Improved methods for screening, diagnosis and / or monitoring of colorectal advanced neoplasia, advanced adenomas and / or colorectal cancer

Abstract

The present invention relates to improved methods for screening, diagnosing and / or monitoring colorectal advanced neoplasia (AN), advanced adenoma (AA) and / or colorectal cancer (CRC), wherein AN includes CRC and AA. In particular, the method provides increased specificity due to a reduction in false positive results in fecal occult blood tests (FOBTs) using characteristics 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 for treatment with anticancer therapy.

Description

Technical Field

[0001] The present invention relates to the field of cancer diagnosis. Specifically, 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), wherein AN includes CRC and AA. In particular, the method provides increased specificity due to the reduction of false positive results in fecal occult blood test (FOBT) using characteristics 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 for anticancer therapy. Background Art

[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 death (Estimated Global Cancer Incidence, Mortality, and Prevalence 2012, http: / / globocan.iarc.fr / Pages / fact_sheets_population.aspx). By age 75, 6% of people will be affected, with incidence rates much higher in developed countries than in developing countries (Wilson, 2005). Although cancers show a strong genetic component, most CRCs are sporadic and develop slowly (Brenner et al., 2014). In most patients, symptoms are absent until advanced disease develops. Regular colorectal cancer screening has shown a 26% reduction in the risk of dying from CRC when stool testing is used and up to a 50% reduction when flexible sigmoidoscopy is used (Zauber, 2015; Elmunzer et al., 2012). However, CRC screening programs are only implemented in a few countries worldwide.

[0003] Guidelines recommend routine CRC screening for asymptomatic adults starting at age 50 years (Rex et al., 2017). Current screening programs are based on two strategies: invasive endoscopy-based procedures and noninvasive stool testing-based procedures (Quintero et al., 2012). There are different endoscopic procedures for CRC screening, such as flexible sigmoidoscopy and colonoscopy. The main advantage of colonoscopy is its ability to directly visualize the entire colon, enabling accurate diagnosis and, therefore, preventing CRC through early detection of precancerous lesions (Young & Womeldorph, 2013). However, this procedure requires bowel preparation and sedation, carries the risk of bowel perforation and other adverse effects, and is time-consuming and expensive (Rutter et al., 2012; Sieg et al., 2001). Flexible sigmoidoscopy is an alternative approach that allows direct visualization of the distal colon, namely the rectum, sigmoid colon, and descending colon. This approach avoids sedation and reduces the risk of bowel perforation (Gatto et al., 2003). Compared with endoscopic strategies, stool testing is noninvasive and less expensive, making it the preferred CRC screening procedure in most countries. Subjects with a positive result on any alternative stool test typically undergo colonoscopy for an accurate diagnosis. Therefore, noninvasive testing is frequently used as an initial step in screening approaches.

[0004] One of the stool 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 the chemical oxidation reaction between heme and α-guaiac acid. The main disadvantage of this type of test is that it requires a prescribed diet to avoid false-positive results caused by the consumption of certain foods, alcohol, or nonsteroidal anti-inflammatory drugs (Sanford, 2009). Although it is non-invasive, it has a low sensitivity for CRC (25%-38%) and precancerous lesions (16%-31%) (Stracci, 2014). 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 FIT screening has higher sensitivity than gFOBT (Brenner & Tao, 2013; Shapiro et al, 2017; Mousavinezhad et al, 2016; Goede et al, 2017). Although the overall sensitivity of FIT for CRC is approximately 61%-91% and for advanced adenomas is 27%-67% (Stracci et al, 2014), these figures still imply a high false-positive rate.

[0005] Recently, reports have shown that the bacterial communities in the intestinal mucosa of CRC patients differ from those in healthy individuals (Borges-Canha et al., 2015; Mira-Pascual et al., 2014). Evidence suggests that the gut microbiota may play an important role in the pathogenesis of CRC (Sobhani et al., 2011; Jun et al., 2010). To date, two possible mechanisms by which the gut microbiota may induce tumorigenesis have been described. On the one hand, the gut microbiota may promote chronic inflammation, which in turn can lead to tumor formation (Kostic et al., 2013; Zackular et al., 2014). On the other hand, there is evidence that some dietary components metabolized by the gut microbiota, such as red meat, can lead to the production of carcinogenic compounds (Joshi et al., 2015). Furthermore, numerous studies have recently been conducted to elucidate whether there is CRC-specific dysbiosis or whether any specific species are associated with CRC development (Dulal & Keku, 2014; Zeller et al., 2014; Marchesi et al., 2011).

[0006] In 2012, a preliminary prospective study by our group of 60 individuals (41 with CRC and 19 with normal colonoscopies) defined a bacterial cluster in mucosal biopsies whose prevalence correlated with CRC risk (Mas de Xaxars Rivero, 2012). Of the 55 phylotypes analyzed, 6 were significantly more frequent in patients with CRC than in controls. Five of these phylotypes shared similarity with sequences of uncultured bacteria isolated from the human gastrointestinal tract or human feces, and one shared 97% similarity with Parabacteroides merdae. In contrast, two phylotypes, B34 (99% similarity to Clostridium nexile) and B35 (97% similarity to Roseburia faecalis), were significantly more prevalent in healthy subjects than in patients with CRC.

[0007] The inventors' team then designed a quantitative polymerase chain reaction (qPCR) system specifically targeting these bacterial markers (Mas de Xaxars Rivero, 2012). Bacterial signatures were then tested on stool samples (7 from healthy controls and 9 from CRC patients), looking for different abundances to determine which ones would be suitable as non-invasive tools for CRC screening (WO 2015 / 132273). A retrospective clinical study involving 46 patients, led by Dr. Josep Trueta of the Hospital Universitari de Girona (Girona, Spain), confirmed the suitability of some bacterial signatures as CRC markers (WO 2015 / 132273).

[0008] Despite recent progress, new tools for early detection of advanced colorectal neoplasia through population-based screening and surveillance strategies are still needed to reduce CRC mortality. The ideal technology should be noninvasive, cost-effective, reproducible, and able to detect precancerous lesions with a high risk of neoplastic development with high sensitivity and specificity. Summary of the Invention

[0009] The present invention provides a novel non-invasive AA, CRC and / or AN screening, diagnosis and / or monitoring test based on a novel bacterial signature, which complements the fecal occult blood test (e.g., FIT) and can reduce FOBT-related false positive results, thereby improving its specificity and positive predictive value (PPV) while maintaining a similar level of sensitivity.

[0010] A first aspect of the present invention relates to a method for screening, diagnosing and / or monitoring a subject for colorectal advanced neoplasia (AN), advanced adenoma (AA) and / or colorectal cancer (CRC) by increasing the specificity of a fecal occult blood test (FOBT) or reducing the false positive rate of a FOBT, wherein the method comprises:

[0011] a) optionally performing a FOBT on a stool sample isolated from said subject, wherein said FOBT comprises determining the presence of occult blood in said stool sample; and

[0012] b) quantifying at least one of the following bacterial markers, or any combination thereof, in an intestinal sample (preferably a stool sample) isolated from the subject:

[0013] - Gemella morbillorum (GMLL),

[0014] - Peptostreptococcus stomatis (PTST), and

[0015] -Bacteroides fragilis (BCTF).

[0016] In a related aspect, the present invention relates to a method for screening, diagnosing and / or monitoring AN, AA and / or CRC in a subject, the method comprising:

[0017] a) optionally, performing a FOBT on a stool sample isolated from said subject, wherein said FOBT comprises determining the presence of occult blood in said stool sample;

[0018] b) quantifying at least one of the following bacterial markers, or any combination thereof, in an intestinal sample (preferably a stool sample) isolated from the subject:

[0019] - Gemini coccus morbilli (GMLL),

[0020] - Peptostreptococcus gastricis (PTST), and

[0021] - Bacteroides fragilis (BCTF).

[0022] In another aspect, the present invention provides a method of treating a subject having AN, AA and / or CRC, wherein the subject is selected by the screening, diagnosis and / or monitoring methods of the other aspects of the invention, and wherein the method further comprises administering an anti-cancer therapy to the subject.

[0023] In a related aspect, the present invention also provides any of the methods described in other aspects, further comprising the step of administering to the patient a therapeutically effective amount of an anti-cancer therapy.

[0024] In another aspect, the invention relates to an anti-cancer therapy for use in a method of treating a cancer patient, wherein the cancer patient is selected by the screening, diagnosis and / or monitoring methods of the other aspects of the invention.

[0025] In another aspect, the present invention also relates to a method for selecting a subject for an exploratory test (the exploratory test is selected from the group consisting of colonoscopy, flexible sigmoidoscopy, double contrast barium enema and computed tomography (CT) colonoscopy), preferably a colonoscopy, wherein the subject is selected by the screening, diagnostic or monitoring method of other aspects of the present invention.

[0026] In yet another aspect, the invention relates to a method of performing exploratory testing in a subject, wherein the subject has been selected by the screening, diagnostic or monitoring methods of the other aspects of the invention, wherein the method further comprises performing exploratory testing on the subject, wherein the test is selected from the group consisting of colonoscopy, flexible sigmoidoscopy, double contrast barium enema and computed tomography (CT) colonoscopy, preferably colonoscopy.

[0027] In another aspect, the present invention provides a kit suitable for quantifying any one of GMLL, PTST, BCTF, or BCTT, wherein the kit comprises one or more of the following reagents:

[0028] i. an oligonucleotide specific for the GMLL genome, preferably an oligonucleotide specific for SEQ ID NO: 1;

[0029] ii. an oligonucleotide specific for the PTST genome, preferably an oligonucleotide specific for SEQ ID NO: 2;

[0030] iii. an oligonucleotide specific for the BCTF genome, preferably an oligonucleotide specific for SEQ ID NO: 3;

[0031] iv. an oligonucleotide specific for the BCTT genome, preferably an oligonucleotide specific for SEQ ID NO: 4; and

[0032] v. Optionally, oligonucleotides suitable for quantification of eubacteria as defined above.

[0033] In another aspect, the present invention also provides a kit as described herein, for use in a method for screening, diagnosing and / or monitoring AN, AA and / or CRC as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1: Relative abundance percentage of biomarkers (B10 (best hit BLAST for Faecalibacterium prausnitzii); B46 (best hit BLAST for Faecalibacterium prausnitzii, Subdoligranulum variabil); B48 (best hit BLAST for Ruminococcus, Roseburia, Coprococcus); Geminicoccus morbilli (GMLL); Peptostreptococcus gastricis (PTST), Bacteroides fragilis (BCTF), Collinsella intestinalis (CINT), Bacteroides thetaiotaomicron (BCTT), and Roseburia intestinalis (RSBI)) analyzed for subjects with normal colonoscopy (NC), non-advance adenoma (NAA), advanced adenoma (AA), and colorectal cancer (CRC);

[0035] Figure 2 : Comparison of biomarker abundance between the following different diagnoses: NC, normal colonoscopy; neoplasia, non-advanced adenoma + advanced adenoma + colorectal cancer; advanced neoplasia, advanced adenoma + colorectal cancer; CRC, colorectal cancer. Significance level: *p value < 0.05, **p value < 0.01, ***p value < 0.001;

[0036] Figure 3 : Percent relative abundance of biomarkers (butyrate-producing species: B10, B46, B48, and Roseburia intestinalis (RSBI); opportunistic pathogens: Gemini coccus morbilli (GMLL), Peptostreptococcus gastricis (PTST), and Bacteroides fragilis (BCTF); H2 and O2 producers: Collinsella intestinalis (CINT); and saccharolytic bacteria: Bacteroides thetaiotaomicron (BCTT)) analyzed for subjects with normal colonoscopy (NC), non-advanced adenoma (NAA), advanced adenoma (AA), and colorectal cancer (CRC). DETAILED DESCRIPTION

[0037] definition

[0038] The terms "subject" or "individual" are used interchangeably herein and refer to all animals classified as mammals, including but not limited to domestic and farm animals, primates, and humans, such as humans, non-human primates, cattle, horses, pigs, sheep, goats, dogs, cats, or rodents. Preferably, the subject is a male or female human of any age or race.

[0039] As used herein, the term "sensitivity" refers to the proportion of subjects who have the target condition (reference standard positive) and give a positive test result (TP / (TP+FN)). It shows how effective the test is in detecting the disease. Sensitivity ("sens") can be in the range of 0 (0%) < sens < 1 (100%), where ideally, the number of false negatives is equal to or close to zero and sensitivity is equal to or close to 1 (100%).

[0040] As used herein, the term "specificity" in relation to a test refers to the proportion of subjects who do not have the target condition (reference standard negative) and give a negative test result (TN / (TN+FP)). It shows how well the test is at identifying normal (negative) conditions. Specificity ("spec") can range from 0 (0%) < spec < 1 (100%), with the ideal case being that the number of false positives is equal to or nearly equal to 0 and the specificity is equal to or nearly equal to 1 (100%).

[0041] As used herein, the term "accuracy" refers to the proportion of true results (true positives or true negatives) in a population. It measures how accurately a screening test is for a condition, i.e., how correctly it can rule in and out a given condition (TN+TP) / (TN+TP+FN+FP). Accuracy ("acc") can range from 0 (0%) < acc < 1 (100%). Ideally, the number of false positives and false negatives is zero or close to zero, and accuracy is 1 (100%) or close to 1 (100%).

[0042] As used herein, the term "Receiver Operating Characteristic (ROC) curve" refers to a diagram illustrating the performance of a binary classifier system as its discrimination threshold changes. This curve is created by drawing the true positive rate and the false positive rate under various threshold settings. The true positive rate is also referred to as sensitivity. The false positive rate is calculated as 1-specificity. Therefore, the ROC curve is a way of graphically displaying the true positive rate and the false positive rate (sensitivity and (1-specificity)) within a range of cutoff values ​​and selecting the best cutoff value for clinical use. The accuracy represented by the area under the ROC curve (AUC) provides a useful parameter for comparing test performance. An AUC close to 1 represents that the test is highly sensitive and highly specific, while an AUC close to 0.5 represents that the test is neither sensitive nor specific. Typically, if AUC is 0.6 to 0.75, then the test is considered to be a suitable discriminator, if AUC is 0.75 to 0.9, then it is considered to have good discrimination ability, and if AUC is 0.9 to 1, then it is considered to be an excellent discriminator. For more 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.

[0043] The term "probe" as used herein refers to a synthetic or biologically produced nucleic acid having a length of 10 to 285 base pairs, containing a specific nucleotide sequence that allows preferential hybridization with a target nucleic acid sequence under predetermined conditions, and optionally containing a portion for detecting or improving test performance. Typically, a minimum of 10 nucleotides are required to statistically obtain specificity and form a stable hybridization product, and a maximum of 285 nucleotides typically represent an upper limit to the length of the reaction parameters that can be easily adjusted to determine mismatched sequences and preferential hybridization. The probe may optionally contain certain components that contribute to the proper functioning or optimal effect of the probe under certain assay conditions. For example, the probe may be modified to improve its resistance to nuclease degradation (e.g., by blocking), to carry a detection ligand (e.g., fluorescein), to carry a ligand (e.g., biotin) for purification or enrichment purposes, or to facilitate capture thereof onto a solid support (e.g., polydeoxyadenosine "tail").

