Improved method for screening, diagnosis and / or monitoring of colorectal advanced neoplasia, advanced adenoma and / or colorectal cancer

Abstract

To provide a method for increasing specificity or decreasing false positive results in the fecal occult blood test (FOBT) for the screening, diagnosis and / or monitoring of colorectal advanced neoplasia (AN), advanced adenoma (AA) and / or colorectal cancer (CRC), wherein AN encompasses CRC and AA.SOLUTION: The present invention provides a method that comprises: a. conducting an FOBT in a feces sample isolated from a subject, wherein the FOBT comprises the determination of presence of occult blood in the feces sample; and b. quantifying in a feces sample isolated from the subject at least the following bacterial markers: Peptostreptococcus stomatis (PTST), Bacteroides fragilis (BCTF), and Bacteroides thetaiotaomicron (BCTT).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the field of cancer diagnosis. In particular, the present invention relates to an improved method for screening, diagnosing, and / or monitoring colorectal advanced neoplasia (AN), advanced adenoma (AA), and / or colorectal cancer (CRC), where AN includes CRC and AA. In particular, the method provides increased specificity by reducing false positive results in fecal occult blood testing (FOBT) using a signature based on bacterial markers. The present invention further relates to the use of the method in selecting subjects for exploratory testing (e.g., colonoscopy) or treatment with anti-cancer therapeutics. [Background technology]

[0002] CRC is the third most common cancer in men and the second most common cancer in women worldwide, and is the leading cause of cancer deaths (Non-Patent Document 1). By the age of 75, 6% of individuals are affected by CRC, and the incidence rate is much higher in developed countries than in developing countries (Non-Patent Document 2). Although the cancer shows a strong genetic component, most CRCs are sporadic and progress slowly (Non-Patent Document 3). Most patients do not show symptoms until advanced disease appears. Regular bowel cancer screening has been shown to reduce the risk of death from CRC by 26% when fecal testing is used and by up to 50% when flexible sigmoidoscopy is applied (Non-Patent Document 4, Non-Patent Document 5). However, CRC screening programs are only implemented in a few countries around the world.

[0003] Guidelines recommend regular screening for CRC in asymptomatic adults starting at age 50 (Non-Patent Document 6). Current screening programs are based on two strategies: invasive methods based on endoscopy and non-invasive methods based on fecal testing (Non-Patent Document 7). There are various endoscopic techniques for CRC screening, such as flexible sigmoidoscopy and colonoscopy. The main advantage of colonoscopy is that it allows direct visualization of the entire colon, which allows for accurate diagnosis and therefore the prevention of CRC through early detection of precancerous lesions (Non-Patent Document 8). Nevertheless, this technique requires bowel preparation and sedation, has the risk of intestinal perforation and other adverse effects, and is time-consuming and expensive (Non-Patent Document 9, Non-Patent Document 10). Flexible sigmoidoscopy is an alternative approach that allows direct visualization of the distal colon, i.e., the rectum, sigmoid colon, and descending colon. This method avoids sedation and reduces the risk of intestinal perforation (Non-Patent Document 11). Fecal testing is becoming the preferred CRC screening method in most countries due to its non-invasive nature and low cost compared to endoscopic strategies. Subjects with positive results from any of the alternative fecal tests generally undergo colonoscopy to make an accurate diagnosis. Therefore, non-invasive testing is often used as a preliminary step in the screening approach.

[0004] One of the fecal tests used in screening programs is the fecal occult blood test using the guaiac method (guaiac fecal occult blood test, gFOBT), which is based on a chemical oxidation reaction between heme and α-guaiaconic acid. The main disadvantage of these types of tests is that they require a regulated diet to avoid false positive results that may occur due to the intake of certain foods, alcohol, or nonsteroidal anti-inflammatory drugs (Non-Patent Document 12). Although the test is non-invasive, it has been found to have low sensitivity for CRC (25%-38%) and precancerous lesions (16%-31%) (Non-Patent Document 13). The fecal immunochemical test (FIT) was developed to overcome the low sensitivity of gFOBT. This test is specific for human blood hemoglobin and does not require dietary restrictions. Several studies have shown that the FIT screening is more effective than gFOBT. It has been demonstrated that FIT has a higher sensitivity (Non-Patent Document 14, Non-Patent Document 15, Non-Patent Document 16, Non-Patent Document 17). The overall sensitivity of FIT is about 61%-91% for CRC and 27%-67% for advanced adenomas (Non-Patent Document 13), but these figures still represent a high false positive rate.

[0005] Recently, it has been reported that the bacterial communities in the intestinal mucosa of CRC patients are different from those of healthy individuals (Non-Patent Document 18, Non-Patent Document 19). Evidence suggests that the gut microbiota may play an important role in the pathogenesis of CRC (Non-Patent Document 20, Non-Patent Document 21). So far, two possible mechanisms by which gut microbiota may induce tumor development have been described. On the one hand, gut microbiota may promote chronic inflammation, which may further lead to tumor formation (Non-Patent Document 22, Non-Patent Document 23). On the other hand, it has been demonstrated that some dietary components metabolized by gut microbiota, such as red meat, lead to the production of carcinogenic compounds (Non-Patent Document 24). Furthermore, many studies have been conducted recently to elucidate whether there is a CRC-specific gut dysbiosis or any specific species that can be associated with CRC progression (Non-Patent Document 25, Non-Patent Document 26, Non-Patent Document 27).

[0006] In 2012, a preliminary prospective study conducted by our group on 60 individuals (41 CRC patients and 19 patients with normal colonoscopy) defined bacterial clusters in mucosal biopsy samples whose prevalence correlated with CRC risk (28). Among the 55 phylotypes analyzed, six showed significantly higher frequencies in CRC patients than in control subjects. Five of them shared similarity with uncultured bacterial sequences recovered from the human gastrointestinal tract or human feces, and one shared 97% similarity with Parabacteroides merdae. In contrast, there were two phylotypes, B34 (99% similarity with Clostridium nexile) and B35 (97% similarity with Roseburia faecalis), which were more prevalent in healthy subjects than in CRC patients.

[0007] Our group then designed a quantitative polymerase chain reaction system (qPCR) specifically targeting these bacterial markers (Non-Patent Document 28). Later, we tested the bacterial signatures on stool samples (from healthy controls to seven and nine CRC patients) at different abundances to see which ones were suitable for use as a non-invasive tool for CRC screening (Patent Document 1). Dr. Josep Trueta, Hospital Universitari de Girona, Girona, Spain A retrospective clinical study including 46 patients from the National Cancer Institute (NHI) and the National Cancer Center (NCI) confirmed that several bacterial signatures are suitable as CRC markers (Patent Document 1). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2015 / 132273 [Non-patent literature]

[0009] [Non-licensed document 1] Estimated Cancer Incidence, Mortality and Prevalence Worldwide in 2012, http: / / globocan.iarc.fr / Pages / fact_sheets_population.aspx. [Non-licensed document 2] Wilson, 2005 [Non-licensed document 3] Brenner et al, 2014

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Non-licensed literature 9

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[0010] Despite recent advances, there is still a need to find new tools for the early detection of advanced colorectal neoplasms through population-based screening and surveillance strategies to reduce CRC mortality. An ideal technology should be noninvasive, cost-effective, reproducible, and capable of detecting precancerous lesions with high risk of tumor progression with high sensitivity and specificity values. [Means for solving the problem]

[0011] The present invention provides a new non-invasive AA, CRC, and / or AN screening, diagnostic, and / or monitoring test based on a new bacterial signature that can complement fecal occult blood tests (e.g., FITs) and reduce the false positive results associated with FOBTs, thus increasing their specificity and positive predictive value (PPV) while maintaining a similar level of sensitivity.

[0012] A first aspect of the present invention provides a method of increasing the specificity or reducing the false positive rate of a fecal occult blood test (FOBT) for screening, diagnosing, and / or monitoring advanced colorectal neoplasia (AN), advanced adenoma (AA), and / or colorectal cancer (CRC) in a subject, comprising: a) optionally performing a FOBT on a fecal sample isolated from the subject; wherein said FOBT involves determining the presence of occult blood in said fecal sample; b) In an intestinal sample (preferably a fecal sample) isolated from said subject, the following bacterial markers are detected: Gemella Morbillorum (GMLL), Peptostreptococcus stomatis (PT ST), and Bacteroides fragilis (BCTF), or any combination thereof; and The present invention relates to a method comprising the steps of:

[0013] In a related aspect, the invention provides a method of screening, diagnosing, and / or monitoring AN, AA, and / or CRC in a subject, comprising: a) optionally performing a FOBT on a fecal sample isolated from the subject; wherein the FOBT includes determining the presence of occult blood in the fecal sample; b) In an intestinal sample (preferably a fecal sample) isolated from said subject, the following bacterial markers are detected: Gemella morbilorum (GMLL), Peptostreptococcus stomatis (PTST), and Bacteroides fragilis (BCTF), or any combination thereof; and The present invention relates to a method comprising the steps of:

[0014] In a further aspect, the present invention provides a method of treating a subject having AN, AA, and / or CRC, wherein the subject has been selected by the screening, diagnostic, and / or monitoring methods of other aspects of the present invention, and the method further comprises administering to the subject an anti-cancer therapeutic agent.

[0015] In a related aspect, the invention also provides any of the methods described in the other aspects, further comprising the step of administering to said patient a therapeutically effective amount of an anti-cancer therapy.

[0016] In another aspect, the invention relates to an anti-cancer therapeutic for use in a method of treating a cancer patient, said cancer patient having been selected by the screening, diagnostic and / or monitoring methods of other aspects of the invention.

[0017] In an additional embodiment, the present invention also relates to a method for selecting a subject to perform an exploratory test selected from the group consisting of colonoscopy, flexible sigmoidoscopy, double contrast barium enema, and computed tomography (CT) colonography, preferably to perform a colonoscopy, wherein said subject has been selected by the screening, diagnostic, or monitoring method of the other embodiment of the present invention.

[0018] In yet a further aspect, the present invention relates to a method of performing an exploratory test in a subject, said subject having been selected by the screening, diagnostic or monitoring method of the other aspects of the present invention, said method further comprising performing an exploratory test in the subject, said test being selected from the group consisting of colonoscopy, flexible sigmoidoscopy, double contrast barium enema, and computed tomography (CT) colonography, preferably colonoscopy.

[0019] In another aspect, the present invention relates to a kit suitable for quantifying any of GMLL, PTST, BCTF, or BCTT, comprising the following reagents: i. an oligonucleotide specific for the GMLL genome, preferably an oligonucleotide specific for SEQ ID NO:1; ii. an oligonucleotide specific for the PTST genome, preferably an oligonucleotide specific for SEQ ID NO:2; iii. an oligonucleotide specific for the BCTF genome, preferably specific for SEQ ID NO:3; iv. an oligonucleotide specific for the BCTT genome, preferably an oligonucleotide specific for SEQ ID NO: 4, and v. optionally an oligonucleotide suitable for the quantification of eubacteria as defined herein above, The kit includes one or more of the following:

[0020] In further aspects, the present invention also provides kits as described herein for use in the methods of screening, diagnosing, and / or monitoring AN, AA, and / or CRC described herein. [Brief description of the drawings]

[0021] [Figure 1]Analyzed biological markers for subjects with normal colonoscopy (NC), non-advanced adenoma (NAA), advanced adenoma (AA), and colorectal cancer (CRC) were: B10 (Best match BLAST for Faecalibacterium prausnitzii), B46 (Best match BLAST for Faecalibacterium prausnitzii, Subdoligranulum variabile), B48 (Best match BLAST for Ruminococcus sp., Roseburia sp., Coprococcus sp.), Gemella Morbillorum (GMLL), Peptostreptococcus stomatis (PTST), Bacteroides fragilis (Bacteroides Figure 1 shows the relative abundance as percentages of Bacteroides fragilis (BCTF), Collinsella intestinalis (CINT), Bacteroides thetaiotaomicron (BCTT), and Roseburia intestinalis (RSBI). [Diagram 2] Figure 1. Comparison of biomarker abundance between different diagnoses. NC, normal colonoscopy; neoplasm, non-advanced adenoma + advanced adenoma + colorectal cancer; advanced neoplasm, advanced adenoma + colorectal cancer; CRC, colorectal cancer. Significance levels: * if p-value < 0.05, ** if p-value < 0.01, *** if p-value < 0.001. [Diagram 3]Diagram showing the relative abundances in percentage of the analyzed biological markers (butyrate-producing species: B10, B46, B48, and Roseburia intestinalis (RSBI); opportunistic pathogens: Gemella morbillorum (GMLL), Peptostreptococcus stomatis (PTST), and Bacteroides fragilis (BCTF); H2- and O2-producing bacteria: Collinsella intestinalis (CINT); and saccharolytic bacteria: Bacteroides thetaiotaomicron (BCTT)) in subjects with normal colonoscopy (NC), non-advancing adenoma (NAA), advancing adenoma (AA), and colorectal cancer (CRC).

Mode for Carrying Out the Invention

[0022] Definitions The terms "subject" or "individual" are used interchangeably herein to refer to all animals classified as mammals, including but not limited to laboratory animals and livestock, primates and humans, e.g., humans, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents. The subject is preferably a male or female human of any age or race.

[0023] As used herein, the term "sensitivity" refers to the proportion of subjects (TP / (TP+FN)) having the target condition (reference standard positive) and showing a positive test result. Sensitivity indicates how well the test is at detecting the disease. Sensitivity ("sens") can range from 0 (0%) < sens < 1 (100%), and ideally the number of false negatives is equal to or close to zero and the sensitivity is equal to or close to 1 (100%).

[0024] As used herein in connection with a test, the term "specificity" refers to the proportion of subjects (TN / (TN+FP)) not having the target condition (reference standard negative) and showing a negative test result. Specificity indicates how well the test is at identifying the normal (negative) condition. Specificity ("spec") can range from 0 (0%) < spec < 1 (100%), and false... It is desirable that the number of positives is equal to or close to zero and the specificity is equal to or close to 1 (100%).

[0025] As used herein, the term "accuracy" refers to the proportion of true results, either true positives or true negatives, in the population. Accuracy is a measure of the degree of correctness of a screening test for a condition, i.e., how accurate the determination and exclusion of a given condition are (TN+TP) / (TN+TP+FN+FP). Accuracy ("acc") can be in the range of 0 (0%) < acc < 1 (100%), and it is desirable that the number of false positives and false negatives is equal to or close to zero and the accuracy is equal to or close to 1 (100%).

[0026] As used herein, the term "receiver operating characteristic (ROC) curve" refers to a graphical plot that shows the performance of a binary classifier system as its discrimination threshold changes. This curve is created by plotting the true positive rate against the false positive rate for various threshold settings. The true positive rate is also known as sensitivity. The false positive rate is calculated as 1 - specificity. Thus, the ROC curve graphically represents the true positive rate versus the false positive rate (sensitivity versus (1 - specificity)) over the entire range of cutoffs and is a way to select the optimal cutoff for clinical use. The accuracy, expressed as the area under the ROC curve (AUC), provides a useful parameter for comparing test performance. As the AUC approaches 1, it indicates that the test is not only highly sensitive but also highly specific, while as the AUC approaches 0.5, it indicates that the test is neither highly sensitive nor highly specific. Generally, a test is considered an appropriate discriminator when the AUC is between 0.6 and 0.75, has good discriminatory ability when the AUC is between 0.75 and 0.9, and is an excellent discriminator when the AUC is between 0.9 and 1. For further details, see, for example, Zweig MH and Campbell G, Clinical Chemistry 1993; 39:561 - 577, or Greiner et al. Preventive Veterinary Medicine 2000; 45:23 - 41.

