WO2024156754A1

WO2024156754A1 - Cancer associated microbiota and its use in predicting cancer progression

Info

Publication number: WO2024156754A1
Application number: PCT/EP2024/051662
Authority: WO
Inventors: Paolo Giovanni NUCIFORO; Stefania NAPOLI; Garazi SERNA ALONSO
Original assignee: Fundacio Privada Institut dInvestigacio Oncologica Vall dHebron
Current assignee: Fundacio Privada Institut dInvestigacio Oncologica Vall dHebron
Priority date: 2023-01-24
Filing date: 2024-01-24
Publication date: 2024-08-02
Anticipated expiration: 2025-07-24
Also published as: EP4655421A1

Abstract

The present invention relates to a method for the prediction of cancer progression which determines the microorganism content of a cancer sample, and wherein changes in the content of said microorganisms provide an indication of the risk of progression of the cancer.

Description

CANCER ASSOCIATED MICROBIOTA AND ITS USE IN PREDICTING CANCER PROGRESSION

Field of the Invention

The present invention relates to methods for predicting the progression of cancer by determining the microbiota associated with samples of said cancer.

of the Invention

Global cancer statistics have ranked colorectal cancer (CRC) as the fourth most commonly diagnosed cancer and associated with high mortality. Colorectal carcinogenesis represents a heterogeneous process with a differing set of somatic molecular alterations, influenced by diet, environmental, host immunity and microbiota. It is now known that the cancer-associated microbiota influence cancer development and progression with multiple mechanisms, that mainly involve the interactions between host factors (fat-rich diet, obesity, etc.), oxidative stress, genotoxicity, antigen-presenting cells of either dendritic cells or macrophages, and T cell-mediated adaptive immune responses. Metagenomics analyses have revealed an enrichment of Fusobacterium nucleatum (Fn) in human colon cancers and adenomas relative to non-cancerous colon tissues establishing direct relationships between the presence of this bacteria and increased CRC risk. There are many roles that have been associated to Fn in CRC. Studies have shown that a greater amount of Fn in colorectal carcinoma tissue is associated with high degrees of microsatellite instability (MSI-high) and CpG island methylator phenotype (CIMP). Fn has immunosuppressive activities via inhibiting human T-cell responses to mitogens and antigens. Additionally, Fn inhibitory protein has been shown to arrest human T-cells in the G1 phase of the cell cycle. Furthermore, Fn can induce apoptotic cell death in peripheral blood mononuclear cells and Jurkat T-cells. This Fn-induced cell death is mediated through the aggregation of the immune cells, which might have important implications for the pathogenesis of this bacterial species. Fn infection reduced the chemosensitivity of CRC cells to 5-Fu through upregulation of BIRC3 in vitro and in vivo, and high Fn abundance correlates with chemoresistance in advanced CRC patients who received standard 5-Fu-based adjuvant chemotherapy after radical surgery. This evidence suggests that Fn and BIRC3 may serve as promising therapeutic targets for reducing chemoresistance to 5-Fu treatment in advanced CRC.

Different studies have tried to define the associated between Fn and the worse clinical outcome in CRC. The biggest of these studies, published in the 2017 (Mima et all), analysing more than 1000 patients with CRC, showed that the presence of Fn DNA in CRC tissue was positively associated with colorectal cancer-specific mortality.

In locally advanced rectal cancer (LARC), it has been shown that the presence and abundance of Fn in post-treatment, surgically resected tumors correlates with the risk of relapse after radiation therapy and fluoropyrimidine-based concurrent neoadjuvant therapy (nCRT).

Despite of these findings, we still lack good prognostic and predictive biomarkers in CRC, and limited evidences on the utility of Fn, and microbiota in general, as biomarkers are available. Therefore, further methods and biomarkers are required which allow to predict the progression of cancers in patients.