[0044] As used herein, the term "primer" refers to an oligonucleotide that can be used in amplification methods such as polymerase chain reaction ("PCR") to amplify a nucleotide sequence. Primers are designed based on the polynucleotide sequence of a particular target sequence, such as a specific 16S rDNA sequence.

[0045] The term "specific" as used herein in relation to a nucleotide sequence means that the nucleotide sequence will hybridize to / amplify a predetermined target sequence and will not substantially hybridize to / amplify non-target sequences under assay conditions (usually stringent conditions).

[0046] The term "hybridization" as used herein refers to a process by which two partially complementary or completely complementary nucleic acid chains are polymerized in an antiparallel manner under predetermined reaction conditions to form a double-stranded nucleic acid with specific and stable hydrogen bonds, which follows clear rules that nucleic acid bases can pair with each other.

[0047] The term "substantial hybridization" means that the amount of hybridization observed would cause a person observing the result to consider the result to be positive relative to the hybridization data in the positive and negative controls. Data considered to be "background noise" is not substantial hybridization.

[0048] The term "stringent hybridization conditions" refers to conditions at about 35°C to 65°C and in about 0.9 molar NaCl salt solutions. Stringency can also be controlled by reaction parameters such as the concentration and type of ionic species present in the hybridization solution, the type and concentration of the denaturing agent present, and the hybridization temperature. Typically, as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. Generally speaking, the stringency of the conditions under which hybridization occurs will determine certain characteristics of the preferred probes to be used.

[0049] As used herein, the term "therapeutically effective amount" refers to an amount effective in the prophylactic or therapeutic treatment of a disease, disorder, or pathological condition following single or multiple dose administration to a subject (e.g., a human patient).

[0050] As used herein, the term "anti-cancer therapy" refers to a therapy useful in treating cancer. Examples of anti-cancer therapies include, but are not limited to, for example, surgery, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, anti-hormonal agents, agents used for radiation therapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents for treating cancer, such as anti-HER-2 antibodies (e.g., ), anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib), ), platelet-derived growth factor inhibitors (e.g., Gleevec TM(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, ErbB3, ErbB4, PDGFR-β, BlyS, APRIL, BCMA or VEGF receptor, TRAIL / Apo2, and other biologically active and organic chemical agents, etc. Combinations thereof are also included in the present invention.

[0051] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes cell destruction. The term is intended to include 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 (e.g., small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof).

[0052] As used herein, the term "chemotherapeutic agent" refers to a compound that can be used to treat cancer. Examples of chemotherapeutic agents include: alkylating agents, such as thiotepa and Cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, uredopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trishydroxymethylmelamine; annonaceous lactones (especially bullatacine); acin and bullatacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin); nostoc (particularly nostoc 1 and nostoc 8); dolastatin; duocarmycins (including the synthetic analogues KW-2189 and CB1-TM1); acanthopanax eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards (e.g., chlorambucil, naphthiazolin, cholephosphamide, estramustine, ifosfamide, mechlorethamine, methoxychlor hydrochloride, melphalan, novembichin, phenylephrine, prednimustine, trofosfamide, uracil mustard); nitroureas , such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin γ1 and calicheamicin Ω11); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin;and new carcinostaticin chromophores and related pigment protein enediyne antibiotic chromophores), aclacinomycins, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,; Doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrroline-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogamycin, olivomycin, pepriomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomycin, streptozotocin, truffle, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil ( 5-FU); folic acid analogs such as leucovorin, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiopurine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as captestosterone, drostanolone propionate, cyclothiosteroid, melastane, and testolactone; antiadreners such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as frolinic acid. acid; acetylglucosyl ester; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilones; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansine alkaloids, such as maytansine and ansamitocin; mitoguanidine; mitoxantrone; mopidanmol; diamine nitrazolium; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-acetylhydrazine; procarbazine; Polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triazinon; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, baculocin A, and anguidine); urethanes; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g., Paclitaxel, without Cremophor Albumin-engineered nanoparticle formulations of paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Vinorelbine; mitoxantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including regimens of irinotecan with 5-FU and leucovorin); the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including oxaliplatin regimens (FOLFOX); lapatinib PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib) that reduce cell proliferation ) and VEGF-A inhibitors, and pharmaceutically acceptable salts, acids or derivatives of any of the above substances.

[0053] As used herein, the term "antihormonal agent" refers to an agent that regulates or inhibits the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including tamoxifen ( tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and FARESTON toremifene; aromatase inhibitors that inhibit the aromatase enzyme, which regulates estrogen production in the adrenal glands, for example, 4(5)-imidazoles, aminoglutethimide, Megestrol acetate, Exemestane, formestane, fadrozole, vorozole, Letrozole and anastrozole; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, goserelin; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as PKC-α, Ralf, and H-Ras; ribozymes, such as inhibitors of VEGF expression (e.g., ribozymes) and HER2 expression inhibitors; vaccines, such as gene therapy vaccines, such as vaccine, Vaccines and vaccine; rIL-2; Topoisomerase 1 inhibitors; rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

[0054] The term "cytokine" is a general term for proteins released by one cell population that act as intercellular mediators on another cell. 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; hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and tumor necrosis factor-β; Müllerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors, such as NGF-α; platelet-derived growth factor; transforming growth factor (TGF-β); and thrombopoietin (TPO). growth factor (TGF), such as TGF-α and TGF-β; insulin-like growth factor-I and insulin-like growth factor-II; erythropoietin (EPO); osteoinductive factors; interferons, such as interferon-α, interferon-β and interferon-γ colony stimulating factor (CSF), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (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 factor, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (kit ligand, KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

[0055] As used herein, the term "growth inhibitor" refers to a compound or composition that inhibits cell growth in vitro and / or in vivo. Thus, a growth inhibitor can be a substance that significantly reduces the percentage of cells in the S phase. Examples of growth inhibitors include agents that block cell cycle progression (other than in the S phase), such as agents that induce G1 arrest and M phase arrest. Classical M phase blockers include periwinkle (vincristine and vinblastine), and topo II inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Drugs that arrest G1 can also spill over into S phase arrest, such as DNA alkylating agents, such as tamoxifen, prednisone, dacarbazine, nitrogen mustard, cisplatin, methotrexate, 5-fluorouracil, and ara-C. More information can be found in Murakami et al., Chapter 1, "Cell cycle regulation, oncogenes, and antineoplastic drugs," in Mendelsohn and Israel, eds., The Molecular Basis of Cancer (WB Saunders: Philadelphia, 1995), especially page 13.

[0056] As used herein, the term "radiation therapy" or "radiotherapy" refers to the use of directed gamma or beta rays to induce sufficient damage to cells to limit their ability to function normally or to completely destroy the cells. It should be understood that there are many methods known in the art to determine the dosage and duration of treatment. Typical treatment is a one-time administration, with a typical dosage range of 10 to 200 units (Gray) per day.

[0057] As used herein, the term "intestinal diseases" refers to those diseases or conditions that affect the small intestine, colon, and / or rectum.

[0058] Method of the present invention

[0059] In a first aspect, the present invention relates to a method for screening, diagnosing and / or monitoring a subject for colorectal advanced neoplasia (AN), advanced adenoma (AA) and / or colorectal cancer (CRC) by increasing the specificity of a fecal occult blood test (FOBT) or reducing the false positive rate of a FOBT, wherein the method comprises:

[0060] a) optionally performing a FOBT on a stool sample isolated from said subject, wherein said FOBT comprises determining the presence of occult blood in said stool sample; and

[0061] b) quantifying at least one of the following bacterial markers, or any combination thereof, in an intestinal sample (preferably a stool sample) isolated from the subject:

[0062] - Gemini coccus morbilli (GMLL),

[0063] - Peptostreptococcus gastricis (PTST), and

[0064] - Bacteroides fragilis (BCTF).

[0065] In a related aspect, the present invention relates to a method for screening, diagnosing and / or monitoring AN, AA and / or CRC in a subject, the method comprising:

[0066] a) optionally, performing a FOBT on a stool sample isolated from said subject, wherein said FOBT comprises determining the presence of occult blood in said stool sample;

[0067] b) quantifying at least one of the following bacterial markers, or any combination thereof, in an intestinal sample (preferably a stool sample) isolated from the subject:

[0068] - Gemini coccus morbilli (GMLL),

[0069] - Peptostreptococcus gastricis (PTST), and

[0070] - Bacteroides fragilis (BCTF).

[0071] In a preferred embodiment, the FOBT in a) and the quantification of bacterial markers in b) are performed in a stool sample, preferably in the same stool sample.

[0072] The reference strain for GMLL is Gemini cocci strain NCTC11323 (Bacteria; Firmicutes; Bacilli; Bacillales; Family XI. Incertae Sedis; Genus Gemella), whose genome sequence is GenBank Accession No. LS483440.1. In certain embodiments, oligonucleotides specific for SEQ ID NO: 1 can be used to quantify GMLL. SEQ ID NO: 1 corresponds to GenBank Accession No. LS483440.1 REGION: 827110..827892.

[0073]

[0074] The reference strain for PTST is Peptostreptococcus gastricus DSM 17678 (Bacteria; Firmicutes; Clostridia; Clostridiales; Peptostreptococcaceae; Peptostreptococcus), whose genome sequence has GenBank accession number ADGQ01000060.1. In certain embodiments, oligonucleotides specific for SEQ ID NO: 2 can be used to quantify PTST. SEQ ID NO: 2 corresponds to GenBank accession number ADGQ01000060.1. REGION: Complement (142796..143362).

[0075]

[0076] The reference strain for BCTF is Bacteroides fragilis strain NCTC 9343 (Bacteria; Bacteroidetes; Bacteroidia; Bacteroidales; Bacteroidaceae; Bacteroides), whose genome sequence has GenBank accession number CR626927.1. In certain embodiments, oligonucleotides specific for SEQ ID NO: 1 can be used to quantify BCTF. SEQ ID NO: 3 corresponds to GenBank accession number CR626927.1 REGION: 417101..417817.

[0077]

[0078] In a preferred embodiment, these specific oligonucleotides are any of those described in Table 3 for GMLL, PTST and BCTF, respectively, as well as sequences having at least 80% identity to any of these oligonucleotides.

[0079] As shown in Example 3, PTST was found to be highly correlated with neoplastic lesions (non-advanced adenoma + advanced adenoma + CRC; p < 0.001). In addition, 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). In addition, GMLL and PTST were found to be significantly more abundant in CRC than in healthy subjects (p = 0.004 and p < 0.001, respectively).

[0080] As used herein, the term "colorectal cancer," "CRC," refers to cancer that begins in the colon or rectum. Depending on where they originate, these cancers may also be called colon cancer or rectal cancer, respectively. Colon cancer and rectal cancer have many characteristics in common. 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 called adenocarcinoma. These cancers begin in cells that form glands that produce mucus to lubricate the inside of the colon and rectum. Other less common types of tumors may also begin in the colon and rectum. These tumors include: carcinoid tumors, gastrointestinal stromal tumors (GIST), lymphomas, and sarcomas. In a preferred embodiment, the colorectal cancer is an adenocarcinoma.

[0081] Adenomas are noncancerous growths of abnormal glandular cells on the lining of an organ, such as the colon. Adenomas that meet one or more of the following characteristics are generally classified as advanced adenomas: measuring 10 mm or larger in diameter, having a villous architecture, high-grade dysplasia, or intramucosal carcinoma (Quintero E., Castells A., et al., N Engl J Med. 2012, 366(8):697-706; Cubiella J. et al., Cancer Epidemiol Biomarkers Prev. 2014, 23(9):1884-92; Muto T, Bussey HJR MB., Cancer 1975; 36:2251–70).

[0082] As used herein, the term colorectal advanced neoplasia (AN) includes CRC and advanced adenoma (AA). According to the ACS, the test that is most likely to find both AA and CRC is preferred. Cohort studies involving patients with adenoma have shown that polypectomy can prevent approximately 80% of colorectal cancers (Citarda F, et al., Gut 2001, 48: 812-5; Winawer SJ, et al., N Engl J Med 1993, 329: 1977-81).

[0083] As used herein, the term "diagnosis" refers to both the process of attempting to determine and / or identify a possible disease in a subject, i.e., the diagnostic process, and the opinion reached by that procedure, i.e., the diagnostic opinion. It can therefore also be viewed as an attempt to classify an individual's condition into separate and distinct categories in order to make medical decisions about treatment and prognosis. It will be understood that in a preferred embodiment, the method is an in vitro method, i.e., not performed on a human or animal body. In particular, determining the diagnosis of a patient with CRC, AA, and / or AN may be related to the ability to identify and classify patients with CRC, AA, and / or AN.

[0084] In a specific embodiment, the diagnostic methods of the present invention are performed in a subject suspected of having CRC. As used herein, the term "subject suspected of having CRC" refers to a subject that exhibits one or more signs or symptoms that may be indicative of CRC. Subjects suspected of having CRC also include individuals who have received a preliminary diagnosis but have not yet undergone confirmatory testing (e.g., colonoscopy).

[0085] Signs or symptoms that may indicate CRC include, for example, one or more of the following: unexplained weight loss, abdominal pain, unexplained rectal bleeding, iron deficiency anemia, changes in bowel habits, and the 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 following are those situations in which CRC is suspected in the current NICE guidelines (NG12); https: / / www.nice.org.uk / guidance / ng12 / chapter / 1-Recommendations-organised-by-site-of-cancer#lower-gastrointestinal-tract-cancers:

[0086] -Cancer pathway referral for suspected colorectal cancer is recommended for adults (eg, appointment within two weeks) in the following circumstances:

[0087] They are 40 years of age or older and have unexplained weight loss and abdominal pain, or

[0088] They are 50 years of age or older and have unexplained rectal bleeding or

[0089] They are 50 years of age and older and have:

[0090] oIron deficiency anemia or

[0091] oChanges in bowel habits, or

[0092] Tests showed occult blood in their stool.

[0093] -Consider suspected cancer pathway referral for colorectal cancer (eg, appointment within 2 weeks) for adults with a rectal or abdominal mass.

[0094] - Consider colorectal surgery for adults younger than 50 years with rectal bleeding and any of the following unexplained symptoms or findings:

[0095] Referral for suspected cancer pathway (e.g., appointment within 2 weeks):

[0096] ·stomach ache

[0097] Changes in bowel habits

[0098] Weight loss

[0099] Iron deficiency anemia.

[0100] - Offer fecal occult blood testing to evaluate adults without rectal bleeding for colorectal cancer:

[0101] Those aged 50 years and above with unexplained illness:

[0102] o Abdominal pain or

[0103] oWeight loss, or

[0104] Aged 60 or below and have:

[0105] o Changes in bowel habits or

[0106] oIron deficiency anemia, or

[0107] Aged 60 years and over and have anemia, even if they are not iron deficient.

[0108] The method of the present invention can also be used for differential diagnosis between CRC, advanced adenoma and / or advanced tumors and non-advanced adenoma.

[0109] The most effective and economical measure to reduce CRC morbidity and mortality is CRC risk screening and monitoring tests. Screening tests are divided into: tests that primarily detect early-stage cancers; and tests that can detect both early-stage cancers and advanced adenomas, thereby providing greater preventive potential through polypectomy (i.e., polyp removal), as described in the American Cancer Society (ACS) 2008 Screening and Monitoring Guidelines (CA Cancer J Clinicians, 2008; 58(3)130-160) by Levin et al. Different screening guidelines apply to people at average risk and those at increased or high risk (http: / / www.cancer.org / cancer / colonandrectumcancer / moreinformation / colonandrectumcancerearlydetection / col orectal-cancer-early-detection-acs-recommendations).