[0027] The term "probe" as used herein refers to a synthetic or biologically produced nucleic acid of 10 to 285 base pairs in length that contains a specific nucleotide sequence that allows specific and selective hybridization to a target nucleic acid sequence under defined conditions, and optionally includes moieties for improved detection or assay performance. Statistically, a minimum of 10 nucleotides is generally required to obtain specificity and form a stable hybridization product, and a maximum of 285 nucleotides generally represents an upper limit on the length at which reaction parameters can be adjusted to easily determine mismatch sequences and selective hybridization. Probes may optionally contain certain components that contribute to their proper or optimal function under certain assay conditions. For example, probes can be modified to improve their resistance to nuclease degradation (e.g., by end-capping), to carry detection ligands (e.g., fluorescein), to carry ligands for purification or enrichment purposes (e.g., biotin), or to facilitate their capture onto solid supports (e.g., poly-deoxyadenosine "tails").

[0028] As used herein, the term "primer" refers to an oligonucleotide that can be used in an amplification method, such as the polymerase chain reaction ("PCR"), to amplify a nucleotide sequence. Primers are designed based on the polynucleotide sequence of a particular target sequence, for example, a particular 16S rDNA sequence.

[0029] The term "specific" as used herein in reference to a nucleotide sequence means that the nucleotide sequence hybridizes to / amplifies a predetermined target sequence and does not substantially hybridize to / amplify non-target sequences under assay conditions, typically using stringent conditions.

[0030] As used herein, the term "hybridization" refers to the process by which, under defined reaction conditions, two partially or completely complementary strands of nucleic acid come together in an antiparallel manner, following defined rules as to which nucleobases can pair with each other, to form a double-stranded nucleic acid through specific and stable hydrogen bonds.

[0031] The term "substantial hybridization" means that the amount of hybridization observed is such that a person observing the results relative to the hybridization data of positive and negative controls would consider the results positive. Data that is considered "background noise" is not substantial hybridization.

[0032] The term "stringent hybridization conditions" refers to approximately 35°C to 65°C in a salt solution of approximately 0.9 molar NaCl. Stringency may also be influenced by reaction parameters such as the concentration and type of ionic species present in the hybridization solution, the type and concentration of denaturing agents present, and the temperature of hybridization. In general, the more stringent the hybridization conditions, the longer the probe is preferred for forming stable hybrids. As a general rule, the stringency of the conditions under which hybridization is carried out will affect certain characteristics of the preferred probes used.

[0033] As used herein, the term "therapeutically effective amount" refers to an amount effective when administered in single or multiple doses to a subject (such as a human patient) in the prophylactic or therapeutic treatment of a disease, disorder, or pathological condition.

[0034] The term "anti-cancer therapy" as used herein refers to a therapy useful in the treatment of cancer. Examples of anti-cancer therapies include, but are not limited to, surgery, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, anti-hormonal agents, agents used in radiation therapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents that treat cancer, such as anti-HER-2 antibodies (e.g., Herceptin™), anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (Tarceva™), and erythropoietinib (Tregs™). )), platelet derived growth factor inhibitors (e.g., Gleevec™ (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets ErbB2 receptor, ErbB3 receptor, ErbB4 receptor, PDGFRβ receptor, BlyS receptor, APRIL receptor, BCMA receptor, or VEGF receptor, TRAIL / Apo2, and other bioactive and organic chemical agents. Combinations thereof are also encompassed by the present invention.

[0035] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. This term includes radioactive isotopes (e.g., At <211> , I <131> , I <125> , Y <90> , Re <186> , Re <188> , Sm <153> , Bi <212> , P <32> , and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins, including fragments and / or variants thereof, of bacterial, fungal, plant, or animal origin.

[0036] The term "chemotherapeutic agent" as used herein refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as cyclophosphamide under the trademarks thiotepa and CYTOXAN®; alkylsulfonates such as busulfan, improsulfan, and piposulfan; benzodopa, carboquone, metholedopa, and cyclosulfamide; aziridines such as meturedopa, and uredopa; altretamine, thiazolidinone, Ethylenimines and methylamelamines, including triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); doxorubicins (especially cryptophycin 1 and cryptophycin 8); rastatin; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictiin; spongistatin; nitrogen compounds such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard. mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; enediyne antibiotics (e.g., the calicheamicins, especially calicheamicin gamma 1 and calicheamicin omega 1; the dynemicins, including dynemicin A; the bisphosphonates, such as clodronate; esperamicin; and the neocarzinostatin chromophore and related enediyne antibiotic chromophores), aclacinomycin, actinomycin, an Thoramicin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN™ doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin Antibiotics such as mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodrubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; Purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, etc.; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, etc.; androgens such as calsterone, drostanolone propionate, epithiostanol, mepitiostane, testolactone, etc.; anti-adrenals such as aminoglutethimide, mitotane, trilostane, etc.; florin Folic acid supplements such as folic acid;aceglatone;aldophosphamide glycosides;aminolevulinic acid;eniluracil;amsacrine;bestrabulil;bisantrene;edatrexate;defofamine;demecolcine;diaziquone;eflornithine;elliptinium acetate;epothilone;etoglucide;gallium nitrate;hydrochloride xylurea;lentinan;lonidamine;maytansinoids such as maytansine and ansamitocin;mitoguazone;mitoxantrone;mopidanmol;nitraerine;pentostatin;phenamet;pirarubicin;rosoxantrone;podophyllic acid;2-ethylhydrazide;procarbazine;PSK™ polysaccharide complex (JHS Natural Products, Eugene, Oregon); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids such as paclitaxel in TAXOL™, cremophor-free albumin engineered nanoparticle formulations of paclitaxel in ABRAXANE™, and doxetaxel in TAXOTERE™; chlorambucil; gemcitabine in GEMZAR™; 6-thio Guanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including treatment regimens with irinotecan and 5-FU and leucovorin); topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb™); inhibitors of PKCα, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva™)) and VEGF-A that reduce cell proliferation, as well as pharmacologic acceptable salts, acids, or derivatives of any of the above.

[0037] As used herein, the term "antihormonal agent" refers to, for example, tamoxifen (including NOLVADEX™ tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxyphene, ketoxifene, LY. agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including toremifene in 117018, onapristone, and FARESTON™; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, megestrol acetate in MEGASE™, exemestane, formestane, fadrozole, vorozole in RIVISOR™, letrozole in FEMARA™, and anastrozole in ARIMIDEX™; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and toloxacinamide. refers to cytabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as, for example, PKCα, Ralf, and H-Ras; ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME™ ribozymes) and HER2 expression inhibitors; gene therapy vaccines, such as vaccines such as the ALLOVECTIN™ vaccine, the LEUVECTIN™ vaccine, and the VAXID™ vaccine; rIL-2 in PROLEUKIN™; topoisomerase 1 inhibitors in LURTOTECAN™; rmRH in ABARELIX™; and pharma- ceutically acceptable salts, acids, or derivatives of any of the above.

[0038] The term "cytokine" is a general term for proteins released by certain cell populations that act on other cells as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); epidermal growth factor; hepatocyte growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor alpha and tumor necrosis factor beta; Müllerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; Activin; Vascular endothelial growth factor; Integrins; Thrombopoietin (TPO); Nerve growth factors such as NGFα; Platelet growth factors; Transforming growth factors (TGFs) such as TGFα and TGFβ; Insulin-like growth factor I and Insulin-like growth factor II; Erythropoietin (EPO); Bone morphogenetic factors; Interferons such as Interferon α, Interferon β, and Interferon γ; Colony stimulating factors (CSFs) such as Macrophage-CSF (M-CSF); Granulocyte-macrophage-CSF (GM-CSF); and Granulocyte-CSF (Granulocyte-CSF). F (G-CSF); interleukins (IL), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; tumor necrosis factors, such as TNFα or TNFβ; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or recombinant cell culture and biologically active equivalents of the native sequence cytokines.

[0039] The term "growth inhibitor" as used herein refers to a compound or composition that inhibits cell growth in vitro and / or in vivo. Thus, a growth inhibitor may significantly reduce the percentage of cells in S phase. Examples of growth inhibitors include agents that block cell cycle progression (at a position other than S phase), such as agents that induce G1 arrest and M phase arrest. Classical M phase blockers include vincas (vincristine and vinblastine), TAXOL™, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Agents that arrest G1, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C, also extend to S phase arrest. Further information can be found in The Molecular Basis of Cancer, edited by Mendelsohn and Israel, Chapter 1, entitled "Cell cycle regulation, oncogenes, and antitineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), particularly page 13.

[0040] The term "radiotherapy" or "radiation treatment" as used herein refers to the use of directed gamma or beta radiation to induce sufficient damage to cells to limit their ability to function normally or to destroy them completely. It will be appreciated that there are many methods known in the art for determining the radiation dose and duration of treatment. A typical treatment is given as a single application, with typical radiation doses ranging from 10 units to 200 units (grays) per day.

[0041] As used herein, the term "bowel disease" refers to those diseases or disorders that affect the small intestine, colon, and / or rectum.

[0042] Methods of the Invention In a first aspect, the present invention provides a method of increasing the specificity or reducing the false positive rate of a fecal occult blood test (FOBT) for screening, diagnosing, and / or monitoring advanced colorectal neoplasia (AN), advanced adenoma (AA), and / or colorectal cancer (CRC) in a subject, comprising: a) optionally performing a FOBT on a fecal sample isolated from the subject; wherein said FOBT involves determining the presence of occult blood in said fecal sample; b) In an intestinal sample (preferably a fecal sample) isolated from said subject, the following bacterial markers are detected: Gemella morbilorum (GMLL), Peptostreptococcus stomatis (PTST), and Bacteroides fragilis (BCTF), or any combination thereof; The present invention relates to a method comprising the steps of:

[0043] In a related aspect, the invention provides a method of screening, diagnosing, and / or monitoring AN, AA, and / or CRC in a subject, comprising: a) optionally performing a FOBT on a fecal sample isolated from the subject; wherein said FOBT involves determining the presence of occult blood in said fecal sample; b) In an intestinal sample (preferably a fecal sample) isolated from said subject, the following bacterial markers are detected: Gemella morbilorum (GMLL), Peptostreptococcus stomatis (PTST), and Bacteroides fragilis (BCTF), or any combination thereof; The present invention relates to a method comprising the steps of:

[0044] In a preferred embodiment, the quantification of the FOBT in a) and the bacterial marker in b) is performed in a fecal sample, preferably the same fecal sample.

[0045] The reference strain for GMLL is Gemella morvirorum strain NCTC11323 (Kingdom Bacteria; Phylum Firmicutes; Class Bacillales; Order Bacillales; Family XI. Incertae Sedis; Genus Gemella), whose genome sequence has GenBank Accession No. LS483440.1. In a particular embodiment, oligonucleotides specific for SEQ ID NO: 1 can be used for quantification of GMLL. SEQ ID NO: 1 corresponds to the region 827110-827892 of GenBank Accession No. LS483440.1.

[0046] 1 atggcgagtt tattacaaaa aacaagaaaa ataagtacaa ttttacaaga aggacgtcat 61 gataatgtcg attttgaagc aatggcaatg cgcttaagtc ctattttaga ttctgttgtc 121 tatattttag atgtagaagg taatattctt gggtatgatt ctattgtaga ttattctaac 181 gaacgtatgg aagaaattat tcgtgcgagg aaagtgccta aagcttattt agatgcaaca 241 ttaaaagtat atgctacgaa ggttaatatt ccatttcaag atccgctttc tattttccca 301 gatgaagaga aagaaagatt tgatgggaat acctatactg taattttacc aataagaggc 361 gggggagaac gccttggaac tcttgttata ggaagaatgg ataatgactt tcaagatgat 421 gatttggtgt tagcagaata tgcgtcaaca gtagtaggaa ttgaaattct tcacgaaaaa 481 caagataaag aaaaaaatct agctagagac aaagatatgg tgaatatggc tcttaattcg 541 ttatcatatt cagaaaaaga agcaattgaa catattttca gagaattaga tgggacagaa 601 ggactactta tcgcaagtaa aattgcagat agagtaggaa ttactcgctc agttattgtt 661 aatgcactta gaaaattaga aagtgcaggt attatagagt caaaatcatt aggtatgaaa 721 ggtacttata taaaagtact taaagaatat tttttagaat taatgttttc taatgaattt 781 taa

[0047] The reference strain for PTST is Peptostreptococcus stomatis DSM 17678 strain (kingdom Bacteria; phylum Firmicutes; class Clostridia; order Clostridia; family Peptostreptococci; genus Peptostreptococcus), whose genome sequence has GenBank accession number: ADGQ01000060.1. In a particular embodiment, oligonucleotides specific for SEQ ID NO: 2 can be used for quantification of PTST. SEQ ID NO: 2 corresponds to the region: complement (142796-143362) of GenBank accession number: ADGQ01000060.1.

[0048] 1 atggatatta ttcaacttag caacagactt atagaataca gaaaaagtaa caacttaact 61 attaaagatt tctctgacat gtccggaatt agtactgctc tgcttagtca attagaaaga 121 ggtgttggaa accctagcct gagtgtattg aactctatag cagataccat gaatactagc 181 ctctcctctt tattagaaga acctgttgtt ttagaaaacc tggttagaag gtctgacacc 241 ctatcacta ttgtctaccc atgtaggaac aaccttgaat ttcagttgct cacaactagg 301 tctactactc agagcatcaa ccttgtaaga ataatatttc atgctcattc agaaactaat 361 taccagccac ttggaaaaac cgtttctgat gatataatcc acattgaaaa gggcagtatt 421 attgttcaaa ctgatgatgg taagtctatt caactaaaaa aaggtgatac tatgaggata 481 ccacctgacc ttaggtataa attcaagaat atttctgtac acaaggcaca tatgatttgt 541 gccactaata aacttgaagt aagataa

[0049] The reference strain for BCTF is Bacteroides fragilis strain NCTC 9343 (kingdom Bacteria; phylum Bacteroidetes; class Bacteroidetes; order Bacteroidales; family Bacteroidaea; genus Bacteroides), whose genome sequence has GenBank accession number CR626927.1. In a particular embodiment, oligonucleotides specific for SEQ ID NO: 3 can be used for quantification of BCTF. SEQ ID NO: 3 corresponds to the region 417101-417817 of GenBank accession number CR626927.1.

[0050] 1 atggcaaata ctaatatgga acacggagaa ataatactat accaaccaga caatactata 61 aaactggaag tgcgaataga gaatgaaacc gtttggttga cacaagcaca aattgttaac 121 ttattccagt caagtaaagc caatatcagt gagcatataa gaaatatata tgactcagat 181 gaattatctg ctgaatcaac tgttcggaaa ttccgaacag ttcgaatgga aggtaataga 241 aaggttaccc gcattcttga atattataat ctggatatga ttattccgt aggttatcgt 301 gtgaattcca agcgaggagt ccagttccgc caatggtcaa caggagtact taaagaatat 361 ctattaaaag gctacgcaat caatcagcgc gtggagcagt tggaaaacaa agcaaacacc 421 catgaccggc aactggaaga gttaacaaac aaagtcgact ttttcgtccg gacttcatta 481 cctcctattg aaggtgtctt tttcaacgga cagattttcg atgcctatgt cttttccgct 541 cagttgataa agtctgcaaa gtcatcttta gtattgattg acaactttgt cgatgaaagc 601 gtgctcttac tattgagtaa acgtttgccc ggagtgactt ctatcatata taccaagcaa 661 gtaactccac agttagaatt agatttgaca aagcacaaca gtcaataccc ccaatag

[0051] In a preferred embodiment, these specific oligonucleotides are any of those listed in Table 3 for GMLL, PTST, and BCTF, respectively, and sequences having at least 80% identity to any of them.