Summary of the Invention

The inventors have discovered that the microbiota signature of cancer can be used to predict the progression of cancers. Therefore, a first aspect of the invention relates to a method for predicting the progression of cancer in a patient, comprising the steps of:

(i) determining in a sample of said patient the content of at least one type of microorganism belonging to a Fusobacterium cluster-associated microbiota, wherein said cluster-associated microbiota is formed by microorganisms of the Fusobacterium genus, the Porphyromonas genus, the Parvimonas genus, the Campylobacter genus and the Gemella genus and/or the content of at least one type of microorganism belonging to the Enterobacteriaceae family, to the Anaerostipes genus, to the Ruminococcus genus, to the Negativibacillus genus, to the Blautia genus and the Clostridium genus and

(ii) comparing said content to a reference value, wherein an increased content in said at least one type of microorganisms belonging to the Fusobacterium cluster-associated microbiota with respect to the reference value and/or wherein a decreased content of at least one type of microorganism belonging to the Enterobacteriaceae family, to the Anaerostipes genus, to the Ruminococcus genus, to the Negativibacillus genus, to the Blautia genus or the Clostridium genus is indicative of an increased risk of progression of the cancer in said patient.

Another aspect of the present invention relates to a method for selecting a personalized therapy for a patient suffering from cancer, wherein the method comprises determining the risk of progression of the cancer by a method according to the invention and selecting a therapy adequate for the treatment of cancer in those patients in which the cancer is predicted to progress.

One more aspect of the present invention relates to a method for preventing the progression of cancer of a patient or for preventing the relapse of cancer in patient, said method comprising determining the risk of progression of the cancer by a method according to the invention and treating the patient with a therapy adequate for the cancer which is predicted to progress or to relapse

Brief description of the Figures

Figure 1 . A. Heatmap of the relative abundance of the Fusobacterium genus among all the sample based on the 16S rRNA gene sequencing results. B. Correlation between the Fusobacterium RNA-ISH and the 16S rRNA gene sequencing.

Figure 2. Diversity analyses according to intratumoral Fusobacterium relative abundance determined by 16S rRNA gene sequencing. A. Alpha diversity analysis represented by rarefaction plots with the corresponding p value table, B. Visualization of microbial communities’ structure (Beta diversity analysis) by PCoA plots.

Figure 3. Linear discriminant analysis (LDA) effect size (LEfSe)(Kruskal-Wallis test and LDA=2) of differentially abundant taxa between Fusobacterium HIGH vs NEG (A) and between Fusobacterium LOW vs NEG (B) tumors.

Figure 4. Diversity analyses between RELAPSE and NO RELAPSE tumors. A. Alpha diversity analysis represented by rarefaction plots with the corresponding p value table, B. Visualization of microbial communities’ structure (Beta diversity analysis) by PCoA plots.

Figure 5. Linear discriminant analysis (LDA) effect size (LEfSe)(Kruskal-Wallis test and LDA=2) of differentially abundant taxa between tumors with (YES) and without (NO) RELAPSE.

Figure 6. Meta-analysis of differential abundance of genera enriched (Fuso group=YES) or depleted (Fuso group=NO) with Fusobacterium in tissue and stool samples of the different CRC datasets. A) Differential abundance in tumor tissue samples as compared to normal tissue controls. B) Differential abundance in stool samples from cancer patients as compared to healthy controls.

Figure 7. Differential abundance analysis of taxa enriched (Fuso group=yes) or depleted (Fuso group=no) with Fusobacterium in STOOL samples from different cancer patient cohorts as compared to normal controls. A - B) Represents the two pancreatic cancer cohorts. C) Represents the gastric cancer cohort. D) Represents the prostate cancer cohort. E) Represents the ovarian cancer cohort. F) Represents the breast cancer cohort. G-H) Represents the two melanoma cancer cohorts.

Figure 8. Differential abundance analysis of genera enriched (Fuso group=yes) or depleted (Fuso group=no) with Fusobacterium in TISSUE samples from different cancer patient cohorts as compared to normal controls. A) Represents the gastric cancer cohort. B) Represents the breast cancer cohort. C) Represents the prostate cancer cohort. D) Represents the oral cancer cohort.

Detailed Description of the Invention

The inventors of the present invention have been able to classify tumours on the basis of the presence or absence of bacteria belonging to different genus or species, creating a microbiota signature for a cancer which is indicative of poor prognosis and disease progression in a cancer patient.