[0110] The term "screening" is understood herein to mean the examination or testing of a group of asymptomatic individuals belonging to the general population, or of a group of individuals with one or more risk factors (e.g., subjects at moderate or high risk of a disease), with the purpose of distinguishing healthy individuals from individuals who have or are suspected of having a disease. Screening methods are often used for the "early detection" of a disease. The expression "early detection" refers to detection before clinical signs appear.

[0111] The goal of cancer screening is to reduce mortality through early detection and treatment, thereby reducing the incidence of advanced disease, which typically has a poor prognosis. To this end, modern CRC screening can achieve this goal through the detection of adenocarcinomas (preferably early-stage adenocarcinomas) and the detection and removal of advanced adenomas. The screening method of the present invention has therefore been found to be an effective tool for reducing CRC-related mortality by detecting CRC and / or advanced tumors.

[0112] The screening method of the present invention can be performed in individuals who do not present signs and / or symptoms of CRC (referred to herein as "asymptomatic individuals"). It can also be performed in subjects with or without a personal or family history of colon tumors or other CRC risk factors as described below.

[0113] In a specific embodiment, the screening method of the present invention is performed in subjects at moderate risk for CRC. These are typically subjects 50 years of age and older who do not have a personal or family history of CRC. Women and men aged 50 years and older at average risk are encouraged to undergo colorectal cancer screening tests (see Table 2 of Levin et al., CA Cancer J Clinicians, 2008; 58(3): 130-160, which is incorporated herein by reference).

[0114] In another embodiment, the screening method of the present invention is performed in subjects at increased risk and / or high risk. According to the ACS (see Table 3 of Levin et al., CA Cancer J Clinicians, 2008; 58(3): 130-160, which is incorporated herein by reference), subjects at increased risk and high risk may include the following:

[0115] Increased risk

[0116] - Subjects with a history of polyps on previous colonoscopy;

[0117] - Subjects with colorectal cancer; and

[0118] - Subjects with a family history of colorectal cancer or adenomatous polyps, including:

[0119] i. A first-degree relative under the age of 60 or two or more first-degree relatives of any age with CRC or adenomatous polyps;

[0120] and / or

[0121] ii. A first-degree relative aged ≥60 years or two second-degree relatives with CRC or adenomatous polyps.

[0122] High risk

[0123] - Patients diagnosed with familial adenomatous polyposis (FAP) through genetic testing or suspected of having FAP without genetic testing;

[0124] - Subjects with hereditary non-polyposis colon cancer (HNPCC or Lynch syndrome) or an increased risk of HNPCC based on family history who have not undergone genetic testing; and

[0125] - Subjects suffering from inflammatory bowel disease, such as chronic ulcerative colitis or Crohn's disease.

[0126] Another object of the present invention is to provide a pre-diagnostic tool. Specifically, the method of the present invention can be used to screen and / or identify subjects with susceptibility to CRC or increased risk of CRC, for example by detecting the presence of advanced adenomas.

[0127] As used herein, the term "monitoring" refers to determining the evolution of a disease and / or the efficacy of surgical and / or therapeutic treatment, e.g., determining whether the disease is in remission; or conversely, whether the disease is progressing or recurring. One of the goals of the monitoring methods of the present invention is to detect recurrence early. In a specific embodiment, the methods of the present invention are used to monitor a subject with cancer who has undergone surgical resection, optionally in combination with adjuvant and / or neoadjuvant therapy.

[0128] In a preferred embodiment, the methods of the present invention are used to screen, diagnose and / or monitor AN, AA and / or CRC.

[0129] As used herein, the term "sample" or "biological sample" refers to a biological material isolated from a subject. A biological sample can comprise any biological material suitable for detecting a desired biomarker and can comprise cells and / or non-cellular material from the subject. In a preferred embodiment, the sample is an intestinal sample. These intestinal samples can be, for example, biopsies of mucosal tissue from the colon and / or rectum. Preferably, the intestinal sample is a fecal sample. Fecal samples are routinely used in clinical practice, and those skilled in the art will know how to determine the most appropriate method for obtaining and preserving them. Once a sample is obtained, it can be used fresh, frozen, or stored using appropriate means.

[0130] The method of the present invention optionally comprises, in step a), performing a FOBT on a stool sample isolated from a subject, wherein the FOBT comprises determining the presence of occult blood in a stool sample isolated from the subject. Blood in the stool is a non-specific finding that may arise from CRC or larger (>1 to 2 cm) polyps. Because small adenomatous polyps do not bleed, and bleeding from cancerous or large polyps may be intermittent or not always detectable in a single stool sample, proper use of fecal blood testing typically requires annual testing, involving the collection of samples from consecutive bowel movements (2 or 3, depending on the product). Fecal blood tests are often referred to as fecal occult blood tests (FOBTs) because they are intended to detect the presence of occult blood in the stool. FOBTs are divided into two main categories based on the analyte detected: gFOBTs and FITs. Guaiac-based tests detect blood in the stool via the pseudoperoxidase activity of heme or hemoglobin, whereas immunochemical-based tests react to human globin (Levin et al. CA Cancer J Clinicians, 2008; 58(3): 130-160).

[0131] The usual gFOBT protocol involves collecting two samples from each of three consecutive bowel movements. Because the gFOBT is a qualitative test, it is usually read visually by a trained laboratory technician using the naked eye to interpret the visual results. Prior to testing with a sensitive guaiac-based test, individuals are typically instructed to avoid aspirin and other nonsteroidal anti-inflammatory drugs, vitamin C, red meat, poultry, fish, and some raw vegetables because diet-test interactions may increase the risk of false-positive and false-negative (particularly for vitamin C) results. An example of a commercially available gFOBT is the Hemoccult SENSA (Beckman Coulter, USA).

[0132] The FIT detects human globin, a protein that, along with heme, makes up human hemoglobin. Therefore, compared to guaiac-based tests that rely on detecting peroxidase in human blood, the FIT is more specific for human blood and also reacts with peroxidases present in dietary ingredients such as rare red meats, cruciferous vegetables, and some fruits. Furthermore, unlike the gFOBT, the FIT does not produce false-negative results in the presence of high-dose vitamin C supplements (which block the peroxidase reaction). Furthermore, because globin is degraded by digestive enzymes in the upper gastrointestinal tract, the FIT is also more specific for lower gastrointestinal bleeding, thereby increasing their specificity for CRC. Finally, some variations of the FIT have less demanding sample collection requirements for the patient than the gFOBT, requiring smaller samples or less direct stool processing. There are a variety of FITs based on different detection techniques (e.g., reversed passive hemagglutination (RPHA), latex agglutination, ELISA, and immunoturbidimetry) and are included herein (Levin B et al., Gastroenterology 2008; 134: 1570-95). Preferably, the immunological test is based on latex agglutination, such as the OC-SENSOR assay (Eiken Chemical Co., Japan) used in the Examples.

[0133] Preferably, the FOBT is a fecal immunochemical test (FIT) that quantifies human hemoglobin (hHb). A subject is determined to be FIT-positive when the hHb level in a stool sample is above a predetermined threshold. For example, a subject may be determined to be FIT-positive when the hHb level is equal to or greater than 10 μg hHb / g stool (or equal to or greater than 50 ng hemoglobin / mL; FIT50 cutoff) or when the hHb level is equal to or greater than 20 μg hHb / g stool (or equal to or greater than 100 ng hemoglobin / mL; FIT100 cutoff). The FIT100 cutoff is a commonly used cutoff for FIT in CRC screening in Catalonia and other parts of Spain. As shown in Table 5, the method of the present invention achieves improved specificity when two thresholds are used, compared to using only the corresponding FIT test. In a preferred embodiment, a FIT-positive subject has an hHb level equal to or greater than 10 μg hHb / g stool (FIT50 cutoff), for which slightly higher sensitivity is achieved.

[0134] As described above, the method of the present invention may include: 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 include quantifying Bacteroides thetaiotaomicron (BCTT) in the intestinal sample (preferably, a fecal sample). The reference strain of BCTT is Bacteroides thetaiotaomicron VPI-5482 strain (bacteria; Bacteroidetes; Bacteroidetes; Bacteroidales; Bacteroidaceae; Bacteroides genus), and the GenBank accession number of its genome sequence is: AE015928.1. In a specific embodiment, an oligonucleotide specific for SEQ ID NO: 4 can be used for the quantification of BCTT. SEQ ID NO: 4 corresponds to GenBank accession number: AE015928.1REGION: 326008..326649. In a preferred embodiment, these specific oligonucleotides are any of those of the BCTT described in Table 3, as well as sequences having at least 80% identity with any of the oligonucleotides therein.

[0135]

[0136]

[0137] Preferably, step b) comprises or consists of quantifying PTST, BCTF and BCTT.

[0138] The molecular biology method for determining the amount of target nucleic acid sequence is well known in the art. These methods include, but are not limited to, endpoint PCR, competitive PCR, quantitative PCR (qPCR), PCR-pyrophosphate sequencing, PCR-ELISA, DNA microarray, nucleic acid sequencing (e.g., next generation sequencing method), in situ hybridization assay (e.g., dot immunoblotting (dot-blot) or fluorescence in situ hybridization assay (Fluorescence In Situ Hybridization assay, FISH), mass spectrometry, branched DNA (Nolte, Adv. Clin. Chem. 1998, 33: 201-235) and multiplex forms of the method (see, e.g., Andoh et al., Current Pharmaceutical Design, 2009; 15, 2066-2073) and the next generation of any listed technology and combinations thereof, all of which are within the scope of the present invention.

[0139] A variety of next generation sequencing methods have been described and are well known to those skilled in the art. These next generation sequencing methods include, for example, cyclic reversible termination (e.g., Illumina, SEQLL, Qiagen) synthesis sequencing, single nucleotide addition method synthesis sequencing (e.g., Roche-454, Thermo Fisher-Ion Torrent), connection sequencing (e.g., ThermoFisher SOLiD and BGI-Complete Genomics), real-time long read sequencing (e.g., Pacific Biosciences, Oxford Nanopore Technologies), synthetic long read sequencing (e.g., Illumina, 10X Genomics, iGenomeX), for example, referring to Goodwin S, et al., Nat Rev Genet.2016, 17 (6): 333-51.

[0140] In some embodiments, the molecular biology quantitative method is based on sequence-specific amplification. This amplification-based assay includes an amplification step comprising contacting a sample (preferably an isolated DNA sample) with two or more amplification oligonucleotides specific for the target sequence in the target nucleic acid to produce an amplified product (if the target nucleic acid sequence is present in the sample). The amplified product will contain 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.

[0141] A particularly preferred quantitative method is quantitative PCR (qPCR), also known as real-time PCR. It involves a PCR that amplifies and simultaneously quantifies target DNA molecules. Its main feature is the real-time detection of amplified DNA as the reaction proceeds. Different instruments can be used, for example, Applied Biosystems' ABI Prism 7700 SDS, GeneAmp 5700 SDS, ABI Prism 7900HT SDS; Bio-Rad's iCycler iQ; Cepheid's Smart Cycler; Corbett Research's Rotor-Gene; Roche Molecular Biochemicals' LightCycler, Agilent's AriaMx, and Stratagene's Mx4000 Multiplex. The qPCR process enables accurate real-time quantification of PCR products by measuring the accumulation of PCR products very early in the exponential phase of the reaction, thereby reducing the quantitative bias associated with PCR amplification efficiency that occurs in endpoint PCR. Real-time PCR is well known in the art and will not be described in detail herein. A technical overview and protocol for qPCR can be obtained from the aforementioned suppliers, for example, at 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 a review of qPCR methods, see Wong ML and Medrano JF, Biotechniques 2005, 39(1):75-85. In a specific embodiment, the quantitative method is multiplex qPCR.

[0142] Different detection chemistries are available for qPCR. All of them can be used with the qPCR instruments mentioned above. The term "detection chemistry" refers to the method that reports the amplification of a specific PCR product in real-time PCR. These detection chemistries can be divided into two 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 based on the type of fluorescent molecule used in the PCR reaction: (i) primer-probes (Scorpions, LUX TM、Cyclicons、 ); (ii) probes; hydrolysis (TaqMan, MGB-TaqMan, Snake assays) and hybridization (Hybprobe or FRET, Molecular Beacons, HyBeacon TM ,MGB-Pleiades,MGB-Eclipse, Yin-Yang or substitution); and (iii) nucleic acid analogs (PNA, ZNA TM , Unnatural bases: Plexor TM Primer, Tiny-Molecular Beacon), see E. Navarro et al., Acta Clinical Chemica Sinica, Vol. 439, Jan. 15, 2015, pp. 231-250.

[0143] The probe can be a dual-labeled oligonucleotide, such as a hydrolysis probe or a molecular beacon. The 5' end of the oligonucleotide is usually 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 in the amplified target molecule. In a more preferred embodiment, the probe is a hydrolysis probe, which is designed so that the length of the sequence places the 5' fluorophore and the 3' quencher in a position close enough to suppress fluorescence. Several reporter molecules and quenchers used for qPCR probes are well known in the art.

[0144] Typically, in order to quantify a nucleotide sequence, specific oligonucleotides, such as probes and / or primers, are used. The term "primer and / or probe" specifically includes "one or more primers and / or one or more probes". The two expressions are used interchangeably herein and include, for example, a primer; a probe; a primer and a probe; a pair of primers; and a pair of primers and a probe. The design and verification of primers and probes are well 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 for the method of the present invention are described below under the kit of the present invention.

[0145] Preferably, the oligonucleotides used in the methods of the present invention are about 5 to about 50 nucleotides in length, about 10 to about 30 nucleotides in length, or about 20 to about 25 nucleotides in length. In certain embodiments, the oligonucleotides that specifically hybridize to the target sequence are about 19 to about 21 nucleotides in length. In a specific embodiment, the oligonucleotides have been modified for detection purposes or to enhance assay performance as described herein.

[0146] These oligonucleotides can be ribonucleotides or deoxyribonucleotides. In a specific embodiment, the oligonucleotide can have at least one chemical modification. For example, suitable oligonucleotides can be composed of one or more "conformation-restricted" or bicyclic sugar nucleoside modifications, such as "locked nucleic acids." "Locked nucleic acid" (Locked nucleic acid, LNA) is a modified ribonucleotide that includes an extra bridge between the 2' carbon and 4' carbon of the ribose moiety, resulting in a "lock" conformation, which imparts enhanced thermal stability to the oligonucleotide containing LNA. In other embodiments, the oligonucleotide can include a peptide nucleic acid (PNA), which includes a peptide-based backbone rather than a sugar-phosphate backbone. Other chemical modifications that the oligonucleotide can 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; and backbone modifications, such as one or more phosphorothioate, morpholino or phosphonocarboxylate bonds. 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, LNAs, bicyclic nucleosides, phosphonoformates, 2'O-alkyl groups, etc. In some embodiments, oligonucleotides may be chemically modified, for example, to increase their resistance to nuclease degradation (e.g., by end-capping), to carry detection ligands (e.g., fluorescein), or to facilitate their capture on a solid support (e.g., a polydeoxyadenosine "tail").

