently been proposed as an optimal method for standardized evaluation of the metabolic tumor response rates [37].

[00214] In the present study, the inventors observed significant differences between response classifications and F. nucleatum levels in ESCC tissues. Interestingly, PET response and tumor regression grade (TRG) were more strongly associated with F. nucleatum levels compared to RECIST, in ESCC patients receiving NAC treatment. While RECIST in ESCC patients primarily evaluates shrinkage of lymph nodes, PET and TRG reflect the response of the primary tumor itself. In this study, since F. nucleatum levels in tumor tissues correlate with higher T factor, the inventors’ data imply that this bacterium might be involved in modulating chemotherapeutic response more directly.

[00215] In conclusion, the inventors for the first time demonstrate that high intratumoral F. nucleatum levels associated with tumor recurrence and poor RFS in two large, independent cohorts of ESCC patients. More importantly, the inventors provide a novel evidence that high burden of F. nucleatum in ESCC is predictive of response to neoadjuvant chemotherapy. Collectively, the inventors’ data highlight that F. nucleatum is not only an important predictive biomarker of chemotherapeutic response, but might be a potential target of antibiotic intervention for improving the therapeutic response rates in ESCC patients. E. TABLES

Table 1. F. nucleatum expression level in ESCC and clinicopathological features

Table 2. Cox proportional hazards regression analysis for prediction of RFS

Table 3. Logistic regression analysis for chemoresponse

[00216] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. All references and publications referred to throughout the disclosure are incorporated by reference for all purposes. REFERENCES

[00217] The following references and the publications referred to throughout the specification, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. 1. Bray F, Ferlay J, Soerjomataram I et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018.

2. Pennathur A, Gibson MK, Jobe BA, Luketich JD. Oesophageal carcinoma. Lancet 2013; 381: 400-412.

3. Smyth EC, Lagergren J, Fitzgerald RC et al. Oesophageal cancer. Nat Rev Dis Primers 2017; 3: 17048.

4. Rustgi AK, El-Serag HB. Esophageal carcinoma. N Engl J Med 2014; 371: 2499-2509. 5. Arnold M, Soerjomataram I, Ferlay J, Forman D. Global incidence of oesophageal cancer by histological subtype in 2012. Gut 2015; 64: 381-387.

6. van Hagen P, Hulshof MC, van Lanschot JJ et al. Preoperative chemoradiotherapy for esophageal or junctional cancer. N Engl J Med 2012; 366: 2074-2084.

7. Ando N, Kato H, Igaki H et al. A randomized trial comparing postoperative adjuvant chemotherapy with cisplatin and 5-fluorouracil versus preoperative chemotherapy for localized advanced squamous cell carcinoma of the thoracic esophagus (JCOG9907). Ann Surg Oncol 2012 19 68 74 chemoradiotherapy or chemotherapy in oesophageal carcinoma: a meta-analysis. Lancet Oncol 2007; 8: 226-234.

9. Yamashita K, Hosoda K, Moriya H et al. Prognostic Advantage of Docetaxel/Cisplatin/ 5-Fluorouracil Neoadjuvant Chemotherapy in Clinical Stage II/III Esophageal Squamous Cell Carcinoma due to Excellent Control of Preoperative Disease and Postoperative Lymph Node Recurrence. Oncology 2017; 92: 221-228.

10. Ancona E, Ruol A, Santi S et al. Only pathologic complete response to neoadjuvant chemotherapy improves significantly the long term survival of patients with resectable esophageal squamous cell carcinoma: final report of a randomized, controlled trial of preoperative chemotherapy versus surgery alone. Cancer 2001; 91: 2165-2174.

11. Hayashi K, Ando N, Watanabe H et al. Phase II evaluation of protracted infusion of cisplatin and 5-fluorouracil in advanced squamous cell carcinoma of the esophagus: a Japan Esophageal Oncology Group (JEOG) Trial (JCOG9407). Jpn J Clin Oncol 2001; 31: 419-423. 12. Watanabe M, Baba Y, Yoshida N et al. Outcomes of preoperative chemotherapy with docetaxel, cisplatin, and 5-fluorouracil followed by esophagectomy in patients with resectable node-positive esophageal cancer. Ann Surg Oncol 2014; 21: 2838-2844.