[0052] As shown in Example 3, PTST was found to be highly correlated with neoplastic lesions (non-advanced adenoma + advanced adenoma + CRC; p<0.001). Furthermore, GMLL, PTST, and BCTF were each identified as potential biomarkers for detecting advanced neoplastic (AN) lesions (p=0.006, p<0.001, and p=0.030, respectively). GMLL and PTST were also found to be significantly more abundant in CRC than in healthy subjects (p=0.004 and p<0.001, respectively).

[0053] The term "colorectal cancer", "CRC" as used herein refers to cancer that begins in the colon or rectum. These cancers may also be referred to individually as colon cancer or rectal cancer, depending on where they begin. Colon cancer and rectal cancer have many common characteristics. According to the ACS, several types of cancer may begin in the colon or rectum. More than 95% of colorectal cancers are a type of cancer known as adenocarcinoma. These cancers begin in cells that form glands that make mucus that lubricates the inside of the colon and rectum. Other less common types of tumors may also begin in the colon and rectum. These include carcinoid tumors, gastrointestinal stromal tumors (GIST), lymphomas, and sarcomas. In a preferred embodiment, the colorectal cancer is an adenocarcinoma.

[0054] Adenomas are non-cancerous growths of abnormal glandular cells in the lining of organs such as the colon. Adenomas that meet one or more of the following characteristics are typically classified as advanced adenomas (Quintero E., Castells A., et al., N Engl J Med. 2012, 366(8):697-706 (Non-Patent Document 7); Cubiella J. et al., Cancer Epidemiol Biomarkers Prev. 2014, 23(9):1884-92; Muto T, Bussey HJR MB., Cancer 1975;36:2251-70).

[0055] The term colorectal advanced neoplasia (AN) used herein includes CRC and advanced adenoma (AA).According to ACS, the test that is most likely to detect both AA and CRC is preferred.Cohort studies including patients with adenoma suggest that polypectomy can prevent approximately 80% of colorectal cancer (Citarda F, et al., Gut 2001, 48:812-5; Winawer SJ, et al., N Engl J Med 1993, 329:1977-81).

[0056] The term "diagnosis" as used herein refers to both the process of attempting to determine and / or identify possible diseases in a subject, i.e., diagnostic procedure, and the opinion reached by this process, i.e., diagnostic opinion. Thus, diagnosis can also be considered as an attempt to classify an individual's condition into separate and distinct categories that allow medical decisions to be made about treatment and prognosis. It should be understood that in a preferred embodiment, this method is a method carried out in vitro, i.e., a method that is not carried out on the human or animal body. In particular, diagnosis of determining CRC patients, AA patients, and / or AN patients can relate to the ability to identify and classify CRC patients, AA patients, and / or AN patients.

[0057] In certain embodiments, the diagnostic method of the present invention is carried out in a subject suspected of having CRC. As used herein, the term "subject suspected of having CRC" refers to a subject exhibiting one or more signs or symptoms that may be indicative of CRC. A subject suspected of having CRC also includes an individual who has received a preliminary diagnosis but has not yet undergone a confirmatory test (e.g., colonoscopy).

[0058] Signs or symptoms that may be indicative of CRC include, for example, one or more of the following: unexplained weight loss, abdominal pain, unexplained rectal bleeding, iron deficiency anemia, change in bowel habits, and presence of occult blood in the stool (see, for example, Jellema et al, BMJ 2010, 340: c1269, or Adelstein et al. BMC Gastroenterology 2011, 11:65). For illustrative purposes, the current NICE guidelines (NG12); https: / / www.nice.org.uk / guidance / ng12 / chapter / 1-Recommendations-organised-by-site-of-cancer#lower-gastrointestinal-tract-cancers are provided herein below for cases where CRC is suspected:

[0059] Suspected cancer pathway referral for colorectal cancer (For example, if you are reserving an appointment within two weeks) If you are over 40 years old and have unexplained weight loss and abdominal pain, or If you are over 50 years old and have unexplained rectal bleeding, or Age 60 and older: Iron deficiency anemia, or Changes in bowel habits, or If occult blood is found in the stool during testing, Introduce adults.

[0060] In adults with a rectal or abdominal mass, consider a suspected cancer pathway referral for colorectal cancer (eg, if appointment within 2 weeks).

[0061] Rectal bleeding and any of the following unexplained symptoms or findings: abdominal pain, Changes in bowel habits, weight loss, iron deficiency anemia, Cancer pathways suspected for colorectal cancer in adults under 50 years of age with any of the following: Consider referrals (e.g., for appointments within two weeks).

[0062] No rectal bleeding, Over 50 years old, with unknown cause Abdominal pain, or weight loss, Adults with Under age 60: Changes in bowel habits, or iron deficiency anemia, Adults with Adults aged 60 years or older who have anemia but are not iron deficient provides a test for occult blood in feces to assess colorectal cancer.

[0063] The methods of the invention may also be used in the differential diagnosis between CRC, advanced adenomas, and / or advanced neoplasms versus non-advanced adenomas.

[0064] The most effective and cost-effective means of reducing morbidity and mortality from CRC are CRC risk screening and monitoring tests. Screening tests are classified into those that primarily detect cancer early and those that can detect cancer early and also advanced adenomas, thus offering greater potential for prevention through polypectomy (i.e., polyp removal). American Cancer Society (ACS) 2008 screening and surveillance guidelines, Levin et al. (CA Cancer J Clinicians, 2008;58(3)130-160). Different screening guidelines apply to people at average risk and those at elevated or high risk (http: / / www.cancer.org / cancer / colonandrectumcancer / moreinformation / colonandrectumcancerearlydetection / colorectal-cancer-early-detection-acs-recommendations).

[0065] The term "screening" is understood herein as a medical examination or test aimed at distinguishing healthy individuals from diseased or suspected diseased individuals in a group of asymptomatic individuals related to the general population or a group of individuals with one or more risk factors (e.g. subjects with a moderate or high risk of developing a disease). Screening methods are generally used for the "early detection" of a disease. The term "early detection" refers to detection before the onset of clinical signs.

[0066] The goal of cancer screening is to reduce mortality through early detection and early treatment, and thus reduce the incidence of advanced disease, which generally has a poor prognosis.To this end, modern CRC screening can achieve this goal through the detection of adenocarcinoma (preferably early stage adenocarcinoma), as well as the detection and removal of advanced adenoma.Therefore, the screening method of the present invention has been found to be a powerful tool for reducing the mortality associated with CRC through the detection of CRC and / or advanced neoplasms.

[0067] The screening methods of the invention can be performed in individuals who do not exhibit signs and / or symptoms of CRC (referred to herein as "asymptomatic individuals"). The screening methods of the invention can also be performed in subjects with or without a personal or family history of colon neoplasia, or other risk factors for CRC as described below.

[0068] In a particular embodiment, the screening methods of the invention are carried out in subjects with intermediate risk of CRC. These are typically subjects aged 50 years or older with no personal or familial history of CRC. Average risk women and men aged 50 years or older are It is recommended that patients undergo screening for colorectal cancer (see Table 2 in Levin et al., CA Cancer J Clinicians, 2008;58(3)130-160, which is incorporated herein by reference). (Forms part of the company).

[0069] In another embodiment, the screening methods of the invention are performed in subjects at elevated risk and / or at high risk. See Table 3 of ACS (Levin et al., CA Cancer J Clinicians, 2008;58(3)130-160, which is incorporated herein by reference. According to the International Journal of Clinical Oncology (ICH), subjects at elevated and high risk include: Subjects at Increased Risk Subjects with a history of polyps on previous colonoscopy, A subject having colorectal cancer, and Subjects with a family history of colorectal cancer or adenomatous polyps, And, i. A subject with either CRC or adenomatous polyps in a first-degree relative younger than 60 years or in two or more first-degree relatives of any age; and / or ii. Subjects with either CRC or adenomatous polyps in a first-degree relative aged 60 years or older or two second-degree relatives with CRC; High risk Subjects with familial adenomatous polyposis (FAP) diagnosed by genetic testing or suspected of having FAP without genetic testing, Subjects with hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome) or at elevated risk for HNPCC based on family history without genetic testing, and A subject with inflammatory bowel disease, such as chronic ulcerative colitis or Crohn's disease, may be mentioned.

[0070] Another goal of the present invention may be to provide a pre-diagnostic tool. In particular, the method of the present invention may be used to screen and / or identify subjects with a predisposition to or at increased risk of having CRC, for example by detecting the presence of advanced adenomas.

[0071] The term "monitoring" as used herein refers to assessing the progress of disease and / or the effectiveness of surgical and / or therapeutic treatment, for example, assessing whether there is disease remission, or, conversely, assessing whether there is disease progression or recurrence.One of the goals of the monitoring method of the present invention is to detect recurrence early.In a particular embodiment, the method of the present invention is a method of monitoring subjects suffering from cancer who have undergone surgical resection, optionally in combination with adjuvant and / or neoadjuvant therapy.

[0072] In a preferred embodiment, the method of the invention is a method of screening, diagnosing, and / or monitoring AN, AA, and / or CRC.

[0073] The term "sample" or "biological sample" as used herein refers to biological material isolated from a subject. Biological samples may contain any biological material suitable for detecting the desired biomarkers and may include cellular and / or non-cellular material from the subject. In a preferred embodiment, the samples are intestinal samples. These may be, for example, biopsy samples from mucosal tissue of the colon and / or rectum. The intestinal sample is preferably a fecal sample. Fecal samples are routinely used in clinical practice and the skilled artisan will know how to identify the most appropriate means of their acquisition and storage. Once the sample is obtained, it may be used fresh or may be frozen or stored using appropriate means.

[0074] The method of the present invention optionally comprises, in step a), performing a FOBT on a stool sample isolated from a subject, where the FOBT comprises the determination of the presence of occult blood in the stool sample isolated from the subject. Blood in stool is a non-specific finding that may be due to CRC or larger (greater than 1 cm to 2 cm) polyps. Since small adenomatous polyps do not tend to bleed, and bleeding from cancer or large polyps is intermittent or not always detectable in a single sample of stool, the proper use of stool blood tests typically requires annual testing, consisting of collecting specimens (two or three, depending on the product) from consecutive bowel movements. Stool blood tests are usually known as fecal occult blood tests (FOBTs), since they are designed to detect the presence of occult blood in stool. FOBTs fall into two main categories based on the analyte detected: gFOBTs and FITs. The guaiac-based test detects blood in the stool via the pseudoperoxidase activity of heme or hemoglobin, whereas the immunochemistry-based test reacts to human globin (Levin et al. (CA Cancer J Clinicians, 2008;58(3)130-160).

[0075] A typical gFOBT protocol consists of collecting two samples from each of three consecutive bowel movements. Because gFOBT is a qualitative test, it is generally read visually by a trained laboratory technician using the naked eye, and the visual results are interpreted. Prior to testing with a sensitive guaiac-based test, individuals are usually instructed to avoid aspirin and other nonsteroidal anti-inflammatory drugs, vitamin C, red meat, poultry, fish, and some raw vegetables due to food-test interactions that may increase the risk of both false positive and false negative (specifically vitamin C) results. One example of a commercially available gFOBT is the Hemoccult SENSA (Beckman Coulter, (United States).

[0076] FIT detects human globin, a protein that, along with heme, constitutes human hemoglobin. Thus, FIT is more specific for human blood than guaiac-based tests, which rely on the detection of peroxidase in human blood and also react to peroxidase present in dietary components such as raw red meat, cruciferous vegetables, and some fruits. Furthermore, unlike gFOBT, FIT does not give false negative results in the presence of high doses of vitamin C supplements, which block the peroxidase reaction. Furthermore, because globin is broken down by digestive enzymes in the upper gastrointestinal tract, FIT is also more specific for lower gastrointestinal bleeding, thus improving its specificity for CRC. Finally, sample collection for some variants of FIT is less demanding on the patient than gFOBT, requiring fewer samples or less direct handling of stool. There are various FITs based on different detection techniques (e.g., reverse passive hemagglutination (RPHA), latex agglutination, ELISA, and immunoturbidimetry), which are encompassed herein (Levin B et al., Gastroenterology 2008;134:1570-95). Preferably, the immunological test is the OC-SENSOR assay used in the examples. It is based on the latex agglutination reaction of I (Eiken Chemical Co., Ltd., Japan) and others.

[0077] Preferably, the FOBT is a fecal immunochemical test (FIT) that quantifies human hemoglobin (hHb). If the level of hHb in the fecal sample is above a predefined threshold, the subject is determined to be FIT positive. For example, a subject may be determined to be FIT positive if the hHb level is 10 μg hHb per gram of feces or more (or 50 ng hemoglobin per mL or more; FIT50 cutoff), or if the hHb level is 20 μg hHb per gram of feces or more (or 100 ng hemoglobin per mL or more; FIT100 cutoff). The FIT100 cutoff is the commonly used cutoff value of FIT in CRC screening in Catalonia and other parts of Spain. As shown in Table 5, when both thresholds are used in the method of the present invention, an improvement in specificity is obtained over the corresponding FIT test alone. In a preferred embodiment, FIT positive subjects have hHb levels of 10 μg hHb / g stool or greater (FIT50 cutoff), which allows for slightly higher sensitivity. .

[0078] As mentioned above, the method of the present invention may comprise, in step b), quantifying any of GMLL, PTST, or BCTF, and any combination thereof including i) GMLL and PTST, ii) GMLL and BCTF, iii) PTST and BCTF, and / or iv) GMLL, PTST, and BCTF. Optionally, the method as defined in any of these embodiments may further comprise quantifying Bacteroides thetaiotaomicron (BCTT) in the intestinal sample (preferably a fecal sample). A reference strain for BCTT is Bacteroides thetaiotaomicron VPI-5482 strain (kingdom Bacteria; phylum Bacteroidetes; class Bacteroidales; order Bacteroidales; family Bacteroidaea; genus Bacteroides), whose genome sequence has GenBank accession number: AE015928.1. In a particular embodiment, an oligonucleotide specific to SEQ ID NO: 4 may be used for quantifying BCTT. SEQ ID NO: 4 corresponds to the region 326008-326649 of GenBank Accession No. AE015928.1. In a preferred embodiment, these specific oligonucleotides are any of those listed in Table 3 for BCTT, as well as sequences having at least 80% identity to any of them.