[0147] The term "quantification level" can be concentration (amount of DNA per unit volume), amount of DNA per unit 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 quantitative level is expressed as a Ct value. The Ct (cycle threshold) value is defined as the number of qPCR cycles required for the fluorescence signal to exceed the threshold value. The Ct level is inversely proportional to the amount of target nucleic acid in the sample (i.e., the lower the Ct level, the greater the amount of target nucleic acid in the sample).

[0148] Quantification of the abundance of a target nucleic acid sequence (e.g., SEQ ID NO: 1) in a stool sample can be absolute or relative. Relative quantification is based on one or more internal reference genes, such as the 16S rRNA gene from a reference strain, e.g., total bacteria (eubacteria) measured using universal primers and expresses the abundance of the target nucleic acid sequence relative to eubacteria (e.g., SEQ ID NO: 1 / eubacteria ratio). Absolute quantification gives the exact amount of the target molecule by comparison to a DNA standard.

[0149] In a specific embodiment, optionally in combination with one or more embodiments or features as described herein, the quantification of the bacterial marker in step b) is relative to that of true bacteria and is expressed as a ratio of the corresponding quantitative values ​​(e.g., a ratio of Ct values). Specific oligonucleotides that can be used to quantify true bacteria are any of those listed in Table 3 for EUB, and sequences that have at least 80% identity to any of these oligonucleotides. Alternatively, any of the following oligonucleotides, or sequences that have at least 80% identity to any of these oligonucleotides, may also be used:

[0150] EUB2 forward (SEQ ID NO: 5): ACTCCTACGGGAGGCAGCAGT

[0151] EUB2 reverse (SEQ ID NO: 6): GTATTACCGCGGCTGCTGGCAC

[0152] In a specific embodiment of the method of the present invention, DNA is extracted from the stool sample before quantifying the bacterial sequences. Several methods for extracting DNA from stool samples are known, all of which rely on chemical or mechanical disruption, lysis using detergents, or a combination of these methods (Kennedy A. et al., PLoS One, 2014; 9(2): e88982). Methods for extracting bacterial DNA from stool samples are known, for example, from 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 WO2003 / 068788.

[0153] A preferred method uses a combination of mechanical disruption, such as high-speed bead beating extraction, chemical lysis and a final purification step, preferably using a silica membrane column, such as that contained in the commercially available DNA extraction kit "MobioPower DNA extraction procedure" (Mo-Bio Laboratories Inc.,), for soil procedures SPIN Kit (MP biomedicals) and Soil (Macherey-Nagel Gmbh & Co. KG). The presence of PCR inhibitors in DNA extracted from stool samples, such as bilirubin, bile salts, and complex carbohydrates, is one of the difficulties faced in determining DNA biomarkers in DNA extracted from stool (Fleckna et al., Mol Cell Probes, 2007; 21(4): 282-7). Preferred DNA extraction methods are those that provide stool extracts with low levels of PCR inhibitors, such as less than 5%, preferably less than 2%, more preferably less than 1%, even more preferably less than 0.5%, such as less than 0.25%, 0.1%, 0.05%, or 0.01%.

[0154] In a specific embodiment, the method of the present invention further comprises:

[0155] c) calculating a combined score from the levels of the bacterial markers determined in b); and

[0156] d) categorizing FOBT-positive subjects as having or presenting an increased risk of having AN, AA, and / or CRC based on the combined score obtained in c).

[0157] The combined score of step (c) is a value obtained according to a given mathematical algorithm, for example, wherein the quantitative value of each bacterial marker determined in step b) is a variable of the mathematical algorithm. Preferably, the bacterial markers are PTST, BCTF and BCTT.

[0158] In certain embodiments, when a combined score is calculated, it is proportional to the absolute or relative amount of any of GMLL, PTST, or BCTF in the sample; and inversely proportional to the absolute or relative amount of BCTT; wherein the higher the score, the higher the likelihood of having CRC, AA, and / or AN. In other words, a high score indicates the presence of disease.

[0159] For example, the combined score can be calculated as the sum of the products of the standardized beta coefficients obtained in the regression analysis of each marker, wherein the bacterial marker value is used as a variable. In a specific embodiment, the beta coefficients of the markers GMLL, PTST and / or BCTF are positive; the beta coefficient of BCTT is negative. A positive sign indicates that an increase in the amount of an individual marker is associated with a higher likelihood of having CRC, AA and / or AN or having CRC, AA and / or AN, and a negative sign indicates that a decrease in the amount of an individual marker is associated with a higher likelihood of having CRC, AA and / or AN or having CRC, AA and / or AN.

[0160] Typically, in step d), the method comprises comparing the combined score in the subject sample to a reference value (e.g., a reference combined score); and wherein an increase in the combined score in the subject sample relative to the reference value (e.g., the reference combined score) is indicative of AN, AA, and / or CRC.

[0161] As used herein, the term "reference combination score" is a reference value obtained according to a given mathematical algorithm, wherein the reference expression value of each bacterial marker used in the method of the present invention is a variable of the mathematical algorithm.

[0162] As used herein, the term "reference value" refers to a predetermined standard used as a reference for evaluating values ​​or data obtained from a sample collected from a subject. This "reference value" may also be referred to as a "cutoff value" or "threshold value."

[0163] A reference value or reference level can be an absolute value, a relative value, a value with an upper or lower limit, a range of values, an average value, a median value, a mean, a z-score value (e.g., mean + 1SD or -1SD), a tertile value, or a value compared to a specific control value or a baseline value. In a specific embodiment, optionally in combination with one or more embodiments or features described above or below, the reference value is a mean or tertile value.

[0164] In addition, it should be noted that a variety of statistical and mathematical methods for establishing threshold or cut-off levels of expression are known in the art. A threshold or cut-off expression level for a particular biomarker can be selected, for example, based on data from a receiver operating characteristic (ROC) plot. One skilled in the art will appreciate that these thresholds or cut-off expression levels can be altered, for example, by moving along the ROC plot for a particular biomarker or combination thereof to obtain different sensitivity or specificity values, thereby affecting overall assay performance.

[0165] The reference value can be based on a single sample value, but is typically based on a large number of samples, including or excluding the sample to be tested. For example, the reference value can be derived from one of the screening populations defined above (e.g., asymptomatic adults in the general population, asymptomatic adults over 50 years of age, or asymptomatic adults with one or more risk factors) or from a population of individuals with CRC-related symptoms. In addition, the reference value can be derived from a collection of tissue samples from a reference AN, AA, and / or CRC patient population for which historical information related to the actual clinical outcomes of the corresponding cancer patients is available.

[0166] Alternatively, the reference values ​​according to the method of the present invention may be obtained from one or more subjects who do not have CRC or advanced adenomas, do not have gastrointestinal diseases (i.e. healthy control subjects), subjects who have advanced adenomas, subjects who have early CRC, e.g. asymptomatic (preclinical stage), or the same subject diagnosed as having CRC and / or advanced adenomas but at an earlier time point.

[0167] In a specific embodiment, the method of the present invention is a method for screening or diagnosing AN, AA and / or CRC, and the reference value is obtained from a subject who does not have AN, AA and / or CRC or a subject who does not have a gastrointestinal disease. In another embodiment, the method of the present invention is a screening method, and the reference value is obtained from a screening population as defined herein. In another embodiment, the method of the present invention is a diagnostic method, and the reference value is obtained from an individual with symptoms associated with CRC.

[0168] In another specific embodiment, the method of the present invention is a method for screening or diagnosing AN, AA and / or CRC, and the reference value is obtained from a subject with non-advanced adenoma. For example, this may be the case when a differential diagnosis between AN, AA and / or CRC relative to non-advanced adenoma (NAA) is required.

[0169] In another specific embodiment, the method of the present invention is a method of monitoring AN, AA and / or CRC and the reference value is obtained from the same subject diagnosed as having AN, AA and / or CRC but at an earlier time point.

[0170] In other embodiments, the method of the present invention may include steps a) and b) as described above and further include:

[0171] e) comparing the amount of any of GMLL, PTST, BCTF, and / or BCTT in the stool sample with the corresponding reference values;

[0172] f) wherein an increased amount of GMLL, PTST and / or BCTF and a decreased 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] Preferably, an increased amount of PTST and BCTF and a decreased amount of BCTT in a sample from a FOBT-positive subject relative to corresponding reference values ​​is indicative of AN, AA and / or CRC.

[0174] In the methods of the present invention, a combined score (or amount of bacterial marker) is considered to be "reduced" when the combined score (or amount of bacterial marker) is lower than a reference combined score (or reference value). Preferably, the combined score is considered to be lower than a reference combined score (or reference value) when the combined score 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 a reference combined score (or reference value).

[0175] Likewise, in the context of the methods of the present invention, a combined score (or amount of bacterial marker) is considered to be "increased" when the combined score is higher than a reference combined score (or reference value). Preferably, a combined score (or amount of bacterial marker) is considered to be higher than a reference combined score (or reference value) when the combined score (or amount of bacterial marker) 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 a reference combined score (or reference value).

[0176] Alternatively or additionally, a subject having a level deviation (i.e., an increase or decrease) of greater than about 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20-fold relative to a reference combined score (or reference value) described herein is described herein.

[0177] As will be appreciated by those skilled in the art, the methods of the present invention do not claim to be correct in 100% of the samples analyzed. However, they do claim to correctly classify a statistically significant number of the analyzed samples. Statistically significant amounts can be determined by those skilled in the art using various measures of statistical significance obtained through statistical tests; illustrative, non-limiting examples of such measures include determining confidence intervals, determining p-values, and the like. Preferred confidence intervals are 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. The teachings of the present invention preferably allow for correct classification of at least 60%, at least 70%, at least 80%, or at least 90% of the subjects in the assay group or population analyzed.

[0178] Validity studies address the agreement between the proposed (index) test and the reference standard regarding the ability to identify the target condition (see Florkowski MC, Clin Biochem Rev. 2008, 29 (Suppl 1): S83–S87). Sensitivity, specificity, accuracy, positive likelihood ratio, negative likelihood ratio, positive predictive value, and negative predictive value are statistical values ​​that can be defined to evaluate test performance. Definitions of acronyms and more detailed information are provided in Table 1 below.

[0179]

[0180] Depending on the intended use of the test in clinical practice, tests are typically calibrated according to the desired specificity and sensitivity. High sensitivity corresponds to a high negative predictive value and is generally considered a desirable property for a "rule out" test (e.g., a screening test that is typically followed by a confirmatory test). High specificity corresponds to a high positive predictive value and is generally considered a desirable property for a "rule in" test (e.g., a companion diagnostic test).

[0181] In a preferred embodiment, the methods of the invention have a sensitivity, specificity and / or accuracy value of at least about 60%, preferably at least about 70%, and can be, for example, 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 analyzed, or preferably in at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the groups or populations analyzed.

[0182] It is further noted that the accuracy of the method of the present invention can be further improved by additionally considering other genetic or bacterial markers, biochemical parameters and / or clinical characteristics (e.g., age, sex, smoking and / or other risk factors) of the patient. The determination of these other markers, parameters and / or characteristics can be performed sequentially or simultaneously with any or all steps of the method of the present invention as described herein.

[0183] In a specific embodiment, optionally in combination with one or more features or embodiments described herein, the method of the present invention further comprises quantifying one or more bacterial markers selected from the group consisting of: 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 strain in its phylogroup (see WO 2017 / 025617A1 and Lopez-Siles et al. Appl Environ Microbiol. 2012, 78: 420-428), and Escherichia coli.

[0184] B10 has the sequence SEQ ID NO: 7 (shown below), which corresponds to the partial sequence of the 16S ribosomal RNA gene of an uncultured bacterial isolate DGGE gel band Eub_10 (GenBank: GQ411118.1). In certain embodiments, oligonucleotides specific for SEQ ID NO: 7 can be used to quantify B10.

[0185]

[0186] B46 has the sequence SEQ ID NO: 8 (shown below), which corresponds to the partial sequence of the 16S ribosomal RNA gene of an uncultured bacterial isolate DGGE gel band Eub_46 (GenBank: GQ411150.1). In certain embodiments, oligonucleotides specific for SEQ ID NO: 8 can be used to quantify B46.

[0187]

[0188] B48 has the sequence SEQ ID NO: 9 (shown below), which corresponds to the partial sequence of the 16S ribosomal RNA gene of an uncultured bacterial isolate DGGE gel band Eub_48 (GenBank: GQ411152.1). In certain embodiments, oligonucleotides specific for SEQ ID NO: 9 can be used to quantify B48.

[0189]

[0190]

[0191] The reference strain for RSBI is Roseburia enterica L1-82 (Bacteria; Firmicutes; Clostridiales; Lachnospiraceae; Genus Roseburia), whose genome sequence is set forth in GenBank Accession No. LR027880.1. In certain embodiments, an oligonucleotide specific for SEQ ID NO: 10 can be used to quantify RSBI. SEQ ID NO: 10 corresponds to GenBank Accession No. LR027880.1 REGION: 599208..600101.

[0192]

[0193] The reference strain for CINT (Bacteria; Actinobacteria; Coriobacteriia; Coriobacteriales; Coriobacteriaceae; Collinsella) is Collinsella enterica DSM 13280, whose genomic sequence is NCBI Reference Sequence GG692711.1. In certain embodiments, an oligonucleotide specific for SEQ ID NO: 11 can be used to quantify CINT. SEQ ID NO: 11 corresponds to NCBI Reference Sequence: GG692711.1 REGION: 297314..298120.

[0194]

[0195] A reference strain of Faecalibacterium prausnitzii (Bacteria; Firmicutes; Clostridiales; Ruminococcaceae; Genus Faecalibacterium) is Faecalibacterium prausnitzii A2-165 (GenBank Accession No. AJ270469.2), which is also a representative strain of its phylogroup II (PHGII). Faecalibacterium prausnitzii M21 / 2 (GenBank Accession No. DS483503.1) is a representative strain of its phylogroup I (PHG1). In certain embodiments, an oligonucleotide specific for SEQ ID NO: 12 (shown below), which corresponds to GenBank Accession No. AJ270469.2, can be used to quantify Faecalibacterium prausnitzii.

[0196]

[0197]

[0198] A reference strain of Escherichia coli (Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacterales; Enterobacteriaceae; Escherichia) is E. coli strain K-12 substrain MG1655, and its genome sequence has GenBank accession number CP032667.1. In certain embodiments, oligonucleotides specific for SEQ ID NO: 13 (shown below) can be used for quantification of E. coli.

[0199]

[0200]

[0201] In a preferred embodiment, any of the specific oligonucleotides described in Table 3, and sequences having at least 80% identity thereto, can be used for the quantification of B10, B46, B48, CINT, or RSBI, respectively. In addition, in a preferred embodiment, any of the specific oligonucleotides described in Table 2 of WO2017 / 025617A1, and sequences having at least 80% identity thereto, can be used for the quantification of Faecalibacterium prausnitzii, its phylogroups (i.e., PHGI and PHGII), and Escherichia coli.

[0202] The method of the present invention or any step thereof can be implemented by a computer. As used herein, the term computer can 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 determination of step d) is performed using a computer.

[0203] Therefore, another aspect of the present invention relates to a computer-implemented method, wherein the method is any method disclosed herein or any combination thereof.