13. McColl KE. Clinical practice. Helicobacter pylori infection. N Engl J Med 2010; 362: 1597-1604.

14. Arthur JC, Perez-Chanona E, Muhlbauer M et al. Intestinal inflammation targets cancer-inducing activity of the microbiota. Science 2012; 338: 120-123.

15. Garrett WS. Cancer and the microbiota. Science 2015; 348: 80-86.

16. Schwabe RF, Jobin C. The microbiome and cancer. Nat Rev Cancer 2013; 13: 800-812. 17. Routy B, Le Chatelier E, Derosa L et al. Gut microbiome influences efficacy of PD-1- based immunotherapy against epithelial tumors. Science 2017.

18. Iida N, Dzutsev A, Stewart CA et al. Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment. Science 2013; 342: 967-970.

19. Castellarin M, Warren RL, Freeman JD et al. Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma. Genome Res 2012; 22: 299-306.

20. Kostic AD, Chun E, Robertson L et al. Fusobacterium nucleatum potentiates intestinal tumorigenesis and modulates the tumor-immune microenvironment. Cell Host Microbe 2013; 14: 207-215.

21. Mima K, Nishihara R, Qian ZR et al. Fusobacterium nucleatum in colorectal carcinoma nucleatum in Esophageal Cancer Tissue Is Associated with Prognosis. Clin Cancer Res 2016; 22: 5574-5581.

23. Yu T, Guo F, Yu Y et al. Fusobacterium nucleatum Promotes Chemoresistance to Colorectal Cancer by Modulating Autophagy. Cell 2017; 170: 548-563.e516.

24. Rice TW, Blackstone EH, Rusch VW.7th edition of the AJCC Cancer Staging Manual: esophagus and esophagogastric junction. Ann Surg Oncol 2010; 17: 1721-1724.

25. Eisenhauer EA, Therasse P, Bogaerts J et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009; 45: 228-247.

26. Izumi D, Yoshida N, Watanabe M et al. Tumor/normal esophagus ratio in (18)F- fluorodeoxyglucose positron emission tomography/computed tomography for response and prognosis stratification after neoadjuvant chemotherapy for esophageal squamous cell carcinoma. J Gastroenterol 2016; 51: 788-795.

27. Chirieac LR, Swisher SG, Ajani JA et al. Posttherapy pathologic stage predicts survival in patients with esophageal carcinoma receiving preoperative chemoradiation. Cancer 2005; 103: 1347-1355.

28. Griffen AL, Beall CJ, Campbell JH et al. Distinct and complex bacterial profiles in human periodontitis and health revealed by 16S pyrosequencing. Isme j 2012; 6: 1176-1185. 29. Yamaoka Y, Suehiro Y, Hashimoto S et al. Fusobacterium nucleatum as a prognostic marker of colorectal cancer in a Japanese population. J Gastroenterol 2018; 53: 517-524. 30. Flanagan L, Schmid J, Ebert M et al. Fusobacterium nucleatum associates with stages of colorectal neoplasia development, colorectal cancer and disease outcome. Eur J Clin Microbiol Infect Dis 2014; 33: 1381-1390.

31. Viaud S, Saccheri F, Mignot G et al. The intestinal microbiota modulates the anticancer immune effects of cyclophosphamide. Science 2013; 342: 971-976.

32. Sivan A, Corrales L, Hubert N et al. Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy. Science 2015; 350: 1084-1089.

33. Vetizou M, Pitt JM, Daillere R et al. Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota. Science 2015; 350: 1079-1084.

34. Wang W, Kryczek I, Dostal L et al. Effector T Cells Abrogate Stroma-Mediated Chemoresistance in Ovarian Cancer. Cell 2016; 165: 1092-1105.

35. Lehouritis P, Cummins J, Stanton M et al. Local bacteria affect the efficacy of chemotherapeutic drugs. Sci Rep 2015; 5: 14554. 81: 773-782.

37. Wahl RL, Jacene H, Kasamon Y, Lodge MA. From RECIST to PERCIST: Evolving Considerations for PET response criteria in solid tumors. J Nucl Med 2009; 50 Suppl 1: 122s- 150s.

38. Juweid ME, Cheson BD. Positron-emission tomography and assessment of cancer therapy. N Engl J Med 2006; 354: 496-507.

39. Sloof GW. Response monitoring of neoadjuvant therapy using CT, EUS, and FDG-PET. Best Pract Res Clin Gastroenterol 2006; 20: 941-957.