[0079] 1 atggcagtac aatttgaatt atataagact ccgatgccaa aggagaaaaa gaacaagacg 61 cgttatcatg cccgtccggt aagttttgag accgtcaata ccgagaaact ggcttatcgc 121 atccatgatc gctccacatt aagagtatcg gacattattt caaccttgga agaactgaaa 181 aatgaagtag ctcagtgcct cctggaaggt aaaaaggtgc atgtcgatgg attaggattc 241 tttcaggtca ctctttcctg cgaagaagaa atacgtaacc cgaaagacaa acgtgtgcac 301 cgggtaaaat tgaaagctat aaagtttaaa gctgataaag aattgaaagg ggaattgtgt 361 cacatgaaat tccagcgttc taaaatcaga ccgcactccg ccaatttatc agaagtagaa 421 atagacatga aactaactga atattttgct gaaaatcaga ttatcacccg gaaagatttt 481 caatatctct gcggaatgac acaaatcact gcatatcgcc atatcaagag gttaatggca 541 gaaaagaaat tgcaaaataa agggacgatc tatcaaccga tatatactcc ggttccgggt 601 aattacaggg tatcggtgga tttaaaatat aaagaacaat ga

[0080] Preferably, step b) comprises or consists of quantifying PTST, BCTF and BCTT.

[0081] Molecular biology methods for measuring the amount of target nucleic acid sequences are known in the art. These methods include, but are not limited to, end-point PCR, competitive PCR, quantitative PCR (qPCR), PCR pyrosequencing, PCR-ELISA, DNA microarrays, nucleic acid sequencing such as next-generation sequencing, in situ hybridization assays such as dot blots or fluorescent in situ hybridization assays (FISH), mass spectrometry, branched DNA (Nolte, Adv. Clin. Chem. 1998,33:201-235), and multiplex versions of the above methods (see, for example, Andoh et al., Current Pharmaceutical Design, 2009;15,2066-2073), as well as any of the listed techniques. This includes next generation types and combinations thereof, all of which are within the scope of the present invention.

[0082] A variety of next-generation sequencing methods have been described and are known to those skilled in the art, including, for example, sequencing by synthesis using a cyclic reversible termination approach (e.g., Illumina, SEQLL, Qiagen), sequencing by synthesis using a single-nucleotide addition approach (e.g., Illumina, SEQLL, Qiagen), and sequencing by synthesis using a single-nucleotide addition approach (e.g., Illumina, SEQLL, Qiagen). sequencing (e.g., Roche-454, Thermo Fisher-Ion Torrent); Sequencing by ligation (e.g., Thermo Fisher's SOLiD and BGI-Complete Genomics), real-time long-read sequencing (e.g., Pacific Biosciences, Oxford Nanopore Technologies), synthetic sequencing (e.g., Sigma-Aldrich ... Examples of such methods include synthetic long-read sequencing (e.g., Illumina, 10X Genomics, iGenomeX) (see, e.g., Goodwin S, et al., Nat Rev Genet. 2016, 17(6):333-51).

[0083] In some embodiments, the molecular biology quantification method is based on sequence-specific amplification. Such an amplification-based assay includes an amplification step that includes contacting a sample (preferably an isolated DNA sample) with two or more amplification oligonucleotides specific to a target sequence in a target nucleic acid to produce an amplification product if the target nucleic acid sequence is present in the sample. The amplification product will include a cDNA sequence corresponding to the target sequence. Suitable amplification methods include, for example, replicase-mediated amplification, ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA), and polymerase chain reaction (PCR), including quantitative PCR.

[0084] One particularly preferred quantification method is quantitative PCR (qPCR), also known as real-time PCR. Quantitative PCR refers to a type of PCR that simultaneously amplifies and quantifies a target DNA molecule. Its key feature is that the amplified DNA is detected as the reaction proceeds in real time. ABI Prism 7700 SDS, GeneAmp 5700 SDS, ABI Prism 7900 HT SDS from Applied Biosystems; iCycler iQ from Bio-Rad; Smart Cycler from Cepheid; Rotor-Gene from Corbett Research; LightCycler from Roche Molecular Biochemicals, AriaMx from Agilent, and ABI Prism 7900 HT SDS from Stratagene are some of the most popular quantification methods. Various instruments are available, such as the Mx4000 Multiplex. The qPCR method allows accurate quantification of PCR products in real time by measuring the accumulation of PCR products very early in the exponential phase of the reaction, thus reducing the quantification bias associated with PCR amplification efficiency that occurs in end-point PCR. Real-time PCR is known in the art and will not be described in detail herein. Technical overviews and protocols for qPCR are available, for example, from the suppliers mentioned above (e.g., http: / / www.sigmaaldrich.com / technical-documents / protocols / biology / sybr-green-qpcr.html or http: / / www.sigmaaldrich.com / life-science / molecular-biology / pcr / quantitative-pcr / qpcr-technical-guide.html). For an overview of the qPCR method, see Wong ML y Medrano JF, Biotechniques 2005, 39(1):75-85. In certain embodiments, the quantification method is multiplex qPCR.

[0085] There are various detection chemistries available for qPCR. All of them can be used in the qPCR instruments mentioned above. The term "detection chemistry" refers to the method of reporting the amplification of a specific PCR product in real-time PCR. These detection chemistries can be categorized into two main groups. The first group includes double-stranded DNA intercalating molecules such as SYBR Green I and EvaGreen, while the second group includes fluorophore-labeled oligonucleotides. The latter are further divided into three subgroups according to the type of fluorescent molecule used in the PCR reaction: (i) primer-probes (Scorpions, Amplifluor™, LUX™, Cyclicons, Angler™); (ii) probes; hydrolysis (TaqMan, MGB-TaqMan, Snake assay) and hybridization (Hybprobe or FRET, molecular beacons, HyBeacon™, MGB-Pleiades, MGB-Eclipse, ResonSense™, Yin-Yang or substitution); and (iii) nucleic acid analogues (PNA, LNA™, ZNA™, non-natural bases: Plexor™ primers, Tiny-Molecular Beacons) (see E. Navarro et al., Clinica Chimica Acta, Volume 439, 15 January 2015, Pages 231-250).

[0086] The probe may be a dual-labeled oligonucleotide such as a hydrolysis probe or a molecular beacon. The 5' end of the oligonucleotide is typically labeled with a fluorescent reporter molecule, while the 3' end is labeled with a quencher molecule. The sequence of the probe is specific to the region of interest within the amplified target molecule. In a more preferred embodiment, the probe is a hydrolysis probe whose sequence length is designed to place the 5' fluorophore and the 3' quencher close enough to suppress fluorescence. Several reporter molecules and quenchers used in qPCR probes are known in the art.

[0087] Generally, specific oligonucleotides such as probes and / or primers are used for quantifying nucleotide sequences. The term "primers and / or probes" includes in particular "multiple primers and / or multiple probes". In the present specification, both expressions are used interchangeably, and include, for example, primers; probes; primers and probes; primer pairs; and primer and probe pairs. The design and validation of primers and probes are known in the art. For the design of primers and probes in quantitative real-time PCR methods, see, for example, Rodriguez A et al. (Methods Mol Biol., 2015, 1275:31-56). Preferred primers and / or probes that can be used in the method of the present invention are described in the kit of the present invention below.

[0088] Preferably, oligonucleotides useful in the methods of the invention are from about 5 to about 50 nucleotides in length, from about 10 to about 30 nucleotides in length, or from about 20 to about 25 nucleotides in length. In certain embodiments, oligonucleotides that specifically hybridize to a target sequence are from about 19 to about 21 nucleotides in length. In certain embodiments, the oligonucleotides are modified for detection purposes or to enhance assay performance, as described herein.

[0089] These oligonucleotides may be ribonucleotides or deoxyribonucleotides. In certain embodiments, the oligonucleotides may have at least one chemical modification. For example, suitable oligonucleotides may be composed of one or more "conformationally restricted" or bicyclic sugar nucleoside modifications, such as "locked nucleic acids." "Locked nucleic acids" (LNAs) are modified ribonucleotides that contain an additional bridge between the 2' and 4' carbons of the ribose sugar moiety, resulting in a "locked" conformation that confers improved thermal stability to oligonucleotides containing LNAs. In other embodiments, the oligonucleotides may include peptide nucleic acids (PNAs), which contain a peptide-based backbone rather than a sugar-phosphate backbone. Other chemical modifications that the oligonucleotides may include include, but are not limited to, sugar modifications such as 2'-O-alkyl (e.g., 2'-O-methyl, 2'-O-methoxyethyl), 2'-fluoro, and 4'-thio modifications, as well as backbone modifications such as one or more phosphorothioate, morpholino, or phosphonocarboxylate linkages. For example, these oligonucleotides, particularly those of shorter length (e.g., less than 15 nucleotides), may contain one or more affinity-enhancing modifications such as, but not limited to, LNA, bicyclic nucleosides, phosphonoformates, 2'O-alkyls, etc. In some embodiments, oligonucleotides can be chemically modified to, for example, improve their resistance to nuclease degradation (e.g., by end-capping), to carry detection ligands (e.g., fluorescein), or to facilitate their capture onto solid supports (e.g., poly-deoxyadenosine "tails").

[0090] The term "quantification level" can be concentration (amount of DNA per unit volume), amount of DNA per cell number, cycle threshold (Ct value), or any mathematical transformation thereof. In a preferred embodiment, the quantification of the bacterial sequence is performed by qPCR, and the quantification level is expressed as a Ct value. The Ct value (cycle threshold) is defined as the number of qPCR cycles required for the fluorescent signal to exceed a threshold. The Ct level is the concentration of the target in the sample. It is inversely proportional to the amount of nucleic acid (i.e., the lower the Ct level, the higher the amount of target nucleic acid in the sample).

[0091] Quantification of the abundance of target nucleic acid sequence (e.g., SEQ ID NO:1) in fecal samples can be absolute or relative. Relative quantification is based on one or more internal reference genes, i.e., 16S rRNA genes from reference strains, for example, by using universal primers to determine total bacteria (eubacteria), and expressing the abundance of target nucleic acid sequence relative to eubacteria (e.g., SEQ ID NO:1 / eubacteria ratio). Absolute quantification provides the exact number of target molecules by comparison with DNA standards.

[0092] In certain embodiments, optionally in combination with one or more of the embodiments or features described herein, the quantification of the bacterial marker in step b) is related to eubacteria and expressed as a ratio of corresponding quantification values ​​(e.g., ratio of Ct values). Particular oligonucleotides that can be used for the quantification of eubacteria are any of those listed in Table 3 for EUB and sequences having at least 80% identity to any of them. Alternatively, the following oligonucleotides: EUB2 forward (SEQ ID NO: 5): ACTCCTACGGGAGGCAGCAGT EUB2 reverse (SEQ ID NO: 6): GTATTACCGCGGCTGCTGGCAC or a sequence having at least 80% identity to any of them may also be used.

[0093] In a particular embodiment of the method of the present invention, DNA is extracted from the fecal sample before quantifying the bacterial sequence.Several methods for DNA extraction from fecal samples are known, all of which rely on chemical or mechanical disruption, lysis using detergents, or a combination of these approaches (Kennedy A. et al., PLoS One, 2014;9(2):e88982). Methods for extracting bacterial DNA from fecal samples are described, for example, in M ​​Corist et al., Journal of Microbiological Methods, 2002; 50(2):131-139, Whitney D et al., Journal of Molecular Diagnostics, American Society for Investigative Pathology, 2004;6(4):386-395, and from WO 2003 / 068788.

[0094] Preferred methods include mechanical disruption, such as rapid bead-beating extraction, chemical lysis, and preferably silica membrane columns such as those included in the commercially available DNA extraction kits "MobioPowerSoil™ DNA extraction procedure" (Mo-Bio Laboratories Inc.), FastDNA™ SPIN kit for soil treatment (MP biomedicals), and NucleoSpin™ Soil (Macherey-Nagel Gmbh & Co. KG). A combination of final purification steps was used. The presence of PCR inhibitors such as bilirubin, bile salts, and complex carbohydrates in DNA extracts from fecal samples is one of the difficulties faced in determining DNA biomarkers in DNA extracts from feces (Fleckna et al., Mol Cell Probes, 2007;21(4):282-7). The preferred DNA extraction method is to obtain a DNA concentration of less than 5%. The method results in a fecal extract having a low amount of PCR inhibitors, preferably less than 2%, more preferably less than 1%, and even more preferably less than 0.5%, for example less than 0.25%, less than 0.1%, less than 0.05%, or less than 0.01%.

[0095] In some embodiments, the method of the present invention further comprises: c) calculating a combined score from the levels of the bacterial markers determined in b); and d) classifying FOBT-positive subjects as having or at increased risk of AN, AA, and / or CRC according to the combined score obtained in c); Further includes:

[0096] The combined score of step (c) is for example a value obtained according to a given mathematical algorithm, in which the respective quantification values ​​of the bacterial markers determined in step b) are the variables of said mathematical algorithm. Preferably, said bacterial markers are PTST, BCTF and BCTT.

[0097] In certain embodiments, when calculating the combined score, the combined score is proportional to the absolute or relative amount of either GMLL, PTST, or BCTF in the sample, and inversely proportional to the absolute or relative amount of BCTT, where the higher the score, the higher the likelihood of having CRC, AA, and / or AN. In other words, a high score is indicative of the presence of disease.

[0098] For example, the combined score can be calculated as the sum of the products of standardized beta coefficients obtained by using bacterial marker values ​​as variables in the regression analysis of each marker. In certain embodiments, the beta coefficients are positive for markers GMLL, PTST, and / or BCTF, and negative for BCTT. A positive sign indicates that an increase in the amount of each marker is associated with having or being more likely to have CRC, AA, and / or AN, and a negative sign indicates that a decrease in the amount of each marker is associated with having or being more likely to have CRC, AA, and / or AN.

[0099] Typically, in step d), the method comprises comparing the combination score in the subject's sample with a reference value (e.g., a reference combination score), wherein an increase in the combination score in the subject's sample relative to the reference value (e.g., the reference combination score) is indicative of AN, AA, and / or CRC.

[0100] The term "reference combination score" as used herein is a reference value obtained according to a given mathematical algorithm, where the reference expression values ​​of each of the bacterial markers used in the method of the present invention are variables of said mathematical algorithm.

[0101] The term "reference value" as used herein refers to a predetermined standard used as a reference to evaluate values ​​or data obtained from a sample collected from a subject. This "reference value" may also be referred to as a "cut-off value" or "threshold value."

[0102] The reference value or reference level may be an absolute value, a relative value, a value with upper or lower limits, a range of values, a representative value, a median, a mean, a z-score value (e.g., the mean + or -1 SD), a tertile, or a value compared to a particular control or baseline value. In certain embodiments, optionally in combination with one or more of the above or below embodiments or features, said reference value is a mean or a tertile.

[0103] It is further noted that various statistical and mathematical methods of establishing expression threshold or cut-off levels are known in the art. Threshold or cut-off expression levels for a particular biomarker can be selected, for example, based on data from a receiver operating characteristic (ROC) plot. Those skilled in the art will understand that these threshold or cut-off expression levels can be varied, for example by moving along the ROC plot for a particular biomarker or combinations thereof, to obtain different sensitivity or specificity values, thereby affecting the overall assay performance.

[0104] The reference value may be based on the value of an individual sample, but is generally based on a number of samples, including or excluding the sample being tested. For example, the reference value may be based on one of the screening populations defined herein above (e.g., asymptomatic adults of the general population, over 50 years of age). The reference value may be derived from a population of individuals with symptoms associated with CRC (asymptomatic adults, or asymptomatic adults with one or more risk factors), or from a population of individuals with symptoms associated with CRC. The reference value may also be derived from a collection of tissue samples from a population of reference AN, AA, and / or CRC patients for which historical information relating to actual clinical outcomes for the corresponding cancer patients is available.