[0204] It is noted that any method capable of implementing the present invention or any computer program for implementing any one of these methods or any combination thereof also forms part of the present invention.

[0205] The computer program is typically loadable directly into the internal memory of a digital computer and comprises software code portions for performing the steps of comparing a combined score (e.g., obtained from the levels of one or more target markers as described herein) from one or more biological samples from a subject with a reference value (e.g., a reference combined value) when the product is run on the computer and determining a diagnosis for the subject.

[0206] It should also be noted that any device or apparatus comprising a device for performing the steps of any method or any combination thereof of the present invention, or carrying a computer program capable of implementing or for implementing any method or any combination thereof of the present invention, is included as part of this description.

[0207] The method of the present invention may further comprise storing the method result in a data carrier, preferably wherein the data carrier is a computer-readable medium. The present invention also relates to a computer-readable storage medium having stored thereon the computer program of the present invention or the result of any method of the present invention.

[0208] As used herein, "computer-readable medium" can be any device that can contain, store, communicate, propagate, or transmit the results determined in the methods of the present invention. The medium can be an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system (or apparatus or device) or a propagation medium.

[0209] Treatment methods and related medical uses

[0210] In another aspect, the present invention provides a method of treating a subject having AN, AA and / or CRC, wherein the subject is selected by a method for screening, diagnosis and / or monitoring as described herein above, and wherein the method further comprises administering an anti-cancer therapy to the subject.

[0211] In a related aspect, the present invention also provides any of the methods described herein above, further comprising the step of administering to the patient a therapeutically effective amount of an anti-cancer therapy.

[0212] In another aspect, the present invention relates to an anti-cancer therapy for use in a method of treating a cancer patient, wherein the cancer patient is selected by the screening, diagnosis and / or monitoring methods described herein.

[0213] In another aspect, the present invention also relates to a method of selecting a subject for an exploratory test selected from the group consisting of a colonoscopy, a flexible sigmoidoscopy, a double contrast barium enema, and a computed tomography (CT) colonoscopy, preferably a colonoscopy, wherein the subject is selected by the screening, diagnostic, or monitoring method described herein.

[0214] In yet another aspect, the present invention relates to a method of performing exploratory testing in a subject, wherein the subject has been selected by a screening, diagnostic or monitoring method as described herein, wherein the method further comprises performing an exploratory test on the subject, wherein the test is selected from the group consisting of colonoscopy, flexible sigmoidoscopy, double contrast barium enema and computed tomography (CT) colonoscopy, preferably colonoscopy.

[0215] Kit and use of the kit in the method of the present invention

[0216] In another aspect, the present invention provides a kit suitable for quantifying any one of GMLL, PTST, BCTF, or BCTT, wherein the kit comprises one or more of the following reagents:

[0217] i. an oligonucleotide specific for the GMLL genome, preferably an oligonucleotide specific for SEQ ID NO: 1;

[0218] ii. an oligonucleotide specific for the PTST genome, preferably an oligonucleotide specific for SEQ ID NO: 2;

[0219] iii. an oligonucleotide specific for the BCTF genome, preferably an oligonucleotide specific for SEQ ID NO: 3;

[0220] iv. an oligonucleotide specific for the BCTT genome, preferably an oligonucleotide specific for SEQ ID NO: 4; and

[0221] v. Optionally, oligonucleotides suitable for quantification of eubacteria as defined above.

[0222] Optionally, instructions are also included for using the reagent to determine the level of the bacterial marker in a stool sample isolated from a subject.

[0223] The term "kit" or "test kit" denotes the combination of reagents and auxiliaries required for an assay. Although in most cases test kits consist of several units, one-piece analytical components can also be used and must likewise be considered test kits.

[0224] In a preferred embodiment, the kit is suitable for quantifying PTST, BCTF and BCTT and comprises reagents ii), iii) and iv) as defined above. Preferably, it further comprises reagent v) as defined above.

[0225] In certain embodiments, optionally in combination with one or more features or embodiments as described herein,

[0226] - the oligonucleotide specific for SEQ ID NO: 1 is an oligonucleotide comprising or consisting of any of the following sequences: SEQ ID NO: 22 or SEQ ID NO: 23, or a sequence having at least 80% identity to any of these sequences; and / or

[0227] - the oligonucleotide specific for SEQ ID NO: 2 is an oligonucleotide comprising or consisting of any of the following sequences: SEQ ID NO: 24 or SEQ ID NO: 25, or a sequence having at least 80% identity to any of these sequences; and / or

[0228] - the oligonucleotide specific for SEQ ID NO: 3 is an oligonucleotide comprising or consisting of any of the following sequences: SEQ ID NO: 26 or SEQ ID NO: 27, or a sequence having at least 80% identity to any of these sequences; and / or

[0229] - the oligonucleotide specific for SEQ ID NO: 4 is an oligonucleotide comprising or consisting of any of the following sequences: SEQ ID NO: 30 or SEQ ID NO: 31, or a sequence having at least 80% identity to any of these sequences; and / or

[0230] - Oligonucleotides suitable for quantifying eubacteria are oligonucleotides comprising or consisting of any of the following sequences: 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 these sequences.

[0231] A preferred embodiment relates to a kit suitable for quantifying PTST, BCTF and BCTT, wherein the kit comprises the following reagents:

[0232] - an oligonucleotide comprising or consisting of any of the following sequences: SEQ ID NO: 24 or SEQ ID NO: 25, or a sequence at least 80% identical to any of them;

[0233] - an oligonucleotide comprising or consisting of any of the following sequences: SEQ ID NO: 26 or SEQ ID NO: 27, or a sequence at least 80% identical to any of them; and

[0234] - an oligonucleotide comprising or consisting of any of the following sequences: SEQ ID NO: 30 or SEQ ID NO: 31, or a sequence at least 80% identical to any of them; and

[0235] - optionally, an oligonucleotide suitable for quantifying eubacteria (preferably an oligonucleotide comprising or consisting of any of the following sequences: 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 these sequences);

[0236] - Optionally, further comprising instructions for using the reagent to determine the level of the bacterial marker in a stool sample isolated from a subject.

[0237] The specific oligonucleotide is as described above. Preferably, the oligonucleotide is a primer and / or a probe.

[0238] Another preferred embodiment relates to a kit suitable for quantifying PTST, BCTF and BCTT, wherein the kit comprises the following reagents:

[0239] - a primer comprising or consisting of SEQ ID NO: 24, a primer comprising or consisting of SEQ ID NO: 25, or a sequence at least 80% identical to any of them;

[0240] - a primer comprising or consisting of SEQ ID NO: 26, a primer comprising or consisting of SEQ ID NO: 27, or a sequence at least 80% identical to any of them; and

[0241] - a primer comprising or consisting of SEQ ID NO: 30, a primer comprising or consisting of SEQ ID NO: 31, or a sequence at least 80% identical to any of them; and

[0242] - optionally, an oligonucleotide 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 sequence having at least 80% identity to any of them);

[0243] - Optionally, further comprising instructions for using the reagent to determine the level of the bacterial marker in a stool sample isolated from a subject.

[0244] In a preferred embodiment, the sequence with 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 percent identity between two sequences can be determined by any means known in the art, such as the Needleman and Wunsch global alignment algorithm.

[0245] In addition, the kit further comprises oligonucleotides specific for other genes or bacterial markers as described above. For example, wherein the other bacterial markers are selected from the group consisting of: B10, B46, B48, CINT, RSBI, Faecalibacterium prausnitzii, F. prausnitzii PHGI, F. prausnitzii PHGII and Escherichia coli.

[0246] The kit may include additional reagents. In a specific embodiment, the kit includes reagents for performing real-time PCR reactions, which typically include a DNA polymerase, such as TaqDNA polymerase (e.g., hot start TaqDNA polymerase), a buffer, magnesium, dNTPs, and optional other reagents (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 the amplification product as described herein above.

[0247] Optionally, the kit may also include suitable tubes and solvents for DNA extraction, such as a chloroform / methanol solution. Furthermore, the kit may also include a container for collecting stool samples and, optionally, any buffer and / or additives sufficient for sample storage, such as the FIT tube collector used in the Examples. Other preferred features and embodiments of the kit of the present invention are described throughout the specification.

[0248] In another aspect, the present invention also provides a kit as described herein, for use in a method for screening, diagnosing and / or monitoring AN, AA and / or CRC as described herein.

[0249] It is contemplated that any feature described herein may be optionally combined with any embodiment of any method, medical use, kit, and kit use of the invention; and any embodiment discussed in this specification may be implemented with respect to any of these. It should be understood that the specific embodiments described herein are presented as illustrations and not as limitations of the invention.

[0250] All publications and patent applications herein are 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.

[0251] The use of "a" or "an" can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "another" can also mean one or more. The term "or" as used in the claims is intended to mean "and / or" unless explicitly stated to refer to only alternatives or the alternatives are mutually exclusive.

[0252] As used in this specification and claims, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “include” and “includes”), or “containing” (and any form of containing, such as “contain” and “contains”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The term “comprising” also encompasses and expressly discloses the terms “consisting of” and “consisting essentially of. As used herein, the phrase “consisting essentially of limits the scope of a claim to the specified materials or steps, and those materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. As used herein, the phrase “consisting of excludes any element, step, or ingredient not specified in the claim, except for impurities normally associated with that element or limitation.

[0253] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, specifically included are combinations containing repetitions of one or more items or terms, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so on. Those skilled in the art will understand that, unless otherwise apparent from the context, there is generally no limit on the number of items or terms in any combination.

[0254] As used herein, approximating words such as, but not limited to, "about," "approximately," and "approximately" refer to a situation that, when so modified, is understood not to be necessarily absolute or exact, but will be considered close enough for one of ordinary skill in the art to warrant designating such a situation as existing. The extent to which the description can vary will depend on how much change can be made and still allow one of ordinary skill in the art to recognize that the modified feature still has the properties and capabilities required of the unmodified feature. Generally speaking, but in accordance with the foregoing discussion, numerical values ​​modified by approximate words such as "about" herein can vary from the stated value by ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%. Thus, the term "about" can refer to the indicated value ±5% of its value, preferably the indicated value ±2% of its value, and most preferably, the term "about" refers to the exact indicated value (±0%).

[0255] The following examples are intended to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0256] Terms

[0257] 1. A method for screening, diagnosing and / or monitoring a subject for colorectal advanced neoplasia (AN), advanced adenoma (AA) and / or colorectal cancer (CRC) by increasing the specificity of a fecal occult blood test (FOBT) or reducing the false positive rate of the FOBT, wherein the FOBT comprises determining the presence of occult blood in a stool sample isolated from the subject; and wherein the method further comprises quantifying at least the following bacterial markers in the stool sample isolated from the subject:

[0258] - Peptostreptococcus gastricis (PTST),

[0259] - Bacteroides fragilis (BCTF), and

[0260] - Bacteroides thetaiotaomicron (BCTT);

[0261] The term colorectal advanced neoplasia (AN) includes CRC and advanced adenoma (AA).

[0262] 2. A method for screening, diagnosing and / or monitoring AN, AA and / or CRC in a human subject, the method comprising:

[0263] a) performing a FOBT on a stool sample isolated from the subject, wherein the FOBT comprises determining the presence of occult blood in the stool sample;

[0264] b) quantifying a bacterial marker consisting of at least Peptostreptococcus gastricus (PTST), Bacteroides fragilis (BCTF), and Bacteroides thetaiotaomicron (BCTT) in a stool sample isolated from the subject:

[0265] c) calculating a combined score from the levels of the bacterial markers determined in b); and

[0266] d) categorizing FOBT-positive subjects as presenting an increased risk of developing AN, AA, and / or CRC based on the combined score obtained in c).

[0267] 3. The method according to clause 1, wherein the combined score calculated in c) is directly proportional to the amount of PTST and BCTF; and inversely proportional to the amount of BCTT; wherein the higher the score, the higher the likelihood of having AN, AA and / or CRC.

[0268] 4. A method according to any one of clauses 2 or 3, wherein in step d), the method comprises comparing the combined score in the sample from the subject with a reference value; and wherein an increase in the combined score in the sample from the subject relative to the reference combined score is indicative of AN, AA and / or CRC.

[0269] 5. A method for screening, diagnosing and / or monitoring AN, AA and / or CRC in a human subject, said method comprising steps a) and b) according to clause 2, wherein said method further comprises:

[0270] e) comparing the amounts of PTST, BCTF, and BCTT in the stool sample with corresponding reference values;

[0271] f) wherein an increased amount of PTST and BCTF and a decreased amount of BCTT in a sample from a FOBT-positive subject relative to corresponding reference values ​​is indicative of AN, AA and / or CRC.

[0272] 6. The method according to any one of clauses 1 to 5, wherein the subject is a subject suspected of having CRC.

[0273] 7. The method according to any one of clauses 1 to 5, wherein the subject is an asymptomatic subject.

[0274] 8. A method according to clause 7, wherein the subject is a subject with an intermediate risk of CRC or a high risk of CRC; wherein the subject with an intermediate risk of CRC is 50 years of age or older and has no personal or family background of CRC; and wherein the subject has an increased and / or high risk of CRC.

[0275] 9. The method of any one of clauses 1 to 8, wherein the CRC is an adenocarcinoma.

[0276] 10. The method according to any one of clauses 1 to 9, wherein the FOBT is a guaiac FOBT or a fecal immunochemical test (FIT) for quantification of human hemoglobin (hHb).

[0277] 11. The method according to any one of clauses 1 to 10, wherein the FOBT is a fecal immunochemical test (FIT) for quantification of human hemoglobin (hHb).

[0278] 12. The method according to clause 11, wherein the hHb level of a FIT-positive subject is equal to or higher than 10 μg hHb / g stool (FIT50 cutoff) or equal to or higher than 20 μg hHb / g stool (FIT100 cutoff), preferably wherein the hHb level of a FIT-positive subject is equal to or higher than 10 μg hHb / g stool (FIT50 cutoff).

[0279] 13. The method of any one of clauses 1 to 12, wherein the method further comprises 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), Gemini coccus morbilli (GMLL), Roseburia intestinalis (RSBI), Collinsella intestinalis (CINT), Faecalibacterium prausnitzii and / or any strain of its phylogroup, and Escherichia coli (ECO).

[0280] 14. A method according to any one of clauses 1 to 13, wherein the levels of PTST, BCTF and BCTT and optionally the bacterial marker of clause 13 are determined by a molecular biological method selected from the group consisting of: next generation sequencing, quantitative PCR (qPCR), PCR-pyrosequencing, PCR-ELISA, DNA microarray, branched DNA, dot immunoblotting, fluorescence in situ hybridization assay (FISH), and multiplexed forms of said methods.

[0281] 15. The method according to clause 14, wherein the molecular biology method is qPCR, preferably wherein the quantitative level is expressed as a Ct value.

[0282] 16. A method according to any one of clauses 1 to 15, wherein the levels of PTST, BCTF and BCTT and optionally the level of the bacterial marker according to clause 12 are expressed as relative abundance, preferably relative to the level of true bacteria (EUB).

[0283] 17. The method according to any one of clauses 1 to 16, wherein DNA is extracted from the stool sample prior to quantification of PTST, BCTF and BCTT and optionally the bacterial markers according to clause 12.

[0284] 18. The method according to any one of clauses 1 to 17, wherein the quantification of FOBT, and PTST, BCTF and BCTT and optionally the bacterial markers according to claim 12 are performed in the same stool sample.