40. Yang Y, Weng W, Peng J et al. Fusobacterium nucleatum Increases Proliferation of Colorectal Cancer Cells and Tumor Development in Mice by Activating Toll-Like Receptor 4 Signaling to Nuclear Factor-kappaB, and Up-regulating Expression of MicroRNA-21. Gastroenterology 2017; 152: 851-866.e824.

41. Rubinstein MR, Wang X, Liu W et al. Fusobacterium nucleatum promotes colorectal carcinogenesis by modulating E-cadherin/beta-catenin signaling via its FadA adhesin. Cell Host Microbe 2013; 14: 195-206.

42. Geller LT, Barzily-Rokni M, Danino T et al. Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine. Science 2017; 357: 1156-1160.

Claims

CLAIMS 1. A method involving a esophageal or colorectal cancer patient comprising measuring the number of Fusobacterium nucleatum in a biological sample from an esophageal or colorectal cancer patient.

2. The method of claim 1, wherein the biological sample is a stool sample, a tissue sample, a tumor sample, or blood.

3. The method of claim 1, further comprising administering chemotherapy to the patient after measuring the number of F. nucleatum in the biological sample.

4. The method of claim 3, wherein the chemotherapy comprises i) 5-fluorouracil or a 5- FU-based compound and ii) cisplatin or other platinum-based compound.

5. The method of claim 4, wherein the chemotherapy further comprises docetaxel or other taxel-based compound.

6. The method of claim 1 or 3, further comprising administering an antibiotic for F. nucleatum.

7. The method of claim 5, wherein the antibiotic comprises metronidazole, piperacillin, ticarcillin, clavulanate, amoxicillin, sulbactam, ampicillin, ertapenem, imipenem, metropenem, clindamycin, cefoxitin, salts thereof, and/or combinations thereof.

8. The method of claim 6 or 7, wherein the one or more antibiotics is administered within 1, 2, 3, or 4 weeks prior to administering chemotherapy to the patient.

9. The method of any of claims 1-7, wherein a low number of F. nucleatum is measured as compared to a control level or sample.

10. The method of claim 9, wherein the control level or sample is representative of the number of F. nucleatum in a cohort of esophageal cancer patients with increased relapse-free survival.

11. The method of claim 9, wherein the control level or sample is representative of the number of F. nucleatum in a cohort of esophageal cancer patients with decreased relapse-free survival.

12. The method of any of claims 1-7, wherein a high number of F. nucleatum is measured as compared to a control level or sample.

13. The method of claim 12, wherein the control level or sample is representative of the number of F. nucleatum in a cohort of esophageal cancer patients with increased relapse-free

14. The method of claim 12, wherein the control level or sample is representative of the number of F. nucleatum in a cohort of esophageal cancer patients with decreased relapse-free survival.

15. The method of any of claims 1-14, further comprising performing positron emission tomography on the patient to evaluate any tumor.

16. The method of any of claims 1-15, wherein the patient has stage I, II, III, or IV cancer.

17. The method of claim 16, wherein the patient has stage II or III cancer.

18. The method of any of claims 1-17, wherein a cohort comprises at least 50, 100, 200, 300, 400, 500 or more patients.

19. A method for predicting length of relapse free survival in an esophageal cancer patient comprising measuring the number of F. nucleatum in a biological sample from the patient, wherein a patient with a low number of F. nucleatum is predicted to have a longer time of relapse free survival than a patient with a high number of F. nucleatum.

20. The method of claim 19, wherein the biological sample is a stool sample, a tissue sample, a tumor sample, or blood.

21. The method of claim 19 or 20, further comprising administering chemotherapy to the patient after measuring the number of F. nucleatum in the biological sample.

22. The method of claim 21, wherein the chemotherapy comprises i) 5-fluorouracil or a 5- FU-based compound and ii) cisplatin or other platinum-based compound.

23. The method of claim 22, wherein the chemotherapy further comprises docetaxel or other taxel-based compound.

24. The method of claim 19 or 21, further comprising administering one or more antibiotic(s) for F. nucleatum.

25. The method of claim 23, wherein the antibiotic(s) comprises metronidazole, piperacillin, ticarcillin, clavulanate, amoxicillin, sulbactam, ampicillin, ertapenem, imipenem, metropenem, clindamycin, cefoxitin, salts thereof, and/or combinations thereof.