[0105] Alternatively, the reference value according to the methods of the invention can be obtained from one or more subjects who do not have CRC or advanced adenoma and do not have gastrointestinal disease (i.e. healthy control subjects), subjects with advanced adenoma, subjects with CRC at an early stage (preclinical stage), such as asymptomatic, or the same subjects who have been diagnosed with CRC and / or advanced adenoma, but at an earlier time point.

[0106] In a particular embodiment, the method of the invention is a method of screening or diagnosing AN, AA, and / or CRC, and the reference value is obtained from a subject without AN, AA, and / or CRC, or from a subject without a gastrointestinal disorder. In another embodiment, the method of the invention is a method of screening, and said reference value is obtained from a screening population as defined herein. In a further embodiment, the method of the invention is a method of diagnosis, and said reference value is obtained from an individual with a symptom associated with CRC.

[0107] In yet further particular embodiments, the method of the invention is a method for screening or diagnosing AN, AA, and / or CRC, and the reference value is obtained from a subject suffering from non-advanced adenoma. This may be the case, for example, when differential diagnosis between AN, AA, and / or CRC versus non-advanced adenoma (NAA) is desired.

[0108] In additional specific embodiments, the method of the invention is a method of monitoring AN, AA, and / or CRC, and the reference value is obtained from the same subject diagnosed with AN, AA, and / or CRC, but at an earlier time point.

[0109] In another embodiment, the method of the present invention may comprise steps a) and b) as described herein above, e) comparing the amount of any of GMLL, PTST, BCTF, and / or BCTT in the fecal sample with a corresponding reference value; wherein: f) An increase in the amount of GMLL, PTST, and / or BCTF and a decrease in the amount of BCTT in samples of FOBT positive subjects relative to the corresponding reference values ​​is indicative of AN, AA, and / or CRC.

[0110] Preferably, an increase in the amount of PTST and BCTF, and a decrease in the amount of BCTT in samples of FOBT positive subjects relative to the corresponding reference values, is indicative for AN, AA, and / or CRC.

[0111] In the method of the present invention, a combination score (i.e., bacterial marker amount) is considered to be "reduced" if said combination score (i.e., bacterial marker amount) is lower than a reference combination score (i.e., reference value). Preferably, a combination score is considered to be lower than a reference combination score (i.e., reference value) if it is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% or more lower than the reference combination score (i.e., reference value).

[0112] Similarly, in the context of the method of the present invention, a combination score (i.e., bacterial marker amount) is considered to be "increased" if said combination score is higher than a reference combination score (i.e., reference value).Preferably, a combination score (i.e., bacterial marker amount) is considered to be higher than a reference combination score (i.e., reference value) if it is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% or more higher than the reference combination score (i.e., reference value).

[0113] Alternatively or additionally, subjects having a level deviation (i.e., increase or decrease) of more than about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or 20-fold greater than a reference combination score (i.e., reference value) described herein.

[0114] The method of the present invention does not claim to be correct for 100% of the samples analyzed, as understood by those skilled in the art. However, it is necessary that a statistically significant amount of the samples analyzed is correctly classified. A statistically significant amount can be established by those skilled in the art by using various statistical significance measures obtained by statistical tests, and illustrative and non-limiting examples of the statistical significance measures include determining a confidence interval, determining a p-value, etc. A preferred confidence interval is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%. The p-value is preferably less than 0.1, less than 0.05, less than 0.01, less than 0.005, or less than 0.0001. According to the teachings of the present invention, preferably at least 60%, at least 70%, at least 80%, or at least 90% of the subjects of the determined group or determined population analyzed can be correctly classified.

[0115] Validation studies address the agreement between a proposed (index) test and a reference standard for its ability to identify the target condition (see Florkowski MC, Clin Biochem Rev. 2008, 29 (Suppl 1): pp. 83-87). Sensitivity, specificity, accuracy, positive likelihood ratio, negative likelihood ratio, positive predictive value, and negative predictive value are statistics that can be defined to evaluate test performance. Acronym definitions and further details are provided in Table 1 below.

[0116] TIFF2025004152000001.tif64170

[0117] Tests are usually calibrated in terms of the desired specificity and sensitivity depending on the target use of the test in clinical practice. High sensitivity corresponds to a high negative predictive value and is generally followed by a confirmatory test. High specificity corresponds to a high positive predictive value and is generally considered a desirable characteristic for a "definitive" test, such as a comparative diagnostic test.

[0118] In a preferred embodiment, the methods of the invention have sensitivity, specificity and / or accuracy values ​​of at least about 60%, preferably at least about 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, in at least 60% of the groups or populations assayed, or preferably in at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the groups or populations assayed.

[0119] It is further noted that the accuracy of the methods of the invention can be further enhanced by further taking into account other genetic or bacterial markers, biochemical parameters, and / or clinical characteristics of the patient (e.g., age, sex, smoking, and / or other risk factors). The determination of these other markers, parameters, and / or characteristics can be sequential or simultaneous with any or all of the steps of the methods of the invention described herein.

[0120] In certain embodiments, optionally in combination with one or more of the features or embodiments described herein, the methods of the invention comprise the step of using B10, B46, B48, Roseburia intestinalis (RSBI), Collinsella intestinalis (CINT), Faecalibacterium prausnitzii (Duncan et al., Int. J. Syst. Evol. Microbiol 2002, 52:2141-2146) and / or any of their phylogenetic groups (WO 2017 / 025617). , and Lopez-Siles et al. Appl Environ Microbiol. 2012, 78:420-428) and The method further comprises quantifying one or more bacterial markers selected from the group consisting of Escherichia coli.

[0121] B10 has the sequence SEQ ID NO: 7 (shown below), which corresponds to uncultured bacterium isolate DGGE gel band Eub_10 16S ribosomal RNA gene, partial sequence (GenBank: GQ411118.1). In certain embodiments, oligonucleotides specific for SEQ ID NO: 7 can be used to quantify B10.

[0122] 1 cttcggattg taaactcctg ttgttgagga gataatgacg gtactcaaca aggtaagtga 61 cggctaacta cgtgccagca gccgcggtaa aacgtaggtc acaagcgttg tccggaatta 121 ctgggtgtaa agggagcgca ggcgggaaga caagttggaa gtgaaatcca tgggctcaac 181 ccatgaactg ctttcaaaac tgtttttctt gagtagtgca caggtaggcg gaattcccgg 241 tgtagcggtg gaatgcgtag atatcgggag gaacaccagt ggcgaaggcg gcctactggg 301 caccaactga cgctgaggct cgaaagtgtg ggtagcaaac aggattagat accctggtag 361 tccacactgt aaacgatgat tactaggtgt tggaggattg accccttcag tgccgcagtt 421 aacacaataa gtaatccacc tggggagtac gaccgcaagg ttgaaactca aaggaattga 481 cggac

[0123] B46 has the sequence of SEQ ID NO:8 (shown below), which corresponds to the uncultured bacterial isolate DGGE gel band Eub_46 16S ribosomal RNA gene, partial sequence (GenBank: GQ411150.1). In certain embodiments, oligonucleotides specific for SEQ ID NO:8 can be used to quantify B46.

[0124] 1 gtaacatttc tgtaacaaga catacagcag tttctcagag ttcccaaact caaaggaatt 61 gacggaagcc gcggttccac gtaagtcaca agcgttgtcc ggaattactg ggtgtaaagg 121 gagcgcaggc gggaagacaa gttggaagtg aaatccatgg gctcaaccca taaactgctt 181 tcctaactgt ttttcttgag tagtgcacag gtaggcggaa ttcccggtgt agcggtggaa 241 tgcctagata tcgggaggaa caccattggc gaaggcggcc tactggactg caactgacgc 301 tgaggctcga aagtgtgggt agcaaacagg attagatacc ctggtagtcc actccgtaaa 361 ccatgattac tacgtgttgg aggattgacc ccttctgtgc cgcagataac acaataagta 421 atccacctgg ggagtacgac cgcccggttg agactcacag gaattgacgg actc

[0125] B48 has the sequence of SEQ ID NO:9 (shown below), which corresponds to the uncultured bacterial isolate DGGE gel band Eub_48 16S ribosomal RNA gene, partial sequence (GenBank: GQ411152.1). In certain embodiments, oligonucleotides specific for SEQ ID NO:9 can be used to quantify B48.

[0126] 1 tctcttacgg ggagcagcag tggggaatat tgcacaatgg gggaaaccct gatgcagcga 61 cgccgcgtga gcgatgaagt atttcggtat gtaaagctct atcagcaggg aagaaaatga 121 cggtacctga ctaagaagcc ccggctaaat acgtgccagc agccgcggta atacgtatgg 181 tgcaagcgtt atccggattt actgggtgta aagggagcgt agacggagtg gcaagtctga 241 tgtgaaaacc cggggctcaa ccccgggact gcattggaaa ctgtgcatct agagtgtcgg 301 agaggtaagc ggaattccta gtgtagcggt gaaatgcgta gatattagga ggaacaccag 361 tggcgaaggc ggcttactgg acgataactg acgctgaggc tcgaaagcgt ggggagcaaa 421 caggattaga taccctggta gtccacgccg taaacgatga ctactaggtg tcggggagca 481 cagctcttcg gtgccgcagc aaacgcaata agtattccac ctggggagta cgttcgcaag 541 aatgaaactc acagaatttg acggag

[0127] The reference strain for RSBI is Roseburia intestinalis strain L1-82 (kingdom Bacteria; phylum Firmicutes; class Clostridia; order Clostridia; family Lachnospiraceae; genus Roseburia), the genome sequence of which is shown in GenBank accession number: LR027880.1. In a particular embodiment, oligonucleotides specific for SEQ ID NO: 10 can be used for the quantification of RSBI. SEQ ID NO: 10 corresponds to the region 599208-600101 of GenBank accession number LR027880.1.

[0128] 1 atgtcttttt caagtgaagt gaaacaggag ttagcaaagc agagtggaaa gagcagacat 61 tgtcagatcg ctgaacttgc ggcgttagtt gcttttgacg gaaagcgtca gcagctgatc 121 ggggatgcgg gagatgtgct ggattcggaa aatccgcttc tgcaggaaaa atatggactg 181 ttactggcac agctgttcca tgttaata gaagagatag atacattgcc accggaacgc 241 attcttgaga caatcaagat gtggaatcag tctttaaggt gtgcagatat cacagagacg 301 gtaaatggta ttttactgca gcagacatgt tgcaggcggg catatatccg gggagcattt 361 ttagcaggtg gatcgatcag tgatccgaat aagtcttatc attttgagat tgtctgccgg 421 gaaattgcgc aggcgaaaca gcttcaggat gcaatcaaca gttttgagat ggaagcaaag 481 atcgtagaac gaaagaaaca ccaggtcgta tatttaaagg aaggtgccca gatcgtggat 541 atgttaaata tcatggaagc acatgtggca ctgatgaatc ttgaaaatgt gcgtatctta 601 aaagaaatga gaaattctgt caaccgaaag gtaaattgtg agacggcaaa tatcagcaag 661 actgtggcgg cagccgtaaa acagcttggc gatattgaat atattaaaca gaatgcaggc 721 ttagacagtc tgcctgaaaa cctaaaagaa atggcgttgc tgcggttaga gtatccggat 781 acaccgctta aggaactggg aacatatctc gatcctccgg tcggaaaatc gggagtgaac 841 cacaggctgc gcaggatcag tgagattgca gatgagctgc gtgaaaaaga ataa

[0129] A reference strain of CINT (kingdom Bacteria; phylum Actinobacteria; class Coriobacteria; order Coriobacteria; family Coriobacteria; genus Collinsella) is Collinsella intestinalis strain DSM 13280, the genome sequence of which has the NCBI reference sequence: GG692711.1. In a particular embodiment, the oligonucleotide specific for SEQ ID NO: 11 is This sequence can be used to quantify CINT. SEQ ID NO: 11 corresponds to the region: 297314-298120 of the NCBI reference sequence: GG692711.1.

[0130] 1 atgtccggac actctaaatg ggcaactacc aagcaccgca agggtgcgca ggacgctaag 61 cgttccgccc tgttctccaa gctgagccgt aacatcaccg tcgcggcccg tcttggcaac 121 gaccccaacc cggacaacaa cgcctccctc gccgctgccg tggccaaggc caaggctcag 181 tccatgccca aggacaagat caaggccgcc atcgacaagg ccttcggttc cggcgccgac 241 gccgccgtgt acgagaacat cgtgtacgag ggctacggcc cggccggtgt tgccgtgtac 301 gtcgagtgcc tgaccgacaa ccgcaaccgc accgccgccg atgtccgctc cgccttctcc 361 cacgctggcg gcaacctggg caccaccggc tccgttgcct tccagttcga gcgcaagggc 421 caggtcgtgg tctccaagca gatcgtcgac ccgaacgaca agaaggagaa cctcatggcc 481 aacggcgctg ccggcgatga ggaagagttc atgatggtca tcgccgaggc cggtggcgac 541 gactacgagg acgccggtga cgagtgggtc gtgtggaccg ccgcgggcga cctcatggcc 601 gtgtccaagg gcatcgaggc tcagggcatc gaggtcaagg gtgccgagct caccatggtc 661 ccgaccaccc cgacggccgt ctccggcgcc gacgccaaga aggtccagcg cctcatcgac 721 cgcctcgagg acctggacga cgtccaggac gtgtactcca ccatggacat gaccgacgag 781 gtcatcgctg ccctcgaaga ggagtaa

[0131] The reference strain of Faecalibacterium prausnitzii (kingdom Bacteria; phylum Firmicutes; class Clostridiales; order Clostridiales; family Ruminococcaceae; genus Faecalibacterium) is Faecalibacterium prausnitzii A2-165 (GenBank accession number: AJ270469.2), which is also a representative strain of its phylogenetic group II (PHGII). Faecalibacterium prausnitzii M21 / 2 (GenBank accession number: DS483503.1) is a representative strain of its phylogenetic group I (PHGI). In a particular embodiment, an oligonucleotide specific to SEQ ID NO: 12 can be used for the quantification of Faecalibacterium prausnitzii. SEQ ID NO: 12 (shown below) corresponds to GenBank accession number: AJ270469.2.