[0285] 19. The method according to any of clauses 1 to 18, wherein the method further comprises storing the method results in a data carrier, preferably wherein the data carrier is a computer-readable medium.

[0286] 20. A computer-implemented method, wherein the method is as defined in any one of clauses 1 to 19.

[0287] 21. A data processing apparatus comprising means for performing the steps of the method of clause 20.

[0288] 22. A kit for quantifying PTST, BCTF, and BCTT, wherein the kit comprises the following reagents:

[0289] - an oligonucleotide specific for SEQ ID NO: 2;

[0290] - an oligonucleotide specific for SEQ ID NO: 3; and

[0291] - an oligonucleotide specific for SEQ ID NO: 4;

[0292] - optionally, oligonucleotides suitable for quantifying eubacteria;

[0293] Optionally, instructions are also included for using the reagent to determine the level of the bacterial marker in a stool sample isolated from a subject.

[0294] 23. The kit according to clause 22, wherein:

[0295] - the oligonucleotide specific for SEQ ID NO: 2 is an oligonucleotide comprising or consisting of any of the following sequences: SEQ ID NO: 24 or SEQ ID NO: 25, or a sequence having at least 80% identity to any of these sequences; and / or

[0296] - the oligonucleotide specific for SEQ ID NO: 3 is an oligonucleotide comprising or consisting of any of the following sequences: SEQ ID NO: 26 or SEQ ID NO: 27, or a sequence having at least 80% identity to any of these sequences; and / or

[0297] - the oligonucleotide specific for SEQ ID NO: 4 is an oligonucleotide comprising or consisting of any of the following sequences: SEQ ID NO: 30 or SEQ ID NO: 31, or a sequence having at least 80% identity to any of these sequences; and / or

[0298] - Oligonucleotides suitable for quantifying eubacteria are oligonucleotides comprising or consisting of any of the following sequences: 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 these sequences.

[0299] 24. Use of a kit according to any one of clauses 22 or 23 in a method for screening, diagnosing and / or monitoring AN, AA and / or CRC according to any one of clauses 1 to 20.

[0300] 25. A method of treating a subject suffering from AN, AA and / or CRC, wherein the subject is selected by a method of screening, diagnosis and / or monitoring according to any one of clauses 1 to 20, and wherein the method further comprises administering an anti-cancer therapy to the subject.

[0301] 26. A method of selecting a subject for an exploratory test selected from the group consisting of a colonoscopy, a flexible sigmoidoscopy, a double contrast barium enema and a computed tomography (CT) colonoscopy, preferably a colonoscopy, wherein the subject is selected by a screening, diagnostic or monitoring method according to any one of clauses 1 to 20.

[0302] Example

[0303] Example 1.-Materials and Methods

[0304] Research group

[0305] A cohort of 333 consecutive patients with CRC-related symptoms who were referred from primary and secondary healthcare facilities to the Ourense General Hospital (Complexo Hospitalario de Ourense) (Ourense, Spain) for a diagnostic colonoscopy was recruited ( Table 1 ). Exclusion criteria were: (1) asymptomatic subjects undergoing colonoscopy for CRC screening; (2) patients with a history of previous colon disease undergoing surveillance colonoscopy; (3) patients requiring hospitalization; (4) patients whose symptoms had ceased within the 3 months before evaluation; and (5) patients who had received antibiotic treatment within the last month before enrollment. The study protocol was approved by the Biobank of the University Hospital Complex of Vigo (Biobanco del Complexo Hospitalario Universitario de Vigo) (Vigo, Spain). Written informed consent was obtained from all study patients.

[0306] Table 1. Patient characteristics classified according to colonoscopy diagnosis. Hb: hemoglobin; FIT100 (20 μg Hb / g stool); CRC: colorectal cancer; AA: advanced adenoma; NAA: non-advanced adenoma; NC: normal colonoscopy.

[0307]

[0308] All subjects underwent colonoscopy to determine their colorectal status. Based on the endoscopic and pathological findings, the diagnosis was divided into four groups: normal colonoscopy (colonoscopy found no or no sigmoid colon hyperplastic polyps and / or rectal hyperplastic polyps <10 mm); non-advanced adenoma (tubular adenoma <10 mm with low-grade dysplasia, serrated polyps <10 mm and no dysplasia); advanced adenoma (adenoma >10 mm or with villous components or high-grade dysplasia, serrated polyps >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 answer questionnaires to record clinical and epidemiological data.

[0309] Table 2. Patients with colorectal cancer according to TNM stage of the tumor. CRC: colorectal cancer.

[0310]

[0311] Stool sample collection

[0312] Participants were asked to collect a stool sample from a single bowel movement in a sterile stool container before colonoscopy and bowel cleansing. The sample was frozen immediately after deposition. The subjects then brought the sample to the hospital, where it was frozen at -20°C for short-term storage and stored at -80°C upon arrival at the GoodGut SL facility in Girona (Spain). A total of 11 subjects were excluded from the study due to errors in stool sample collection.

[0313] DNA extraction from stool samples

[0314] Genomic DNA was extracted from frozen stool samples after homogenization using the NucleoSpin Soil Kit (Macherey-Nagel GMbH&Co., Düren, Germany). DNA was eluted in a final volume of 100 μl of SE elution buffer according to the manufacturer's instructions and stored at −20°C until use. DNA concentration was determined using the Qubit fluorometric assay (ThermoFisher Scientific, Waltham, MA). All samples were adjusted to a final concentration of 8 ng / μl and requantified.

[0315] qPCR detection of CRC biomarkers

[0316] There are ten target-specific bacterial sequences: Eubacterium (EUB), B10 (best match BLAST Faecalibacterium prausnitzii), B46 (best match BLAST Subdoligranulum variabile), B48 (best match BLAST Ruminococcus, Roseburia, Coprococcus), Roseburia intestinalis (RSBI), Geminicoccus morbilli (GMLL), Peptostreptococcus gastricis (PTST), Bacteroides fragilis (BCTF), Collinsella enterica (CINT), and Bacteroides thetaiotaomicron (BCTT).

[0317] Quantification of the different biomarkers was performed by preparing a single reaction for each biomarker using SYBR Green Master Mix (Promega, Madison, USA). Each reaction consisted of 20 μl containing 1× GoTaq qPCR Master Mix, each primer at a concentration of 150 nM to 300 nM, and a maximum of 20 ng of genomic DNA template. The species-specific primers used in this study are shown in Table 3 and were purchased from Macrogen (Macrogen, Seoul, South Korea).

[0318] Table 3. Forward and reverse primers used in this study. EUB: Eubacterium; B10; B46; B48; GMLL: Gemini cocci morbilli; PTST: Peptostreptococcus gastricis; BCTF: Bacteroides fragilis; CINT: Collinsella enterica; BCTT: Bacteroides thetaiotaomicron; RSBI: Roseburia intestinalis.

[0319]

[0320]

[0321] All quantitative PCR were performed on an AriaMx Real-Time PCR System (Agilent Technologies, Santa Clara, USA). The thermal profiles were different depending on the biomarker being analyzed ( Table 4 A melting curve step was added at the end of each qPCR to verify the presence of the expected amplicon size and to control for 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 when the difference between the threshold cycles (Ct) was less than 0.6. A threshold cycle (Ct) was established for each biomarker. 8 A dynamic range of 8 logs of genomic units / μL was used to calculate relative abundance. A no-template control reaction was included in each PCR run.

[0322] Table 4. qPCR conditions for the following bacteria: EUB: Eubacterium; B10; B46; B48; GMLL: Gemini coccus morbilli; PTST: Peptostreptococcus gastricis; BCTF: Bacteroides fragilis; CINT: Collinsella enterica; BCTT: Bacteroides thetaiotaomicron; RSBI: Roseburia intestinalis.

[0323]

[0324] FIT analysis

[0325] The FIT analysis was performed at the Complexo Universitario de Ourense using the same samples used in the CRC-specific biomarker analysis. Fecal samples for fecal hemoglobin determination were analyzed using an OC-Sensor tube collector and assayed using an automated OC-Sensor that detects gastrointestinal bleeding associated with diseases such as CRC, polyps, and diverticulitis (Eiken Chemical Co., Tokyo, Japan), as previously described in Cubiella, J. et al., 2016. The sample buffer in the OC-Sensor sample tube ensures optimal stability and safe transport of the sample from collection to analysis. A positive test refers to a fecal hemoglobin concentration equal to or greater than 100 ng / mL (20 μg Hb / g stool; FIT100).

[0326] Statistical analysis

[0327] In terms of qualitative analysis, if the Ct value obtained was not within its dynamic range, the biomarker was considered absent. All statistical analyses were performed using the SPSS 23.0 statistical software package (IBM, NYC, USA). A significance level was established for P values ​​≤ 0.05.

[0328] Data normality was assessed using 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.

[0329] Receiver operating characteristic (ROC) curve analysis was performed to determine the usefulness of each biomarker in distinguishing different colon tumor states. The accuracy of discrimination was measured by the area under the ROC curve (AUC).

[0330] Machine learning was used to determine which combinations of study variables (sex, age, BMI, smoking, bacterial markers, FIT) could distinguish subjects with advanced neoplastic lesions from subjects with normal colonoscopy or non-advanced adenomas. The method included initial training iterations on 100 random partitions of the dataset and further validation of the prediction models generated using four different machine learning algorithms (neural network, logistic regression, gradient boosted tree, random forest). RAID-CRC was ultimately designed using the four bacterial markers analyzed in combination with FIT results.

[0331] Example 2.- Fecal biomarkers in tumor progression

[0332] The relative abundance of each bacterial marker was determined for each diagnosis (normal colonoscopy, non-advanced adenoma, advanced adenoma, CRC) Figure 1 ). Regardless of colonoscopic 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 compared to colonoscopy-normal individuals (p=0.006 and p<0.001, respectively) or non-advanced adenoma subjects (p=0.047 and p<0.001, respectively). Although there were no significant differences, a trend could be observed for B46, which was more abundant in CRC patients than in patients with advanced adenomas (p=0.087). Interestingly, EUB abundance remained constant regardless of tumor status. Comparison between different CRC stages (0, I, II, III, and IV) did not show significant differences in the abundance of any bacterial markers.

[0333] Example 3.-GMLL, PTST and BCTF can detect advanced tumor lesions

[0334] The relative abundance of bacterial markers was compared after subjects were grouped as follows: (1) normal colonoscopy; (2) neoplastic (non-advanced adenoma + advanced adenoma + CRC); (3) advanced neoplastic (advanced adenoma + CRC); and (4) CRC ( Figure 2 ). PTST was found to be highly correlated with tumor lesions (p < 0.001). Regarding the detection of advanced tumor lesions, GMLL, PTST and BCTF are potential biomarkers for the detection of advanced tumor lesions (p = 0.006, p < 0.001 and p = 0.030, respectively). In terms of prevalence, these three opportunistic pathogens were found to be more common in patients with advanced tumors (GMLL, 64.9%; PTST, 58.4%; and BCTF, 44.7%) than in healthy subjects (GMLL, 53.5%; PTST, 26.1%; and BCTF, 29.8%).

[0335] Our results clearly demonstrate bacterial dysbiosis in CRC patients. The bacterial markers studied were categorized according to gut health-related phenotypes: butyrate producers (B10, B46, B48, RSBI), opportunistic pathogens (GMLL, PTST, BCTF), hydrogen and oxygen producers (CINT), and sugar-decomposing species (BCTT) ( Figure 3The relative abundance of these phenotypes was found to gradually change with the progression of the disease state. In particular, between subjects with normal colonoscopy and those with CRC, we found that the relative abundance of butyrate producers decreased and was replaced by a group of pathogenic bacteria, which were more abundant in individuals with CRC and advanced adenomas than in subjects with normal colonoscopy.

[0336] Our results showed that high abundance of PTST and BCTF was associated with advanced tumors, while high abundance of BCTT was associated with good health. BCTT is a commensal bacterium commonly found in the gut microbiota of healthy individuals. Commensal bacteria have been shown to reduce intestinal inflammation and contribute to colonization resistance (Macfarlane & Macfarlane, 2012; Baümler & Sperandio, 2016). Therefore, high abundance of BCTT is associated with good gut health.

[0337] Example 4.- Combination of CRC bacterial markers and FIT significantly reduces false positive results

[0338] On the one hand, when using FIT100 (20 μg Hb / g stool), a significant difference was observed between subjects with normal colonoscopy or non-advanced adenoma and advanced tumors (p < 0.001). 17.1% (19 subjects) of subjects with normal colonoscopy and 24.3% (27 subjects) of subjects with non-advanced adenoma showed FIT positive values. These results resulted in a sensitivity and specificity of 84.0% and 81.0% for detection of advanced tumors, respectively, and a positive predictive value and negative predictive value of 58.0% and 94.0%, respectively (AUC = 0.828, 95.0% CI (0.773-0.883)). 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 adenoma showed false positive results. FIT50 resulted in a sensitivity and specificity of 91.0% and 76.0%, respectively, for the detection of advanced tumors, and a positive predictive value and negative predictive value of 55.0% and 96.0%, respectively (AUC = 0.836, 95.0% CI (0.787-0.886)). The 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 (0.628-0.7376%), and 33.0% for BCTF (AUC = 0.571, 95.0% CI (0.499-0.656)), while the specificity values ​​were similar.

[0339] The FIT results (FIT100 and FIT50) were combined with fecal bacterial markers to understand which one provided higher performance in terms of sensitivity and specificity values ​​for the detection of advanced neoplastic lesions. The combination of bacterial markers and FIT100 (RAID CRC-FIT100) resulted in a sensitivity of 76.0% and a specificity of 91.0% (Table 5). Nevertheless, FIT50 (RAID CRC-FIT50) also slightly improved these results because it showed a 4.0% higher sensitivity (80.0%) for the detection of advanced tumors with similar specificity values ​​and was therefore the selected cutoff value. Therefore, the preferred test is based on a combination of EUB, PTST, BCTF and BCTT, in which the fecal hemoglobin concentration is equal to or higher than 50 ng / μL (10 μg Hb / g feces). Although BCTT did not show significant differences between patients with normal or non-advanced adenomas and patients with advanced tumors on colonoscopy, BCTT was able to improve the specificity of the test once used in combination with EUB, PTST and BCTF.

[0340] Table 5. Diagnostic performance of RAID-CRC (using FIT100 and FIT50), FIT100, and FIT50 in the studied symptomatic population compared with FIT100 in the screening population. FIT100 (20 μg hemoglobin / g stool); FIT50 (10 μg hemoglobin / g stool); PPV: positive predictive value; NPV: negative predictive value.

[0341]

[0342] The final test (RAID CRC-FIT50 test) includes FIT50 in combination with quantitative levels of three bacterial markers (PTST / EUB, BCTF / EUB, BCTT / EUB) normalized to eubacteria, where the quantitative levels are expressed as Ct values.

[0343] The use of ratios allowed data normalization, which is crucial for controlling for qPCR-related variables in order to distinguish true biological changes from experimentally induced changes (Vandesompele et al, 2002). Lowering the fecal hemoglobin concentration threshold from 100 ng / μL to 50 ng / μL in the RAID CRC-FIT50 test allowed the capture of positive subjects that would otherwise be considered false negatives at the 100 ng / μL cutoff, at the expense of an increased false-positive rate. However, combining with bacterial markers diluted this effect, as the RAID CRC-FIT50 test resulted in a significant reduction in false-positive results due to the increased specificity of FIT50 and FIT100 for advanced tumor detection (Table 5).