26. The method of claim 24 or 25, wherein the one or more antibiotics is administered within 1, 2, 3, or 4 weeks prior to administering chemotherapy to the patient.

27. The method of claim 24 or 25 or 26, further comprising measuring the number of F. nucleatum in a sample from the patient after antibiotic(s) have been administered to the patient.

29. The method of claim 28, wherein the control level or sample is representative of the number of F. nucleatum in a cohort of esophageal cancer patients with increased relapse-free survival.

30. The method of claim 28, wherein the control level or sample is representative of the number of F. nucleatum in a cohort of esophageal cancer patients with decreased relapse-free survival.

31. The method of any of claims 19-25, wherein a high number of F. nucleatum is measured as compared to a control level or sample.

32. The method of claim 31, wherein the control level or sample is representative of the number of F. nucleatum in a cohort of esophageal cancer patients with increased relapse-free survival.

33. The method of claim 31, wherein the control level or sample is representative of the number of F. nucleatum in a cohort of esophageal cancer patients with decreased relapse-free survival.

34. The method of any of claims 19-33, further comprising performing positron emission tomography on the patient to evaluate any tumor.

35. The method of any of claims 19-34, wherein the patient has stage I, II, III, or IV cancer.

36. The method of claim 35, wherein the patient has stage II or III cancer.

37. The method of any of claims 19-36, wherein a cohort comprises at least 50, 100, 200, 300, 400, 500 or more patients.

38. A method for treating a patient with a gastrointestinal cancer comprising

administering one or more antibiotics to the patient prior to administering chemotherapy to the patient, wherein a biological sample from the patient has been measured for F. nucleatum. 39. The method of claim 38, wherein the biological sample is a stool sample, a tissue sample, a tumor sample, or blood.

40. The method of claim 38 or 39, further comprising administering chemotherapy to the patient after measuring the number of F. nucleatum in the biological sample.

41. The method of claim 40, wherein the chemotherapy comprises i) 5-fluorouracil or a 5- FU-based compound and ii) cisplatin or other platinum-based compound.

42. The method of claim 41, wherein the chemotherapy further comprises docetaxel or other taxel-based compound.

44. The method of claim 42, wherein the antibiotic(s) comprises metronidazole, piperacillin, ticarcillin, clavulanate, amoxicillin, sulbactam, ampicillin, ertapenem, imipenem, metropenem, clindamycin, cefoxitin, salts thereof, and/or combinations thereof.

45. The method of claim 43 or 44, wherein the one or more antibiotics is administered within 1, 2, 3, or 4 weeks prior to administering chemotherapy to the patient.

46. The method of claim 43 or 44 or 45, further comprising measuring the number of F. nucleatum in a sample from the patient after antibiotic(s) have been administered to the patient.

47. The method of any of claims 38-44, wherein a low number of F. nucleatum is measured as compared to a control level or sample.

48. The method of claim 47, wherein the control level or sample is representative of the number of F. nucleatum in a cohort of esophageal cancer patients with increased relapse-free survival.

49. The method of claim 47, wherein the control level or sample is representative of the number of F. nucleatum in a cohort of esophageal cancer patients with decreased relapse-free survival.

50. The method of any of claims 38-44, wherein a high number of F. nucleatum is measured as compared to a control level or sample.

51. The method of claim 50, wherein the control level or sample is representative of the number of F. nucleatum in a cohort of esophageal cancer patients with increased relapse-free survival.

52. The method of claim 50, wherein the control level or sample is representative of the number of F. nucleatum in a cohort of esophageal cancer patients with decreased relapse-free survival.

53. The method of any of claims 38-52, further comprising performing positron emission tomography on the patient to evaluate any tumor.

54. The method of any of claims 38-53, wherein the patient has stage I, II, III, or IV cancer.

55. The method of claim 54, wherein the patient has stage II or III cancer.

56. A method for increasing the time of relapse-free survival in an esophageal or colorectal cancer patient comprising:

a) measuring the number of F. nucleatum in a biological sample from the patient; c) administering chemotherapy to the patient, wherein the patient has increased length of time of relapse-free survival.

57. The method of claim 56, wherein the biological sample is a stool sample, a tissue sample, a tumor sample, or blood.

58. The method of claim 56, wherein the chemotherapy comprises i) 5-fluorouracil or a 5- FU-based compound and ii) cisplatin or other platinum-based compound.