[0132] 1 gttgatcctg gctcaggacg aacgctggcg gcgcgcctaa cacatgcaag tcgaacgagc 61 gagagagagc ttgctttctc gagcgagtgg cgaacgggtg agtaacgcgt gaggaacctg 121 cctcaaagag ggggacaaca gttggaaacg actgctaata ccgcataagc ccacagctcg 181 gcatcgagca gagggaaaag gagcaatccg ctttgagatg gcctcgcgtc cgattagcta 241 gttggtgagg taatggccca ccaaggcaac gatcggtagc cggactgaga ggttgaacgg 301 ccacattggg actgagacac ggcccagact cctacgggag gcagcagtgg ggaatattgc 361 acaatggggg aaaccctgat gcagcgacgc cgcgtggagg aagaaggtct tcggattgta 421 aactcctgtt gttgaggaag ataatgacgg tactcaacaa ggaagtgacg gctaactacg 481 tgccagcagc cgcggtaaaa cgtaggtcac aagcgttgtc cggaattact gggtgtaaag 541 ggagcgcagg cgggaagaca agttggaagt gaaatctatg ggctcaaccc ataaactgct 601 ttcaaaactg tttttcttga gtagtgcaga ggtaggcgga attcccggtg tagcggtgga 661 atgcgtagat atcgggagga acaccagtgg cgaaggcggc ctactgggca ccaactgacg 721 ctgaggctcg aaagtgtggg tagcaaacag gattagatac cctggtagtc cacaccgtaa 781 acgatgatta ctaggtgttg gaggattgac cccttcagtg ccgcagttaa cacaataagt 841 aatccacctg gggagtacga ccgcaaggtt gaaactcaaa ggaattgacg ggggcccgca 901 caagcagtgg agtatgtggt ttaattcgac gcaacgcgaa gaaccttacc aagtcttgac 961 atcctgcgac gatgctggaa acagtatttt ccttcgggac gcagagacag gtggtgcatg 1021 gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaaccctta 1081 ctgtcagtta ctacgcaaga ggactctggc aggactgccg ttgacaaaac ggaggaaggt 1141 ggggatgacg tcaaatcatc atgcccttta tgacttgggc tacacacgta ctacaatggc 1201 gttaaacaaa gagaagcaag accgcgaggt ggagcaaaac tcagaaacaa cgtcccagtt 1261 cggactgcag gctgcaactc gcctgcacga agtcggaatt gctagtaatc gtggatcagc 1321 atgccacggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc atgagagccg 1381 gggggacccg aagtcggtag tctaaccgca aggaggacgc cgccgaaggt aaaactggtg 1441 attggggtga agtcgtaaca aggtac

[0133] A reference strain of Escherichia coli (kingdom Bacteria; phylum Proteobacteria; class Gammaproteobacteria; order Enterobacteriale; family Enterobacteriaceae; genus Escherichia) is Escherichia coli K-12 strain substrain MG1655, the genome sequence of which has GenBank accession number: CP032667.1. In a particular embodiment, oligonucleotides specific for SEQ ID NO: 13 (shown below) may be used for quantification of Escherichia coli.

[0134] 1 cgccctcccg aaggttaagc tacctacttc ttttgcaacc cactcccatg gtgtgacggg 61 cggtgtgtac aaggcccggg aacgtattca ccgtggcatt ctgatccacg attactagcg 121 attccgactt catggagtcg agttgcagac tccaatccgg actacgacgc actttatgag 181 gtccgcttgc tctcgcgagg tcgcttctct ttgtatgcgc cattgtagca cgtgtgtagc 241 cctggtcgta agggccatga tgacttgacg tcatccccac cttcctccag tttatcactg 301 gcagtctcct ttgagttccc ggccggaccg ctggcaacaa aggataaggg ttgcgctcgt 361 tgcgggactt aacccaacat ttcacaacac gagctgacga cagccatgca gcacctgtct 421 cacggttccc gaaggcacat tctcatctct gaaaacttcc gtggatgtca agaccaggta 481 agttcttcg cgttgcatcg aattaaacca catgctccac cgcttgtgcg ggccccgtc 541 aattcatttg agttttaacc ttgcggccgt actccccagg cggtcgactt aacgcgttag 601 ctccggaagc cacgcctcaa gggcacaacc tccaagtcga catcgtttac ggcgtggact 661 accagggtat ctaatcctgt ttgctcccca cgctttcgca cctgagcgtc agctttcgtc 721 cagggggccg ccttcgccac cggtattcct ccagatctct acgcatttca ccgctacacc 781 tggaattcta cccccctcta cgagactcaa gcttgccagt atcagatgca gttcccaggt 841 tgagcccggg gatttcacat ctgacttaac aaaccgcctg cgtgcgcttt acgcccagta 901 attccgatta acgcttgcac cctccgtatt accgcggctg ctggcacgga gttagccggt 961 gcttcttctg cgggtaacgt caatgagcaa aggtattaac tttactccct tcctccccgc 1021 tgaaagtact ttacaacccg aaggccttct tcatacacgc ggcatggctg catcaggctt 1081 gcgcccattg tgcaatattc cccactgctg cctcccgtag gagtctggac cgtgtctcag 1141 ttccagtgtg gctggtcatc ctctcagacc agctagggat cgtcgcctag gtgagccgtt 1201 accccaccta ctagctaatc ccatctgggc acatccgatg gcaagaggcc cgaaggtccc 1261 cctctttggt cttgcgacgt tatgcggtat tagctaccgt ttccagtagt tatccccctc 1321 catcaggcag tttcccagac attactcacc cgtccgccac tcgtcagcaa agaagcaagc 1381 ttcttcctgt taccgttcga cttgcatgtg ttaggcctgc cgccagcgtt caatctgagc 1441 catgatcaaa ct

[0135] In a preferred embodiment, any of the specific oligonucleotides listed in Table 3 and sequences having at least 80% identity thereto may be used to quantify B10, B46, B48, CINT, or RSBI, respectively. Also, in a preferred embodiment, any of the specific oligonucleotides listed in Table 2 of WO 2017 / 025617 and sequences having at least 80% identity thereto may be used to quantify Faecalibacterium prausnitzii, its phylogenetic groups (i.e., PHGI and PHGII), and Escherichia coli.

[0136] The method of the present invention, or any of its steps, may be implemented by a computer. The term computer as used herein may refer to any programmable device or system. In certain embodiments, optionally in combination with any of the features or embodiments described herein, the combined score of step c) is calculated using a computer, and / or the selection, classification, and / or decision of step d) is performed using a computer. This is done using

[0137] Thus, a further aspect of the present invention relates to a computer implemented method, which is any of the methods disclosed herein or any combination thereof.

[0138] It is noted that any computer program capable of carrying out any of the methods of the invention or used to carry out any of these methods or any combination thereof also forms part of the invention.

[0139] The computer program is typically directly loadable into the internal memory of a digital computer that contains software code portions that, when the product is executed on a computer, perform the steps of comparing a combination score (e.g., obtained from the levels of one or more target markers described herein) from one or more biological samples of a subject with a reference value (e.g., a reference combination value) and determining a diagnosis for the subject.

[0140] It is also noted that any device or apparatus comprising means for performing the steps of any of the methods of the invention or any combination thereof or capable of performing or having a computer program for performing any of the methods of the invention or any combination thereof is included as forming part of this specification.

[0141] The method of the invention may also comprise storing the result of the method on a data carrier, preferably said data carrier being a computer readable medium. The invention further relates to a computer readable storage medium having stored thereon the computer program of the invention or the result of any of the methods of the invention.

[0142] As used herein, a "computer-readable medium" may be any apparatus that can contain, store, communicate, transmit, or transfer the results of the determinations of the methods of the present invention. The medium may be an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system (i.e., an apparatus or device) or a propagation medium.

[0143] Treatment methods and related medical uses In a further aspect, the present invention provides a method of treating a subject having AN, AA, and / or CRC, wherein said subject has been selected by a screening, diagnostic, and / or monitoring method as described herein above, and said method further comprises administering to the subject an anti-cancer therapeutic agent.

[0144] In a related aspect, the present invention also provides any of the methods described hereinabove, further comprising the step of administering to said patient a therapeutically effective amount of an anti-cancer therapeutic.

[0145] In another aspect, the invention relates to an anti-cancer therapeutic for use in a method of treating a cancer patient, wherein the cancer patient has been selected by the screening, diagnostic, and / or monitoring methods described herein.

[0146] In an additional aspect, the present invention also relates to a method for selecting a subject to perform an exploratory test selected from the group consisting of colonoscopy, flexible sigmoidoscopy, double contrast barium enema, and computed tomography (CT) colonography, preferably to perform a colonoscopy, wherein said subject has been selected by the screening, diagnostic, or monitoring methods described herein.

[0147] In yet a further aspect, the present invention relates to a method of performing an exploratory test in a subject, said subject being selected by the screening, diagnostic or monitoring methods described herein, said method further comprising performing an exploratory test in the subject, said test being selected from the group consisting of colonoscopy, flexible sigmoidoscopy, double contrast barium enema, and computed tomography (CT) colonography, preferably colonoscopy.

[0148] Kits and Use of Kits in the Methods of the Invention In another aspect, the present invention relates to a kit suitable for quantifying any of GMLL, PTST, BCTF, or BCTT, comprising the following reagents: i. an oligonucleotide specific for the GMLL genome, preferably an oligonucleotide specific for SEQ ID NO:1; ii. an oligonucleotide specific for the PTST genome, preferably an oligonucleotide specific for SEQ ID NO:2; iii. an oligonucleotide specific for the BCTF genome, preferably specific for SEQ ID NO:3; iv. an oligonucleotide specific for the BCTT genome, preferably an oligonucleotide specific for SEQ ID NO: 4, and v. optionally an oligonucleotide suitable for the quantification of eubacteria as defined herein above, The kit includes one or more of the following:

[0149] The kit optionally further comprises instructions for use of the reagent in determining the level of the bacterial marker in a fecal sample isolated from a subject.

[0150] The term "kit" or "test kit" refers to a combination of reagents and adjuvants required for an analysis. A test kit most often consists of several units, but integrated analytical elements are also available and should be considered as a test kit as well.

[0151] In a preferred embodiment, said kit is suitable for quantifying PTST, BCTF and BCTT, said kit comprising reagent ii), reagent iii) and reagent iv) as defined herein above. Preferably, said kit further comprises reagent v) as defined herein above.

[0152] In certain embodiments, optionally in combination with one or more of the features or embodiments described herein: The oligonucleotide specific for SEQ ID NO:1 is an oligonucleotide comprising or consisting of either SEQ ID NO:22 or SEQ ID NO:23, or a sequence having at least 80% identity to either of them, and / or An oligonucleotide specific for SEQ ID NO:2 is an oligonucleotide comprising or consisting of either SEQ ID NO:24 or SEQ ID NO:25, or a sequence having at least 80% identity to either of them, and / or An oligonucleotide specific for SEQ ID NO:3 is an oligonucleotide comprising or consisting of either SEQ ID NO:26 or SEQ ID NO:27, or a sequence having at least 80% identity to either of them, and / or An oligonucleotide specific for SEQ ID NO: 4 is an oligonucleotide comprising or consisting of either SEQ ID NO: 30 or SEQ ID NO: 31, or a sequence having at least 80% identity thereto, and / or Suitable oligonucleotides for quantifying eubacteria include any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:14, or SEQ ID NO:15, or any of which have at least 80% identity. The oligonucleotide is an oligonucleotide that contains or consists of a sequence having the same identity.

[0153] A preferred embodiment is a kit suitable for quantifying PTST, BCTF and BCTT, comprising the following reagents: An oligonucleotide comprising or consisting of either SEQ ID NO: 24 or SEQ ID NO: 25, or a sequence having at least 80% identity thereto; An oligonucleotide comprising or consisting of either SEQ ID NO: 26 or SEQ ID NO: 27, or a sequence having at least 80% identity thereto, and An oligonucleotide comprising or consisting of either SEQ ID NO: 30 or SEQ ID NO: 31, or a sequence having at least 80% identity thereto, and Optionally, an oligonucleotide suitable for quantifying eubacteria (preferably an oligonucleotide comprising or consisting of any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:14 or SEQ ID NO:15, or a sequence having at least 80% identity to any of them), Including, Optionally, the kit further comprises instructions for the use of said reagent in determining the level of said bacterial marker in a fecal sample isolated from a subject.

[0154] Said particular oligonucleotides are as described herein above. Preferably, said oligonucleotides are primers and / or probes.

[0155] A further preferred embodiment is a kit suitable for quantifying PTST, BCTF and BCTT, comprising the following reagents: A primer comprising or consisting of SEQ ID NO: 24, a primer comprising or consisting of SEQ ID NO: 25, or a primer comprising or consisting of a sequence having at least 80% identity to any of them; A primer comprising or consisting of SEQ ID NO: 26, a primer comprising or consisting of SEQ ID NO: 27, or a primer comprising or consisting of a sequence having at least 80% identity to any of them, and A primer comprising or consisting of SEQ ID NO: 30, a primer comprising or consisting of SEQ ID NO: 31, or a primer comprising or consisting of a sequence having at least 80% identity to any of them, and Optionally, oligonucleotides suitable for quantifying eubacteria (preferably a primer comprising or consisting of SEQ ID NO:5, a primer comprising or consisting of SEQ ID NO:6, a primer comprising or consisting of SEQ ID NO:14, or a primer comprising or consisting of SEQ ID NO:15, or a primer comprising or consisting of a sequence having at least 80% identity to any of them), Including, Optionally, the kit further comprises instructions for the use of said reagent in determining the level of said bacterial marker in a fecal sample isolated from a subject.

[0156] In a preferred embodiment, the sequence having at least 80% identity is a sequence that is at least about 85%, preferably at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% identical to the reference sequence. The percentage of identity between two sequences can be determined by any means known in the art, such as the Needleman and Wunsch global alignment algorithm.

[0157] Additionally, the kit may further comprise oligonucleotides specific for other genes or bacterial markers as described herein above, for example, where the other bacterial markers are B10, B46, B48, CINT, RSBI, Faecalibacterium prausnidii, or the like. tsii, PHGI of F. prausnitzii, PHGII of F. prausnitzii, and E. coli.

[0158] The kit may include additional reagents. In certain embodiments, the kit typically includes reagents for performing real-time PCR reaction, including DNA polymerase such as Taq DNA polymerase (e.g., hot-start Taq DNA polymerase), buffer, magnesium, dNTPs, and optionally other agents (e.g., stabilizers such as gelatin and bovine serum albumin). In addition, the real-time PCR reaction mixture also includes reagents for real-time detection and quantification of amplification products, as described herein above.

[0159] Optionally, the kit may also include suitable tubes and solvents for DNA extraction, such as chloroform / methanol solutions. In addition, the kit may also include a container for collecting fecal samples, and optionally any buffers and / or additives suitable for sample storage, such as the FIT collection tubes used in the examples. Other preferred features and embodiments of the kit of the present invention are as described herein throughout the present specification.

[0160] In further aspects, the present invention also provides kits as described herein for use in the methods of screening, diagnosing, and / or monitoring AN, AA, and / or CRC described herein.

[0161] It is contemplated that any feature described herein may be combined in any way with any of the method, medical use, kit and kit use embodiments of the present invention, and any embodiment described herein may be implemented with respect to any of them. It is understood that the specific embodiments described herein are presented by way of illustration, not as limitations of the present invention.

[0162] All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0163] The use of the word "a" or "an" may mean "one," but is also consistent with the meanings "one or more," "at least one," and "one or more." The use of the term "another" may also refer to one or more. The use of the word "or" in the claims is used to mean "and / or," unless expressly stated to refer only to alternatives or where the alternatives are mutually exclusive.

[0164] As used in this specification and the claims, "comprising" (and any form of "comprise", such as "comprise" and "comprises"), "having", "Having" (as well as "have" and "has") "including" (and "includes") and any form of "comprise", such as "include", or "containing, (containing)" (and "contains" and "contain" etc.) The words "comprises" and "comprises" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The terms "consists of" and "consists essentially of" are also encompassed and expressly disclosed. As used herein, the phrase "consisting essentially of" limits the scope of a claim to those materials or steps explicitly recited, and which do not materially affect the basic and novel attribute(s) of the claimed invention. As used herein, the phrase "consisting only of" excludes any element, step, or ingredient not explicitly recited in the claim, except, for example, for impurities ordinarily associated with that element or limitation. exclude.