[0344] Applying the RAID CRC-FIT50 test to detect advanced tumors resulted in a reduction in the number of false-positive results for FIT100, with 9.7% of subjects showing normal colonoscopy results and 11.7% of subjects having non-advanced adenomas. In summary, the RAID-CRC-FIT50 test was shown to provide a sensitivity and specificity of 80.0% and 90.0%, respectively (AUC = 0.837, 95.0% CI (0.730-0.944)), with 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 having false-positive results for FIT100 and 23 subjects having false-positive results for the RAID-CRC test-FIT50 test. Interestingly, the sensitivities of the RAID CRC-FIT50 test and the RAID CRC-FIT100 test for CRC and AN were similar to those of the FIT100 protocol, but with higher specificity and positive predictive value (PPV).

[0345] In this work, we developed a novel method suitable for use in national CRC screening programs using stool samples. The bacterial signatures used in this work were originally derived from mucosal samples. Therefore, their presence in stool is not significantly affected by variability caused by diet and some external factors (Conlon & Bird, 2015; Vandeputte et al., 2015) but rather represents a measure of true abundance in the colonic mucosa. This helps overcome the significant background noise present in stool and provides physiological meaning to the biomarkers.

[0346] Our dataset does not contain metadata regarding body mass index (BMI), smoking, or dietary habits. Although these parameters have been reported to influence the microbiota composition of stool samples (Davis et al., 2017; Yun et al., 2017; Rogers et al., 2012; Capurso & Lahner, 2017; Baothman et al., 2016), our biomarkers are derived from mucosal samples, which are less dependent on external factors, as described above (Watt et al., 2016; Durban et al., 2011). Biedermann et al. reported that smoking cessation increases microbial diversity (Biedermann et al., 2013). Other studies have observed that long-term alcohol consumption leads to an increase in Proteobacteria and a decrease in Bacteroidetes (Kakiyama et al., 2013; Rao et al., 2004). Regarding BMI, its effect on the microbiota is controversial (Turnbaugh et al., 2006; Schwiertz et al., 2010; Santacruz et al., 2009). Because the RAID-CRC test described in this article may be used in screening scenarios where the population being screened may be subject to various conditions and habits, a non-stratified strategy was found to be a good way to reproduce the CRC screening scenario with the highest reliability.

[0347] Cost-effectiveness is also a key issue in population-based screening (Sonnenberg et al., 2000; McGrath et al., 2002; Telford et al., 2010). Wong and colleagues compared FIT and colonoscopy in this setting and showed that FIT was cost-effective in average-risk screening, while colonoscopy was cost-effective in high-risk subjects (Wong, 2015). Therefore, combining FIT with fecal bacterial biomarkers may be more cost-effective, as the use of RAID-CRC could save up to 30% of unnecessary colonoscopies. More specifically, implementing RAID-CRC in a CRC screening program would result in 33,000 fewer colonoscopies due to false-positive results compared to a FOBT-based screening program (55,000 vs. 22,000 false-positives) (García et al., 2012). Considering that the cost of RAID-CRC is comparable to that of FOBT, the estimated savings from follow-up colonoscopies after a positive screen would be €77 million per 100,000 screening program participants ( Table 6Furthermore, the use of the CRC biomarkers proposed in this work is feasible in both developed and resource-poor settings with limited colonoscopy facilities, as RAID-CRC represents a potentially feasible and cost-effective tool in CRC screening scenarios.

[0348] In conclusion, RAID-CRC is a promising tool for CRC screening due to its noninvasive nature, low cost, and ability to reduce the number of false-positive results associated with the use of FOBTs (e.g., FIT).

[0349] Table 6. Comparison of costs associated with subsequent colonoscopy in different CRC screening programs.

[0350]

[0351] *Savings calculated assuming combining FOBT with microbiome analysis increases screening sensitivity by more than 45% compared to FOBT alone.

[0352] ** The costs are calculated taking into account a test cost of 25€.

[0353] *** The costs are calculated taking into account that the test costs 10€.

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[0423] 69.Matsuda,K.et al.Bacterial Identification by 16S rRNA Gene PCR-Hybridization as a Supplement to Negative CultureResults.J.Clin.Microbiol.49,2031–2034(2011). Sequence Listing <110> Goodgate Dr. Josep Truta Biomedical Research Foundation, Girona University of Girona <120> Improved methods for screening, diagnosis and / or monitoring of colorectal advanced neoplasia, advanced adenomas and / or colorectal cancer <130> 905 028 <160> 33 <170> PatentIn version 3.5 <210> 1 <211> 783 <212> DNA <213> Gemella morbillorum <220> <221> misc_feature <222> (1)..(783) <223> GenBank accession number: LS483440.1 REGION: 827110..827892 <400> 1 atggcgagtt tattacaaaa aacaagaaaa ataagtacaa ttttacaaga aggacgtcat 60 gataatgtcg attttgaagc aatggcaatg cgcttaagtc ctattttaga ttctgttgtc 120 tatattttag atgtagaagg taatattctt gggtatgatt ctattgtaga ttattctaac 180 gaacgtatgg aagaaattat tcgtgcgagg aaagtgccta aagcttattt agatgcaaca 240 ttaaaagtat atgctacgaa ggttaatatt ccatttcaag atccgctttc tattttccca 300 gatgaagaga aagaaagatt tgatgggaat acctatactg taattttacc aataagaggc 360 gggggagaac gccttggaac tcttgttata ggaagaatgg ataatgactt tcaagatgat 420 gatttggtgt tagcagaata tgcgtcaaca gtagtaggaa ttgaaattct tcacgaaaaa 480 caagataaag aaaaaaatct agctagagac aaagatatgg tgaatatggc tcttaattcg 540 ttatcatatt cagaaaaaga agcaattgaa catattttca gagaattaga tgggacagaa 600 ggactactta tcgcaagtaa aattgcagat agagtaggaa ttactcgctc agttattgtt 660 aatgcactta gaaaattaga aagtgcaggt attatagagt caaaatcatt aggtatgaaa 720 ggtacttata taaaagtact taaagaatat tttttagaat taatgttttc taatgaattt 780 taa 783 <210> 2 <211> 567 <212> DNA <213> Peptostreptococcus stomatis DSM 17678 strain <220> <221> misc_feature <222> (1)..(567) <223> GenBank accession number: ADGQ01000060.1 REGION: complementary (142796..143362) <400> 2 atggatatta ttcaacttag caacagactt atagaataca gaaaaagtaa caacttaact 60 attaaagatt tctctgacat gtccggaatt agtactgctc tgcttagtca attagaaaga 120 ggtgttggaa accctagcct gagtgtattg aactctatag cagataccat gaatactagc 180 ctctcctctt tattagaaga acctgttgtt ttagaaaacc tggttagaag gtctgacacc 240 ctatccacta ttgtctaccc atgtaggaac aaccttgaat ttcagttgct cacaactagg 300 tctactactc agagcatcaa ccttgtaaga ataatatttc atgctcattc agaaactaat 360 taccagccac ttggaaaaac cgtttctgat gatataatcc acattgaaaa gggcagtatt 420 attgttcaaa ctgatgatgg taagtctatt caactaaaaa aaggtgatac tatgaggata 480 ccacctgacc ttaggtataa attcaagaat atttctgtac acaaggcaca tatgatttgt 540 gccactaata aacttgaagt aagataa 567 <210> 3 <211> 717 <212> DNA <213> Bacteroides fragilis <220> <221> misc_feature <222> (1)..(717) <223> GenBank accession number: CR626927.1 REGION: 417101..417817 <400> 3 atggcaaata ctaatatgga acacggagaa ataatactat accaaccaga caatactata 60 aaactggaag tgcgaataga gaatgaaacc gtttggttga cacaagcaca aattgttaac 120 ttattccagt caagtaaagc caatatcagt gagcatataa gaaatatata tgactcagat 180 gaattatctg ctgaatcaac tgttcggaaa ttccgaacag ttcgaatgga aggtaataga 240 aaggttaccc gcattcttga atattataat ctggatatga ttatttccgt aggttatcgt 300 gtgaattcca agcgaggagt ccagttccgc caatggtcaa caggagtact taaagaatat 360 ctattaaaag gctacgcaat caatcagcgc gtggagcagt tggaaaacaa agcaaacacc 420 catgaccggc aactggaaga gttaacaaac aaagtcgact ttttcgtccg gacttcatta 480 cctcctattg aaggtgtctt tttcaacgga cagattttcg atgcctatgt cttttccgct 540 cagttgataa agtctgcaaa gtcatcttta gtattgattg acaactttgt cgatgaaagc 600 gtgctcttac tattgagtaa acgtttgccc ggagtgactt ctatcatata taccaagcaa 660 gtaactccac agttagaatt agatttgaca aagcacaaca gtcaataccc ccaatag 717 <210> 4 <211> 642 <212> DNA <213> Bacteroides thetaiotaomicron <220> <221> misc_feature <222> (1)..(642) <223> GenBank accession number: AE015928.1 REGION: 326008..326649 <400> 4 atggcagtac aatttgaatt atataagact ccgatgccaa aggagaaaaa gaacaagacg 60 cgttatcatg cccgtccggt aagttttgag accgtcaata ccgagaaact ggcttatcgc 120 atccatgatc gctccacatt aagagtatcg gacattattt caaccttgga agaactgaaa 180 aatgaagtag ctcagtgcct cctggaaggt aaaaaggtgc atgtcgatgg attaggattc 240 tttcaggtca ctctttcctg cgaagaagaa atacgtaacc cgaaagacaa acgtgtgcac 300 cgggtaaaat tgaaagctat aaagtttaaa gctgataaag aattgaaagg ggaattgtgt 360 cacatgaaat tccagcgttc taaaatcaga ccgcactccg ccaatttatc agaagtagaa 420 atagacatga aactaactga atattttgct gaaaatcaga tttcacccg gaaagatttt 480 caatatctct gcggaatgac acaaatcact gcatatcgcc atatcaagag gttaatggca 540 gaaaagaaat tgcaaaataa agggacgatc tatcaaccga tatatactcc ggttccgggt 600 attacaggg tatcggtgga tttaaaatat aaagaacaat ga 642 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> EUB2 <220> <221> misc_feature <222> (1)..(21) <400> 5 actcctacgg gaggcagcag t 21 <210> 6 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> EUB2 reverse primer <220> <221> misc_feature <222> (1)..(22) <400> 6 gtattaccgc ggctgctggc ac 22 <210> 7 <211> 485 <212> DNA <213> Artificial Sequence <220> <223> B10; DGGE gel band Eub_10 16S of uncultured bacterial isolate ribosomal RNA genes <220> <221> misc_feature <222> (1)..(485) <400> 7 cttcggattg taaactcctg ttgttgagga gataatgacg gtactcaaca aggtaagtga 60 cggctaacta cgtgccagca gccgcggtaa aacgtaggtc acaagcgttg tccggaatta 120 ctgggtgtaa agggagcgca ggcgggaaga caagttggaa gtgaaatcca tgggctcaac 180 ccatgaactg ctttcaaaac tgtttttctt gagtagtgca caggtaggcg gaattcccgg 240 tgtagcggtg gaatgcgtag atatcgggag gaacaccagt ggcgaaggcg gcctactggg 300 caccaactga cgctgaggct cgaaagtgtg ggtagcaaac aggattagat accctggtag tccacactgt aaacgatgat tactaggtgt tggaggattg accccttcag tgccgcagtt 420 aacacaata gtaatccacc tggggagtac gaccgcaagg ttgaaactca aaggaattga cggac 485 <210> 8 <211> 474 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> B46; Radiation Propellant DGGE Container Eub_46 16S Antioxidant RNA infusion <220> <221> misc_feature <222> (1)..(474) <400> 8 gtaacatttc tgtaacaaga catacagcag tttctcagag ttcccaaact caaaggaatt 120. gcggagcc gcggttccac gtaagtcaca agcgttgtcc ggaattactg ggtgtaaagg 180. gcgcaggc gggagacaa gttggaagtg aaatccatgg gctcaaccca taaactgctt tcctaactgt ttttcttgag tagtgcacag gtaggcgga ttcccggtgt agcggtgga 240 tgcctagata tcgggaggaa caccattggc gaaggcggcc tactggactg caactgacgc 300 tgaggctcga aagtgtgggt agcaaacagg attagatacc ctggtagtcc actccgtaaa 360 ccatgattac tacgtgttgg aggattgacc ccttctgtgc cgcagataac acaataagta 420 atccacctgg ggagtacgac cgcccggttg agactcacag gaattgacgg actc 474 <210> 9 <211> 566 <212> DNA <213> Artificial Sequence <220> <223> B48; Uncultured Bacterial Isolate DGGE Gel Band Eub_48 16S Ribosomal RNA gene <400> 9 tctcttacgg ggagcagcag tggggaatat tgcacaatgg gggaaaccct gatgcagcga 60 cgccgcgtga gcgatgaagt atttcggtat gtaaagctct atcagcaggg aagaaaatga 120 cggtacctga ctaagaagcc ccggctaaat acgtgccagc agccgcggta atacgtatgg 180 tgcaagcgtt atccggattt actgggtgta aagggagcgt agacggagtg gcaagtctga 240 tgtgaaaacc cggggctcaa ccccgggact gcattggaaa ctgtgcatct agagtgtcgg 300 agggtaagc ggaattccta gtgtagcggt gaatgcgta gatattagga ggaacaccag tggcgaaggc ggcttactgg acgataactg acgctgaggc tcgaaagcgt ggggagcaaa 420 cagttaga taccctggta gtccacgccg taaacgatga ctactaggtg tcggggagca cagctcttcg gtgccgcagc aaacgcaata agtattccac ctggggagta cgttcgcaag 540 aatgaaactc acagaatttg acggag <210> 10 <211> 894 <212> DNA <213> Liquid strain (Rosburia intestinalis) L1-82 ingredient <220> <221> misc_feature <222> (1)..(894) <223> GenBank database LR027880.1 REGION: 599208..600101 <400> 10 atgtcttttt caagtgaagt gaaacaggag ttagcaaagc agagtggaaa gagcagacat tgtcagatcg ctgaacttgc ggcgttagtt gcttttgacg gaaagcgtca gcagctgatc ggggatgcgg gagatgtgct ggattcgga aatccgcttc tgcaggaaaa atatggactg ttactggcac agctgttcca tgttatata gaagagatag atacattgcc accggaacgc attcttgaga caatcagat gtggaatcag tctttaaggt gtgcagat cacagagacg 300 gtaaatggta ttttactgca gcagacatgt tgcaggcggg catatatccg gggagcattt 360 ttagcaggtg gatcgatcag tgatccgaat aagtcttatc attttgagat tgtctgccgg 420 gaaattgcgc aggcgaaca gcttcaggat gcaatcaaca gtttgagat ggagcaag 480 atcgtagaac gaagaaaca ccaggtcgta tatttaagg aaggtgccca gatcgtggat 540 atgttaaata tcatggaagc acatgtggca ctgatgaatc ttgaaatgt gcgtatctta 600 aaagaatga gaatttctgt aaccgaag gtaaattgtg agacggcaa tatcagcaag 660 actgtggcgg cagccgtaaa acagcttggc gatattgaat atattaaca gatgcaggc 720 ttagacagtc tgcctgaaaa cctaaaagaa atggcgttgc tgcggttaga gtatccggat 780 acaccgctta aggactggg aacatatctc gatcctccgg tcggaaatc gggagtgaac 840 cacaggctgc gcaggatcag tgagattgca gatgagctgc gtgaaaaaga ata 894 <210> 11 <211> 807 <212> DNA <213> Collinsella intestinalis DSM 13280 <220> <221> misc_feature <222> (1)..(807) <223> NCBIName: GG692711.1 REGION: 297314..298120 <400> 11 atgtccggac actctaaatg ggcaactacc aagcaccgca agggtgcgca ggacgctaag 60 cgttccgccc tgttctccaa gctgagccgt aacatcaccg tcgcggcccg tcttggcaac 120 gaccccaacc cggacaacaa cgcctccctc gccgctgccg tggccaaggc caaggctcag 180 tccatgccca aggacaagat caaggccgcc atcgacaagg ccttcggttc cggcgccgac 240 gccgccgtgt acgagaacat cgtgtacgag ggctacggcc cggccggtgt tgccgtgtac 300 gtcgagtgcc tgaccgacaa ccgcaaccgc accgccgccg atgtccgctc cgccttctcc 360 cacgctggcg gcaacctggg caccaccggc tccgttgcct tccagttcga gcgcaagggc 420 caggtcgtgg tctccaagca gatcgtcgac ccgaacgaca agaaggagaa cctcatggcc 480 aacggcgctg ccggcgatga ggaagagttc atgatggtca tcgccgaggc cggtggcgac 540 gactacgagg acgccggtga cgagtgggtc gtgtggaccg ccgcgggcga cctcatggcc 600. gtgtccaagg gcatcgaggc tcagggcatc gaggtcaagg gtgccgagct caccatggtc 660 ccgaccaccc cgacggccgt ctccggcgcc ccgccaaga aggtccagcg cctcatcgac 720 cgcctcgagg acctggacga cgtccaggac gtgtactcca ccatggacat gaccgacgag 780 gtcatcgctg ccctcgaga 807 <210> 12 <211> 1466 <212> DNA <213> Antimicrobial strain (Faecalibacterium prausnitzii) <220> <221> misc_feature <222> (1)..(1466) <223> GenBank ID: AJ270469.2 <400> 12 gttgatcctg gctcaggacg aacgctggcg gcgcgcctaa cacatgcaag tcgaacgagc gagagagagc ttgctttctc gagcgagtgg cgaacgggtg agtaacgcgt gaggaacctg 180. cctcaaagag ggggacaaca gttggaaacg actgctaata ccgcataagc ccacagctcg gcatcgagca gagggaaaag gagcaatccg ctttgagatg gcctcgcgtc cgattagcta gttggtgagg taatggccca ccaaggcaac gatcggtagc cggactgaga ggttgaacgg 300 ccacattggg actgagacac ggcccagact cctacgggag gcagcagtgg ggaatattgc 360 acaatggggg aaaccctgat gcagcgacgc cgcgtggagg aagaaggtct tcggattgta 420 aactcctgtt gttgaggaag ataatgacgg tactcaacaa ggaagtgacg gctaactacg 480 tgccagcagc cgcggtaaaa cgtaggtcac aagcgttgtc cggaattact gggtgtaaag 540 ggagcgcagg cgggaagaca agttggaagt gaaatctatg ggctcaaccc ataaactgct 600 ttcaaaactg tttttcttga gtagtgcaga ggtaggcgga attcccggtg tagcggtgga 660 atgcgtagat atcgggagga acaccagtgg cgaaggcggc ctactgggca ccaactgacg 720 ctgaggctcg aaagtgtggg tagcaaacag gattagatac cctggtagtc cacaccgtaa 780 acgatgatta ctaggtgttg gaggattgac cccttcagtg ccgcagttaa cacaataagt 840 aatccacctg gggagtacga ccgcaaggtt gaaactcaaa ggaattgacg ggggcccgca 900 caagcagtgg agtatgtggt ttaattcgac gcaacgcgaa gaaccttacc aagtcttgac 960 atcctgcgac gatgctggaa acagtatttt ccttcgggac gcagagacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaaccctta 1080 ctgtcagtta ctacgcaaga ggactctggc aggactgccg ttgacaaaac ggaggaaggt ggggatgacg tcaaatcatc atgcccttta tgacttgggc tacacacgta ctacaatggc gttaaacaaa gagaagcaag accgcgaggt ggagcaaaac tcagaaacaa cgtcccagtt cggactgcag gctgcaactc gcctgcacga agtcggaatt gctagtaatc gtggatcagc atgccacggt gaatacgttc ccggggccttg tacacaccgc ccgtcacacc atgagagccg 1380 gggggacccg aagtcggtag tctaaccgca aggaggacgc cgccgaaggt aaaactggtg attggggtga agtcgtaaca aggtc <210> 13 <211> 1452 <212> DNA <213> Liquid(Escherichia coli) <220> <221> misc_feature <222> (1)..(1452) <400> 13 cgccctcccg aaggttaagc tacctacttc ttttgcaacc cactcccatg gtgtgacggg cggtgtgtac aaggcccggg aacgtattca ccgtggcatt ctgatccacg attactagcg 120 attccgactt catggagtcg agttgcagac tccaatccgg actacgacgc actttatgag 180 gtccgcttgc tctcgcgagg tcgcttctct ttgtatgcgc cattgtagca cgtgtgtagc 240 cctggtcgta agggccatga tgacttgacg tcatccccac cttcctccag tttatcactg 300 gcagtctcct ttgagttccc ggccggaccg ctggcaacaa aggataaggg ttgcgctcgt 360 tgcgggactt aacccaacat ttcacaacac gagctgacga cagccatgca gcacctgtct 420 cacggttccc gaaggcacat tctcatctct gaaaacttcc gtggatgtca agaccaggta 480 aggttcttcg cgttgcatcg aattaaacca catgctccac cgcttgtgcg ggccccgtc 540 aattcatttg agttttaacc ttgcggccgt actccccagg cggtcgactt aacgcgttag 600 ctccggaagc cacgcctcaa gggcacaacc tccaagtcga catcgtttac ggcgtggact 660 accagggtat ctaatcctgt ttgctcccca cgctttcgca cctgagcgtc agctttcgtc 720 cagggggccg ccttcgccac cggtattcct ccagatctct acgcatttca ccgctacacc 780 tggaattcta cccccctcta cgagactcaa gcttgccagt atcagatgca gttcccaggt 840 tgagcccggg gatttcacat ctgacttaac aaaccgcctg cgtgcgcttt acgcccagta 900 attccgatta acgcttgcac cctccgtatt accgcggctg ctggcacgga gttagccggt 960 gcttcttctg cgggtaacgt caatgagcaa aggtattaac tttactccct tcctccccgc 1020 tgaaagtact ttacaacccg aaggccttct tcatacacgc ggcatggctg catcaggctt 1080 gcgcccattg tgcaatattc cccactgctg cctcccgtag gagtctggac cgtgtctcag 1140 ttccagtgtg gctggtcatc ctctcagacc agctagggat cgtcgcctag gtgagccgtt 1200 accccaccta ctagctaatc ccatctgggc acatccgatg gcaagaggcc cgaaggtccc 1260 cctctttggt cttgcgacgt tatgcggtat tagctaccgt ttccagtagt tatccccctc 1320 catcaggcag tttcccagac attactcacc cgtccgccac tcgtcagcaa agaagcaagc 1380 ttcttcctgt taccgttcga cttgcatgtg ttaggcctgc cgccagcgtt caatctgagc 1440 catgatcaaa ct 1452 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> EUB_F forward primer <220> <221> misc_feature <222> (1)..(21) <400> 14 actcctacgg gaggcagcag t 21 <210> 15 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> EUB_R reverse primer <220> <221> misc_feature <222> (1)..(22) <400> 15 gtattaccgc ggctgctggc ac 22 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> B10_F forward primer <220> <221> misc_feature <222> (1)..(19) <400> 16 caacaaggta agtgacggc 19 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> B10_R reverse primer <220> <221> misc_feature <222> (1)..(20) <400> 17 cgcctacctg tgcactactc 20 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> B46_F forward primer <220> <221> misc_feature <222> (1)..(19) <400> 18 tccacgtaag tcacaagcg 19 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> B46_R reverse primer <220> <221> misc_feature <222> (1)..(20) <400> 19 cgcctacctg tgcactactc 20 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> B48_F forward primer <400> 20 gtacggggag cagcagtg 18 <210> twenty one <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> B48_R reverse primer <220> <221> misc_feature <222> (1)..(23) <400> twenty one gacactctag atgcacagtt tcc 23 <210> twenty two <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GMLL_F forward primer <400> twenty two aagagttcca aggcgttctc 20 <210> twenty three <211> 25 <212> DNA <213> Artificial Sequence <220> <223> GMLL_R reverse primer <220> <221> misc_feature <222> (1)..(25) <400> twenty three ccatttcaag atccgctttc tattt 25 <210> twenty four <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> PTST_F forward primer <220> <221> misc_feature <222> (1)..(22) <400> twenty four aggttgatgc tctgagtagt ag 22 <210> 25 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> PTST_R reverse primer <220> <221> misc_feature <222> (1)..(23) <400> 25 atgaatacta gcctctcctc ttt 23 <210> 26 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> BCTF_F forward primer <220> <221> misc_feature <222> (1)..(22) <400> 26 tgaaagcgtg ctcttactat tg 22 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BCFT_R reverse primer <220> <221> misc_feature <222> (1)..(20) <400> 27 tattggctgt tgtgctttgt 20 <210> 28 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> CINT_F forward primer <220> <221> misc_feature <222> (1)..(21) <400> 28 gaccatcatg aactcttcct c 21 <210> 29 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> CINT_R reverse primer <220> <221> misc_feature <222> (1)..(17) <400> 29 ccgttgcctt ccagttc 17 <210> 30 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> BCTT_F forward primer <220> <221> misc_feature <222> (1)..(22) <400> 30 agtgacctga aagaatccta at 22 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BCTT_R reverse primer <220> <221> misc_feature <222> (1)..(20) <400> 31 gaccgtcaat accgagaaac 20 <210> 32 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> RSBI_F forward primer <220> <221> misc_feature <222> (1)..(21) <400> 32 gtgccagtaa cagtccatat t 21 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RSBI_R reverse primer <220> <221> misc_feature <222> (1)..(20) <400> 33 tagcaaagca gagtggaaag 20