59. The method of claim 58, wherein the chemotherapy further comprises docetaxel or other taxel-based compound.

60. The method of claim 56, wherein the antibiotic(s) comprises metronidazole, piperacillin, ticarcillin, clavulanate, amoxicillin, sulbactam, ampicillin, ertapenem, imipenem, metropenem, clindamycin, cefoxitin, salts thereof, and/or combinations thereof.

61. The method of any of claims 56-60, wherein the one or more antibiotics is

administered within 1, 2, 3, or 4 weeks prior to administering chemotherapy to the patient. 62. The method of any of claims 56-61, further comprising measuring the number of F. nucleatum in a sample from the patient after antibiotic(s) have been administered to the patient.

63. The method of any of claims 56-62, wherein a low number of F. nucleatum is measured as compared to a control level or sample.

64. The method of claim 63, wherein the control level or sample is representative of the number of F. nucleatum in a cohort of esophageal cancer patients with increased relapse-free survival.

65. The method of claim 63, wherein the control level or sample is representative of the number of F. nucleatum in a cohort of esophageal cancer patients with decreased relapse-free survival.

66. The method of any of claims 56-60, wherein a high number of F. nucleatum is measured as compared to a control level or sample.

67. The method of claim 66, wherein the control level or sample is representative of the number of F. nucleatum in a cohort of esophageal cancer patients with increased relapse-free survival.

68. The method of claim 66, wherein the control level or sample is representative of the number of F. nucleatum in a cohort of esophageal cancer patients with decreased relapse-free survival.

70. The method of any of claims 56-69, wherein the patient has stage I, II, III, or IV cancer.

71. The method of claim 70, wherein the patient has stage II or III cancer.

72. A method for treating a patient with a esophageal cancer comprising administering one or more antibiotics against F. nucleatum to the patient prior to administering

chemotherapy to the patient, wherein a tumor sample from the patient has been measured for F. nucleatum.

73. The method of claim 72, wherein the biological sample is a stool sample, a tissue sample, a tumor sample, or blood.

74. The method of claim 72 or 73, further comprising administering chemotherapy to the patient after measuring the number of F. nucleatum in the biological sample.

75. The method of claim 74, wherein the chemotherapy comprises i) 5-fluorouracil or a 5- FU-based compound and ii) cisplatin or other platinum-based compound.

76. The method of claim 75, wherein the chemotherapy further comprises docetaxel or other taxel-based compound.

77. The method of claim 72 or 74, further comprising administering one or more antibiotic(s) for F. nucleatum.

78. The method of claim 76, wherein the antibiotic(s) comprises metronidazole, piperacillin, ticarcillin, clavulanate, amoxicillin, sulbactam, ampicillin, ertapenem, imipenem, metropenem, clindamycin, cefoxitin, salts thereof, and/or combinations thereof.

79. The method of claim 77 or 78, wherein the one or more antibiotics is administered within 1, 2, 3, or 4 weeks prior to administering chemotherapy to the patient.

80. The method of claim 77 or 78 or 79, further comprising measuring the number of F. nucleatum in a sample from the patient after antibiotic(s) have been administered to the patient.

81. The method of any of claims 72-78, wherein a low number of F. nucleatum is measured as compared to a control level or sample.

82. The method of claim 81, wherein the control level or sample is representative of the number of F. nucleatum in a cohort of esophageal cancer patients with increased relapse-free survival.

83. The method of claim 81, wherein the control level or sample is representative of the number of F. nucleatum in a cohort of esophageal cancer patients with decreased relapse-free

84. The method of any of claims 72-78, wherein a high number of F. nucleatum is measured as compared to a control level or sample.

85. The method of claim 84, wherein the control level or sample is representative of the number of F. nucleatum in esophageal cancer patients with increased relapse-free survival. 86. The method of claim 84, wherein the control level or sample is representative of the number of F. nucleatum in esophageal cancer patients with increased relapse-free survival. 87. The method of any of claims 72-86, further comprising performing positron emission tomography on the patient to evaluate any tumor.

88. The method of any of claims 72-87, wherein the patient has stage I, II, III, or IV cancer.

89. The method of claim 88, wherein the patient has stage II or III cancer.

90. The method of any of claims 72-89, wherein a cohort comprises at least 50, 100, 200, 300, 400, 500, or more patients.