[0165] The term "or combinations thereof" as used herein refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or combinations thereof" includes at least one of A, B, C, AB, AC, BC, or ABC, and is intended to include BA, CA, CB, CBA, BCA, ACB, BAC, or CAB if order is important in the particular context. Continuing with this example, combinations including one or more repeats of an item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc. are expressly included. Those skilled in the art will appreciate that there is typically no limit to the number of items or terms in any combination, unless otherwise clear from the context.

[0166] As used herein, open-ended approximation terms such as "about", "around", "approximately", etc., when so modified, are not necessarily understood to be absolute or complete, but refer to a condition that would be considered by one of ordinary skill in the art to be close enough to provide a basis for designating the condition as existing. The extent to which the description may vary depends on how much modification can be made, allowing one of ordinary skill in the art to recognize that the modified feature still has the required characteristics and capabilities of the unmodified feature. Generally, subject to the above statements, numerical values ​​herein modified by approximation terms such as "about" may vary from the indicated value by ±1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. Thus, the term "about" may mean ±5% of the stated value of the value, preferably ±2% of the stated value of the value, and most preferably the term "about" means exactly the stated value (±0%).

[0167] The following examples serve to illustrate the invention and should not be construed as limiting its scope.

[0168] section 1. A method for increasing the specificity or reducing the false positive rate of a fecal occult blood test (FOBT) for screening, diagnosing, and / or monitoring colorectal advanced neoplasia (AN), advanced adenoma (AA), and / or colorectal cancer (CRC) in a subject, the FOBT comprising determining the presence of occult blood in a fecal sample isolated from a subject, the method comprising detecting in the fecal sample isolated from the subject at least the following bacterial markers: Peptostreptococcus stomatis (PTST), Bacteroides fragilis (BCTF) and Bacteroides thetaiotaomicron (BCTT), and quantifying the level of colorectal neoplasia (AN), wherein the term colorectal advanced neoplasia (AN) encompasses CRC and advanced adenoma (AA).

[0169] 2. A method for screening, diagnosing, and / or monitoring AN, AA, and / or CRC in a human subject, comprising: a) performing a FOBT on a fecal sample isolated from the subject; wherein FOBT involves determining the presence of occult blood in said fecal sample; b) quantifying at least bacterial markers consisting of Peptostreptococcus stomatis (PTST), Bacteroides fragilis (BCTF), and Bacteroides thetaiotaomicron (BCTT) in a fecal sample isolated from said subject; c) calculating a combination score from the levels of the bacterial markers determined in b); and d) classifying FOBT-positive subjects as representing an elevated risk of having AN, AA, and / or CRC according to the combined score obtained in c); A method comprising:

[0170] The combined score calculated in 3.c) is proportional to the amount of PTST and BCTF, and is proportional to the amount of BCT. The method of claim 1, wherein the score is inversely proportional to the amount of T, where the higher the score, the more likely the patient has AN, AA, and / or CRC.

[0171] 4. The method according to clause 2 or 3, wherein in step d), the method comprises comparing the combination score in the subject's sample with a reference combination score, and an increase in the combination score in the subject's sample relative to the reference combination score is indicative of AN, AA, and / or CRC.

[0172] 5. The method comprises steps a) and b) as described in paragraph 2, wherein the method comprises: e) comparing the amounts of PTST, BCTF, and BCTT in the fecal samples with the corresponding reference values; wherein: f) A method for screening, diagnosing and / or monitoring AN, AA and / or CRC in a human subject, wherein an increase in the amount of PTST and BCTF and a decrease in the amount of BCTT in a sample from a FOBT positive subject relative to corresponding reference values ​​is indicative of AN, AA and / or CRC.

[0173] 6. The method of any of clauses 1-5, wherein said subject is a subject suspected of having CRC.

[0174] 7. The method of any of clauses 1-5, wherein said subject is an asymptomatic subject.

[0175] 8. The method of clause 7, wherein the subject is a subject at moderate or high risk of CRC, wherein a subject at moderate risk of CRC is a subject who is 50 years of age or older, has no personal or familial history of CRC, and has an elevated and / or high risk of CRC.

[0176] 9. The method of any of clauses 1-8, wherein said CRC is adenocarcinoma.

[0177] 10. The method of any of clauses 1-9, wherein said FOBT is a guaiac FOBT or a fecal immunochemical test (FIT) that quantifies human hemoglobin (hHb).

[0178] 11. The method of any of clauses 1-10, wherein said FOBT is a fecal immunochemical test (FIT) that quantifies human hemoglobin (hHb).

[0179] 12. The method according to clause 11, wherein the FIT positive subject has an hHb level of ≧10 μg hHb per gram of faeces (cutoff for FIT50) or ≧20 μg hHb per gram of faeces (cutoff for FIT100), preferably the FIT positive subject has an hHb level of ≧10 μg hHb per gram of faeces (cutoff for FIT50).

[0180] 13. The above method is for B10 (SEQ ID NO: 7), B46 (SEQ ID NO: 8), B48 (SEQ ID NO: 9), Gemella morbillorum (GMLL), Roseburia intermedium (IL-10), and 13. The method of any of clauses 1 to 12, further comprising quantifying one or more bacterial markers selected from the group consisting of Roseburia intestinalis (RSBI), Collinsella intestinalis (CINT), Faecalibacterium prausnitzii, and / or any of its phylogenetic groups, and Escherichia coli (ECO).

[0181] 14. Determination of levels of PTST, BCTF, and BCTT, and optionally levels of bacterial markers in section 13, may be performed by next generation sequencing, quantitative PCR (qPCR), PCR pyrolysis, or other methods. 14. The method according to any of clauses 1 to 13, wherein the method is performed by a molecular biology method selected from the group consisting of PCR sequencing, PCR-ELISA, DNA microarray, branched DNA, dot blot, Fluorescence In Situ Hybridization Assay (FISH), and multiplex forms of the above methods.

[0182] 15. The method according to clause 14, wherein said molecular biology method is qPCR and preferably the quantification level is expressed as a Ct value.

[0183] 16. The method of any of clauses 1-15, wherein the levels of PTST, BCTF, and BCTT, and optionally the levels of the bacterial markers of clause 12, are preferably expressed as relative abundance relative to eubacteria (EUB) levels.

[0184] 17. The method of any of clauses 1 to 16, wherein DNA is extracted from the fecal sample prior to quantification of the PTST, BCTF, and BCTT, and optionally the bacterial markers of clause 12.

[0185] 18. The method of any of clauses 1 to 17, wherein quantification of FOBT, PTST, BCTF, and BCTT, and optionally the bacterial marker of claim 12, is performed on the same fecal sample.

[0186] 19. The method according to any of clauses 1 to 18, wherein the method further comprises storing the result of the method on a data carrier, preferably the data carrier is a computer readable medium.

[0187] 20. A computer-implemented method, the method being as defined in any of clauses 1 to 19.

[0188] 21. A data processing apparatus comprising means for performing the steps of the method of clause 20.

[0189] 22. A kit suitable for quantifying PTST, BCTF, and BCTT, comprising the following reagents: an oligonucleotide specific for SEQ ID NO:2; an oligonucleotide specific for SEQ ID NO:3, and an oligonucleotide specific for SEQ ID NO:4, Optionally, oligonucleotides suitable for the quantification of eubacteria, Including, Optionally, the kit further comprises instructions for use of the reagent in determining the level of the bacterial marker in a fecal sample isolated from a subject.

[0190] twenty three. An oligonucleotide specific for SEQ ID NO:2 is an oligonucleotide comprising or consisting of either SEQ ID NO:24 or SEQ ID NO:25, or a sequence having at least 80% identity to either of them, and / or An oligonucleotide specific for SEQ ID NO:3 is an oligonucleotide comprising or consisting of either SEQ ID NO:26 or SEQ ID NO:27, or a sequence having at least 80% identity to either of them, and / or An oligonucleotide specific for SEQ ID NO: 4 is an oligonucleotide comprising or consisting of either SEQ ID NO: 30 or SEQ ID NO: 31, or a sequence having at least 80% identity thereto, and / or The oligonucleotides suitable for quantifying eubacteria are those comprising or consisting of any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:14, or SEQ ID NO:15, or a sequence having at least 80% identity to any of them, as described in paragraph 22. Kit included.

[0191] 24. Use of the kit of clause 22 or 23 in a method for screening, diagnosing, and / or monitoring AN, AA, and / or CRC according to any of clauses 1 to 20.

[0192] 25. A method of treating a subject having AN, AA, and / or CRC, wherein the subject has been selected by a screening, diagnostic, and / or monitoring method according to any of clauses 1-20, and the method further comprises administering to the subject an anti-cancer therapeutic.

[0193] 26. A method for selecting a subject to perform an exploratory test selected from the group consisting of colonoscopy, flexible sigmoidoscopy, double contrast barium enema, and computed tomography (CT) colonography, preferably a colonoscopy, wherein said subject has been selected by a screening, diagnostic, or monitoring method according to any of clauses 1 to 20. EXAMPLES

[0194] Example 1. Materials and Methods Study population A cohort of 333 consecutive patients with symptoms related to CRC referred from primary and secondary health care to the Complexo Hospitalario de Ourense (Ourense, Spain) for diagnostic colonoscopy was recruited (Table 1). Exclusion criteria were: (1) asymptomatic subjects undergoing colonoscopy for CRC screening, (2) patients with a history of colonic disease undergoing surveillance colonoscopy, (3) patients requiring hospitalization, (4) patients whose symptoms had ceased within 3 months prior to evaluation, and (5) patients who had received antibiotic treatment within the previous month prior to inclusion. The study protocol was approved by the Biobanco del Complexo Hospitalario Universitario de Vigo (Vigo, Spain). Written informed consent was obtained from all study patients.

[0195] TIFF2025004152000002.tif39170

[0196] All subjects underwent colonoscopy to determine colorectal status. According to the results of endoscopy and pathology, the diagnoses were classified into four groups: normal colonoscopy (with no colonoscopy findings or hyperplastic polyps of the sigmoid colon and / or rectum less than 10 mm), non-advanced adenoma (tubular adenoma less than 10 mm with low-grade dysplasia, and serrated polyps less than 10 mm without dysplasia), advanced adenoma (adenoma greater than 10 mm or with ciliary components or high-grade dysplasia, serrated polyps greater than 10 mm or with dysplasia, and pTis adenocarcinoma), and invasive CRC. Patients diagnosed with CRC were also classified according to the stage of the tumor (Table 2). Individuals were also asked to complete a questionnaire to record clinical and epidemiological data.

[0197] TIFF2025004152000003.tif47170

[0198] Fecal sample collection Participants were asked to collect a stool sample from one defecation in a sterile fecal container before colonoscopy and before bowel cleansing. The sample was frozen immediately after being placed in the container. Subjects then brought the sample to the hospital, where it was frozen at -20°C for short-term storage and stored at -80°C after arrival at GoodGut SL's facility in Girona (Spain). A total of 11 subjects were excluded from the study due to incorrect collection of stool samples.

[0199] DNA extraction from stool samples NucleoSpin Soil Kit (Macherey-Nagel GMbH & Co., Dublin, Germany) Genomic DNA was extracted from frozen fecal samples after homogenization using a ELISA kit (Promega). DNA was finally eluted in a final volume of 100 μl SE elution buffer according to the manufacturer's instructions and stored at -20°C until use. DNA concentration was determined using Qubit fluorimetry (ThermoFisher Scientific, Waltham, USA). All samples were diluted to 8 ng / μl. Final concentrations were adjusted and requantified.

[0200] qPCR Assays for CRC Biomarkers The specific bacterial sequences targeted were Eubacteria (EUB), B10 (BestMatch BLAST Faecalibacterium prausnitzii), B46 (BestMatch BLAST The ten species were: Bacteroides fragilis (BCTF), Bacteroides intestinalis (B46), Bacteroides subdoligranulum variabile (B48), B48 (Best Match BLAST Ruminococcus spp., Roseburia spp., Coprococcus spp.), Roseburia intestinalis (RSBI), Gemella morvirorum (GMLL), Peptostreptococcus stomatis (PTST), Bacteroides fragilis (BCTF), Collinsella intestinalis (CINT), and Bacteroides thetaiotaomicron (BCTT).

[0201] SYBR Green master mix (Promega, Madison, USA) was used to Quantification of the different biomarkers was performed by preparing a single reaction per biomarker. Each reaction consisted of 20 μl containing 1× GoTaq qPCR master mix, between 150 nM and 300 nM of each primer, and up to 20 ng of genomic DNA template. The species-specific primers used in this study are listed in Table 3. They were purchased from Macrogen (Macrogen, Seoul, Korea).

[0202] TIFF2025004152000004.tif188170

[0203] All quantitative PCRs were performed on an AriaMx real-time PCR system (Agilent Technologies, Santa Clara, USA). Thermal profiles differed depending on the biomarker analyzed (Table 4). A melting curve step was added at the end of each qPCR to confirm the presence of the expected amplicon size and to reduce primer-dimer formation. Data were collected and analyzed using Aria software version 1.3 (Agilent Technologies, Santa Clara, USA). All samples were amplified in duplicate and were considered valid if the difference between threshold cycles (Ct) was less than 0.6. For each biomarker, a range of 10 genome units / μL to 10 8 A dynamic range of 8 logs down to genome units / μL was established and used to calculate relative abundance. Each PCR run included a control reaction without template.

[0204] TIFF2025004152000005.tif98170

[0205] FIT analysis The FIT analysis was performed at the General Hospital of Ourense using the same samples used for the CRC-specific biomarker analysis. As previously described in Cubiella, J. et al., 2016 The assay was performed using an automated OC sensor (Eiken Chemical Co., Ltd., Tokyo, Japan) to analyze stool samples for fecal hemoglobin determination using the collection tube of the OC sensor to detect gastrointestinal bleeding associated with disorders such as CRC, polyps, and diverticulitis. The sample buffer in the sample tube of the OC sensor ensures optimal stability and safe transport of the sample from collection to analysis. A positive test was one with a fecal hemoglobin concentration of 100 ng / mL (20 μg Hb per gram of feces; FIT100) or greater.

[0206] statistical analysis From a qualitative analysis point of view, a biomarker was considered absent if the obtained Ct value was not included within its dynamic range. All statistical analyses were performed using the SPSS 23.0 statistical package (IBM, New York, USA). Significance levels were determined by P values. Established when ≦0.05.

[0207] Normality of the data was assessed by the Kolmogorov-Smirnov test. The nonparametric Kruskal-Wallis test was used to test for differences in variables with more than two categories. Pairwise comparisons of subcategories of these variables were analyzed using the Mann-Whitney test. Multiple comparisons were corrected using the Bonferroni correction. All comparisons using bacterial markers were performed between relative abundances normalized by the dynamic range of each bacterial marker.

[0208] Receiver operating characteristic (ROC) curve analysis was applied to examine the utility of each biomarker for distinguishing between various colon neoplastic conditions. The discrimination accuracy was measured by the area under the ROC curve (AUC).

[0209] Using machine learning, we will combine any of the variables under study (gender, age, BMI, smoking, bacterial markers, FIT) to differentiate subjects with advanced neoplastic lesions from those with normal colonoscopy or The specific methodology consisted of initial training runs on 100 aleatory partitions of the data set to determine whether subjects with advanced adenomas could be differentiated from those with non-advanced adenomas. The study consisted of multiple iterations of machine learning and further validation of the generated predictive models using four different machine learning algorithms (neural networks, logistic regression, gradient boosting trees, and random forests). Finally, the combination of the four bacterial markers analyzed together with the FIT results was used to design the RAID-CRC.