Claims

1. Use of a reagent for quantifying at least the following bacterial markers in the preparation of a kit for screening, diagnosing and / or monitoring advanced colorectal cancer in a subject: - Peptostreptococcus stomatis, - Bacteroides fragilis, and - Bacteroides thetaiotaomicron, in, The advanced colorectal tumors include colorectal cancer and advanced adenoma. The levels of Peptostreptococcus gastricus, Bacteroides fragilis, and Bacteroides thetaiotaomicron were expressed as relative abundance relative to the levels of eubacteria.

2. The use according to claim 1, wherein The kit is used for a method for screening, diagnosing and / or monitoring a subject for advanced colorectal cancer, the method comprising: a. performing a fecal occult blood test in a stool sample isolated from the subject, wherein the fecal occult blood test comprises determining the presence of occult blood in the stool sample; and b. quantifying at least the following bacterial markers in a stool sample isolated from the subject: - Peptostreptococcus gastricis, - Bacteroides fragilis, and -Bacteroides thetaiotaomicron.

3. The use according to claim 2, wherein The method further comprises: c) calculating a combined score from the levels of said bacterial markers determined in b); and d) comparing the combined score in the subject's sample with a reference value and classifying the fecal occult blood test-positive subject as having advanced colorectal neoplasia based on the combined score obtained in c), wherein the combined score of step c) is obtained according to a mathematical algorithm, and wherein the quantitative value of each bacterial marker determined in step b) is a variable of said mathematical algorithm, and wherein said combined score calculated in step c) is directly proportional to the amount of Peptostreptococcus gastricis and Bacteroides fragilis and inversely proportional to the amount of Bacteroides thetaiotaomicron, wherein an increase in the combined score relative to the reference value is indicative of advanced colorectal neoplasia; and wherein the reference value is obtained from a subject not suffering from advanced colorectal neoplasia or a subject not suffering from a gastrointestinal disease.

4. The use according to claim 2, wherein The method further comprises: i. comparing the amounts of Peptostreptococcus, Bacteroides fragilis, and Bacteroides thetaiotaomicron in the stool sample with corresponding reference values; ii. wherein an increased amount of Peptostreptococcus gastricis and Bacteroides fragilis and a decreased amount of Bacteroides thetaiotaomicron in a sample from a subject who tested positive for fecal occult blood relative to corresponding reference values ​​is indicative of advanced colorectal neoplasia.

5. The use according to claim 1, wherein The subject is a subject suspected of having CRC or an asymptomatic subject.

6. The use according to claim 2, wherein The fecal occult blood test is a fecal immunochemical test that quantifies human hemoglobin.

7. The use according to claim 6, wherein FIT-positive subjects had human hemoglobin levels equal to or higher than 10 μg human hemoglobin / g feces.

8. The use according to claim 7, wherein FIT-positive subjects had human hemoglobin levels equal to or higher than 20 μg human hemoglobin / g feces.

9. The use according to claim 2, wherein The method further comprises quantifying Gemmella morbillorum.

10. The use according to claim 1, wherein Levels of Peptostreptococcus gastricus, Bacteroides fragilis, and Bacteroides thetaiotaomicron were determined by qPCR.

11. The use according to claim 9, wherein Levels of Peptostreptococcus gastricus, Bacteroides fragilis, Geminicoccus morbilli, and Bacteroides thetaiotaomicron were determined by qPCR.

12. The use according to claim 11, wherein The levels of Peptostreptococcus gastricus, Bacteroides fragilis, Bacteroides thetaiotaomicron, and Geminicoccus morbilli were determined by qPCR, with quantitative levels expressed as Ct values.

13. The use according to claim 2 or 9, wherein The method further comprises storing the method result in a data carrier.

14. Use according to claim 13, wherein the data carrier is a computer-readable medium.

15. A kit for quantifying Peptostreptococcus gastricus, Bacteroides fragilis and Bacteroides thetaiotaomicron, wherein the kit comprises the following reagents: - an oligonucleotide consisting of any of the following sequences: SEQ ID NO: 24 or SEQ ID NO: 25; - an oligonucleotide consisting of any of the following sequences: SEQ ID NO: 26 or SEQ ID NO: 27; and - an oligonucleotide consisting of any of the following sequences: SEQ ID NO: 30 or SEQ ID NO: 31; and in, The kit comprises oligonucleotides suitable for quantifying eubacteria.

16. The kit according to claim 15, wherein The oligonucleotide suitable for quantifying eubacteria is an oligonucleotide consisting of any of the following sequences: SEQ ID NO: 5 or SEQ ID NO:

6.

17. The kit according to claim 16, wherein The kit also includes instructions for using the reagent to determine the level of the bacterial marker in a stool sample isolated from a subject.

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