[0210] Example 2. Fecal biomarkers in neoplastic progression The relative abundance of each bacterial marker was determined for each diagnosis (normal colonoscopy, non-advanced adenoma, advanced adenoma, CRC) (Figure 1). Regardless of colonoscopy diagnosis, three different butyrate-producing species (B10, B46, and B48) were the most prevalent biomarkers, with relative abundance values ​​of 20.4%, 19.0%, and 20.0%, respectively. GMLL and PTST were significantly more abundant in the CRC population than normal colonoscopy individuals (p=0.006 and p<0.001, respectively) or non-advanced adenoma subjects (p=0.047 and p<0.001, respectively). Although not significantly different, a trend could be observed for B46 to be more abundant in CRC patients than subjects with advanced adenomas (p=0.087). Interestingly, the abundance of EUB remained constant regardless of neoplastic status. Comparison between the different CRC stages (0, I, II, III, and IV) showed no significant differences in the abundance of any of the bacterial markers.

[0211] Example 3. GMLL, PTST, and BCTF can detect advanced neoplastic lesions The relative abundance of bacterial markers was compared after subjects were grouped as follows: (1) normal colonoscopy, (2) neoplasm (non-advanced adenoma + advanced adenoma + CRC), (3) advanced neoplasm (advanced adenoma + CRC), and (4) CRC (Figure 2). PTST was found to be highly correlated with neoplastic lesions (p<0.001). With regard to the detection of advanced neoplastic lesions, GMLL, PTST, and BCTF were potential biomarkers to detect them (p=0.006, p<0.001, and p=0.030, respectively). With regard to prevalence, these three opportunistic pathogens were more prevalent in patients with advanced neoplasms (GMLL, 64.9%; PTST, 58.4%; and BCTF, 44.7%) than in healthy subjects (GMLL, 53.5%; PTST, 26.1%; and BCTF, 29.8%).

[0212] Our results clearly indicate the presence of bacterial dysbiosis in patients with CRC. The studied bacterial markers were classified according to phenotypes related to gut health: butyrate-producing bacteria (B10, B46, B48, RSBI), opportunistic pathogens (GMLL, PTST, BCTF), hydrogen and oxygen producers (CINT), and saccharolytic species (BCTT) (Figure 3). The relative abundance of these phenotypes was found to change gradually with the progression of the disease state. In particular, a decrease in the relative abundance of butyrate-producing bacteria was found between subjects with normal colonoscopy and those with CRC, replacing pathogenic bacterial groups, which were more abundant in individuals with CRC and advanced adenoma than in subjects with normal colonoscopy.

[0213] Our results show that high abundance of PTST and BCTF correlates with advanced neoplasia, whereas abundance of BCTT correlates with healthy individuals. BCTT is a commensal bacterium commonly found in the gut microbiota of healthy individuals. Commensal bacteria have been found to reduce gastrointestinal inflammation and contribute to colonization resistance (Macfarlane & Macfarlane, 2012; Bauemler & Sperandio, 2016). Thus, high abundance of BCTT is a good predictor of progression of neoplasia. Correlated with better gut health.

[0214] Example 4. Combination of CRC bacterial markers with FIT can significantly reduce false positive results On the other hand, a significant difference was observed between subjects with normal colonoscopy or non-advanced adenomas and those with advanced neoplasms when using FIT100 (20 μg Hb per gram of stool) (p<0.001). 17.1% (19 subjects) of subjects with normal colonoscopy and 24.3% (27 subjects) of subjects with non-advanced adenomas showed positive FIT values. These results led to a sensitivity and specificity of 84.0% and 81.0%, respectively, and a positive and negative predictive value of 58.0% and 94.0%, respectively (AUC=0.828, 95.0% CI (0.773-0.883)) for the detection of advanced neoplasms. On the other hand, when using FIT50 (10 μg Hb / g stool), 21.1% (27 subjects) of subjects with normal colonoscopy and 24.2% (31 subjects) of subjects with non-advanced adenomas showed false-positive results. FIT50 led to a sensitivity and specificity of 91.0% and 76.0%, respectively, and a positive and negative predictive value of 55.0% and 96.0%, respectively, for the detection of advanced neoplasms (AUC=0.836, 95.0% CI (0.787-0.886)). Sensitivity values ​​for bacterial markers alone were much lower, 39.0% for GMLL (AUC=0.622, 95.0% CI (0.541-0.694)), 53.0% for PTST (AUC=0.710, 95.0% CI (0.628-0.776)), and 33.0% for BCTF (AUC=0.571, 95.0% CI (0.499-0.656)), whereas specificity values ​​were comparable.

[0215] Both FIT results, FIT100 and FIT50, were combined with fecal bacterial markers to see which one provides a better performance in terms of sensitivity and specificity values ​​for the detection of advanced neoplastic lesions. The combination of bacterial markers with FIT100 (RAID CRC-FIT100) led to obtaining a sensitivity of 76.0% and a specificity of 91.0% (Table 5). Nevertheless, these results were slightly improved by FIT50 (RAID CRC-FIT50), since it showed a 4.0% higher sensitivity (80.0%) with a similar specificity value for the detection of advanced neoplasms. This was therefore the optimal cut-off value. Thus, the preferred test is based on the combination of EUB, PTST, BCTF, and BCTT with a fecal hemoglobin concentration of 50 ng / μL (10 μg Hb per gram of feces) or higher. Although the BCTT showed no significant differences between subjects with normal colonoscopy or non-advanced adenomas and those with advanced neoplasms, when combined with the EUB, PTST, and BCTF, it could increase the specificity of the test.

[0216] TIFF2025004152000006.tif116170

[0217] The final test (RAID CRC-FIT50 test) contains a combination of FIT50 and the quantification levels of three bacterial markers (PTST / EUB, BCTF / EUB, BCTT / EUB) normalized by eubacteria, where the quantification levels are expressed as Ct values.

[0218] The use of ratios allows for normalization of the data, which is important for controlling variables related to qPCR and distinguishing true biological changes from experimentally induced variations (Vandesompele et al., 2002). Lowering the Robin concentration threshold from 100 ng / μL to 50 ng / μL allowed capturing positive subjects who would otherwise be considered false negatives at the 100 ng / μL cutoff, at the expense of increasing the false positive rate. However, this effect was attenuated when combined with bacterial markers, as the RAID CRC-FIT50 test provided a significant reduction in false positive results due to its increased specificity for detecting advanced neoplasms versus FIT50 and FIT100 (Table 5).

[0219] Application of the RAID CRC-FIT50 test to the detection of advanced neoplasms resulted in a reduction in the number of false positive results relative to FIT100, with 9.7% of subjects showing normal colonoscopy and 11.7% of subjects having non-advanced adenomas. Overall, the RAID-CRC-FIT50 test was shown to provide sensitivity and specificity of 80.0% and 90.0%, respectively (AUC=0.837, 95.0% CI (0.730-0.944)), and positive and negative predictive values ​​of 70.0% and 94.0%, respectively. More importantly, the false positive rate was reduced by 50.0%, with 46 subjects with false positive results for FIT100 and 23 subjects for the RAID-CRC-FIT50 test. Interestingly, both the RAID CRC-FIT50 and RAID CRC-FIT100 tests achieved similar sensitivity for CRC and AN as the FIT100 protocol, but the specificity and The positive predictive value (PPV) was higher.

[0220] In this study, a new methodology was developed that is suitable for use in a national CRC screening program using stool samples. The bacterial signatures used in this study were originally obtained from mucosal samples. Therefore, their presence in faeces is not greatly influenced by the variations caused by diet and some external factors (Conlon & Bird, 2015; Vandeputte et al., 2015), but is a measure of their actual abundance in the colonic mucosa. This is indicative of the presence of bacteria in the stool. It helps to dispel the enormous background noise that exists and gives physiological meaning to the biomarkers.

[0221] Metadata regarding body mass index (BMI), smoking, or dietary habits were not available in our dataset. Although these parameters have been reported to affect the microbiota composition of fecal samples (Davis et al., 2017; Yun et al., 2017; Rogers et al., 2012; Capurso & Lahner, 2017; Baothman et al., 2016), as mentioned above, our biomarkers are derived from mucosal samples that are less dependent on external factors (Watt et al., 2016; Durban et al., 2011). Biedermann et al. reported that smoking cessation increases microbial diversity (Biedermann et al., 2013). Other studies found that chronic alcohol consumption leads to an increase in the phylum Proteobacteria and a decrease in the phylum Bacteroidetes (Kakiyama et al., 2013; Rao et al., 2004). The relationship between BMI and microbiota composition was not clear. However, its impact on the microbiota remains controversial (Turnbaugh et al., 2006; Schwiertz et al., 2010; Santacruz et al., 2009). As the test is likely to be used in screening scenarios where the screened population may be influenced by various conditions and habits, a non-stratified strategy proves to be a good method to reproduce the CRC screening scenario with the greatest reliability.

[0222] Cost-effectiveness is also an important issue in population-based screening (Sonnenberg et al., 2000; McGrath et al., 2002; Telford et al., 2010). Wong and colleagues compared FIT with colonoscopy in this scenario and showed that FIT was cost-effective for average-risk screening, whereas colonoscopy was cost-effective among higher-risk subjects (Wong, 2015). Thus, the combination of FIT with fecal bacterial markers may be cost-effective, as the use of RAID-CRC could reduce unnecessary colonoscopies by up to 30%. More specifically, the implementation of RAID-CRC in a CRC screening program would result in a reduction of 33,000 colonoscopies due to false-positive results when compared to a FOBT-based screening program (55,000 false positives vs. 22,000 false positives, respectively) (Garcia et al., 2012). Considering that the cost of RAID-CRC is comparable to that of FOBT, the estimated savings of follow-up colonoscopy after a positive screening result is 77 million euros per 100,000 participants in a screening program (Table 6). Furthermore, the use of the CRC biomarkers presented in this study could be aimed at both developed and resource-poor regions with limited colonoscopy facilities, since RAID-CRC is a cost-effective tool that is likely to be feasible in CRC screening scenarios.

[0223] In conclusion, RAID-CRC is a promising tool for CRC screening due to its non-invasiveness, low cost, and ability to reduce the number of false-positive results associated with the use of FOBT (eg, FIT).

[0224] TIFF2025004152000007.tif97170

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Claims

1. 1. A method of increasing the specificity or reducing the false positive rate of a fecal occult blood test (FOBT) for screening, diagnosing, and / or monitoring advanced colorectal neoplasia (AN), advanced adenoma (AA), and / or colorectal cancer (CRC) in a subject, comprising: a. performing a FOBT on a fecal sample isolated from said subject; wherein said FOBT comprises determining the presence of occult blood in said fecal sample; b. In a fecal sample isolated from said subject, at least the following bacterial markers are detected: Peptostreptococcus stomatis (PTST), Bacteroides fragilis (BCTF), and Bacteroides thetaiotaomicron (BCTT), Quantifying the wherein the term advanced colorectal neoplasia (AN) includes CRC and advanced adenoma (AA).

2. 1. A method of screening, diagnosing, and / or monitoring advanced colorectal neoplasia (AN), advanced adenoma (AA), and / or colorectal cancer (CRC) in a subject, comprising: a. performing a FOBT on a fecal sample isolated from said subject; wherein said FOBT comprises determining the presence of occult blood in said fecal sample; b. In a fecal sample isolated from said subject, at least the following bacterial markers are detected: Peptostreptococcus stomatis (PTST), Bacteroides fragilis (BCTF), and Bacteroides thetaiotaomicron (BCTT), Quantifying the wherein the term advanced colorectal neoplasia (AN) includes CRC and advanced adenoma (AA).

3. The method comprises: c) calculating a combination score from the levels of said bacterial markers determined in b); and d) classifying FOBT-positive subjects as having AN, AA, and / or CRC according to the combined score obtained in c); 3. The method of screening, diagnosing, and / or monitoring AN, AA, and / or CRC in a human subject according to claim 2, further comprising:

4. 4. The method of claim 3, wherein the combined score calculated in c) is proportional to the amount of PTST and BCTF and inversely proportional to the amount of BCTT, wherein the higher the score, the more likely the patient is to have AN, AA, and / or CRC.

5. 5. The method according to any one of claims 2 to 4, wherein in step d) the method comprises comparing the combined score in the subject's sample with a reference value, wherein an increase in the combined score in the subject's sample relative to the reference value is indicative for AN, AA and / or CRC.

6. The method comprises: i. comparing the amount of GMLL, PTST, and BCTT in the fecal sample with corresponding reference values; wherein: ii. The method of claim 2, wherein an increase in the amount of GMLL and PTST and a decrease in the amount of BCTT in a sample of a FOBT-positive subject relative to the corresponding reference value are indicative of AN, AA, and / or CRC. Methods for testing, diagnosing, and / or monitoring.

7. The method of any one of claims 1 to 6, wherein the subject is a subject suspected of having CRC or an asymptomatic subject.

8. The method of any one of claims 1 to 7, wherein the FOBT is a fecal immunochemical test (FIT) that quantifies human hemoglobin (hHb).

9. 9. The method of claim 8, wherein a FIT positive subject has an hHb level of 10 μg hHb / g stool or more (cutoff for FIT50) or 20 μg hHb / g stool or more (cutoff for FIT100), preferably a FIT positive subject has an hHb level of 10 μg hHb / g stool or more (cutoff for FIT50).

10. 10. The method of any one of claims 1 to 9, further comprising quantifying one or more bacterial markers selected from the group consisting of B10 (SEQ ID NO:7), B46 (SEQ ID NO:8), B48 (SEQ ID NO:9), Gemella morvirorum (GMLL), Roseburia intestinalis (RSBI), Collinsella intestinalis (CINT), Faecalibacterium prausnitzii, and / or any of its phylogenetic groups, and Escherichia coli (ECO).

11. 11. The method according to any one of claims 1 to 10, wherein the determination of the levels of PTST, BCTF and BCTT, and optionally the bacterial markers of claim 10, is performed by qPCR, preferably the level of quantification is expressed as a Ct value.

12. 12. The method of any one of claims 1 to 11, wherein the levels of PTST, BCTF and BCTT and optionally the levels of said bacterial markers of claim 10 are preferably expressed as relative abundance to eubacteria (EUB) levels.

13. The method according to any one of claims 1 to 12, wherein the method further comprises storing the result of the method on a data carrier, preferably the data carrier being a computer readable medium.

14. A computer implemented method, the method as defined in any one of claims 1 to 13.

15. A kit suitable for quantifying PTST, BCTF, and BCTT comprising the following reagents: An oligonucleotide comprising or consisting of either SEQ ID NO: 24 or SEQ ID NO: 25, or a sequence having at least 80% identity thereto; An oligonucleotide comprising or consisting of either SEQ ID NO: 26 or SEQ ID NO: 27, or a sequence having at least 80% identity thereto, and An oligonucleotide comprising or consisting of either SEQ ID NO: 30 or SEQ ID NO: 31, or a sequence having at least 80% identity thereto, and Optionally, an oligonucleotide suitable for the quantification of eubacteria, preferably an oligonucleotide comprising or consisting of any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:14 or SEQ ID NO:15, or a sequence having at least 80% identity to any of them, Including, Optionally, the kit further comprises instructions for use of the reagent in determining the level of a bacterial marker in a fecal sample isolated from a subject.

16. Use of the kit according to claim 15 in a method for screening, diagnosing and / or monitoring AN, AA and / or CRC according to any one of claims 1 to 14.

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