As such, a first aspect of the present invention relates to a method for predicting the progression of cancer in a patient, from here onwards the method of the invention, comprising the steps of:

(ii) comparing said content to a reference value, wherein an increased content in said at least one type of microorganisms belonging to the Fusobacterium cluster-associated microbiota with respect to the reference value and/or wherein a decreased content of at least one type of microorganism belonging to the Enterobacteriaceae family, to the Anaerostipes genus, to the Ruminococcus genus, to the Negativibacillus genus, to the Blautia genus or the Clostridium genus is indicative of an increased risk of progression of the cancer in said patient. As used herein, the expression "predicting the progression of cancer in a patient" refers to the likelihood that said cancer will develop into the later stages of disease in relation to the stage at which the method of the invention was performed in the patient, said patient having been previously diagnosed with said cancer. As will be understood by those skilled in the art, the prediction, although preferred to be, need not be correct for 100% of the subjects to be diagnosed or evaluated. The term, however, requires that a statistically significant portion of subjects can be identified as having an increased probability of having a given outcome. Whether a subject is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney test, etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90% at least 95%. The p-values are, preferably 0.05, 0.02, 0.01 or lower.

The term “cancer” refers to a group of diseases involving abnormal, uncontrolled cell growth and proliferation (neoplasia) with the potential to invade or spread (metastasize) to other tissues, organs or, in general, distant parts of the organism; metastasis is one of the hallmarks of the malignancy of cancer and cancerous tumours. The abnormal growth and/or proliferation of cancerous cells is the result of a combination of genetic and environmental factors that alter their normal physiology. The growth and/or proliferation abnormalities of cancerous cells result in physiological disorders and, in many cases, death of the individual, due to the dysfunctionality or loss of functionality of the cell types, tissues and organs affected.

The term “cancer” includes, but is not restricted to, cancer of the breast, heart, small intestine, colon, spleen, kidney, bladder, head, neck, ovaries, prostate gland, brain, pancreas, skin, bone, bone marrow, blood, thymus, womb, testicles, hepatobiliary system and liver; in addition to tumours such as, but not limited to, adenoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, haemangioendothelioma, hemangiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, hepatobiliary cancer, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma and teratoma. Furthermore, this term includes acrolentiginous melanoma, actinic keratosis adenocarcinoma, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamus carcinoma, astrocytic tumors, Bartholin gland carcinoma, basal cell carcinoma, bronchial gland carcinoma, capillary carcinoid, carcinoma, carcinosarcoma, cholangiocarcinoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, Ewing sarcoma, focal nodular hyperplasia, germ cell tumors, glioblastoma, glucagonoma, hemangioblastoma, hemagioendothelioma, hemagioma, hepatic adenoma, hepatic adenomastosis, hepatocellular carcinoma, hepatobilliary cancer, insulinoma, intraepithelial neoplasia, squamous cell intraepithelial neoplasia, invasive squamous-cell carcinoma, large cell carcinoma, leiomyosarcoma, melanoma, malignant melonoma, malignant mesothelial tumor, medulobastoma, medulloepithelioma, mucoepidermoid carcinoma, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, papillary serous adenocarcinoma, pituitary tumors, plasmacytoma, pseudosarcoma, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, microcytic carcinoma, soft tissue carcinoma, somatostatin secreting tumor, squamous carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vipoma, Wilm tumor, intracerebral cancer, head and neck cancer, rectal cancer, astrocytoma, glioblastoma, microcytic cancer and non-microcytic cancer, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer and breast cancer. In a particular embodiment of the method of the invention the cancer is oral cancer, melanoma, prostate cancer, breast cancer, pancreas cancer, stomach cancer, or ovary cancer.

In a particular embodiment of the method of the invention the cancer is colorectal cancer. The term "colorectal cancer" includes cancer of the colon, rectum, and/or anus, and especially, adenocarcinomas, and may also include carcinomas (e.g., squamous cloacogenic carcinomas), melanomas, lymphomas, and sarcomas. Epidermoid (nonkeratihizing squamous cell or basaloid) carcinomas are also included. The cancer may be associated with particular types of polyps or other lesions, for example, tubular adenomas, tubulovillous adenomas (e.g., villoglandular polyps), villous (e.g., papillary) adenomas (with or without adenocarcinoma), hyperplastic polyps, hamartomas, juvenile polyps, polypoid carcinomas, pseudopolyps, lipomas, or leiomyomas. The cancer may be associated with familial polyposis and related conditions such as Gardner's syndrome or Peutz-Jeghers syndrome. The cancer may be associated, for example, with chronic fistulas, irradiated anal skin, leukoplakia, lymphogranuloma venereum, Bowen's disease (intraepithelial carcinoma), condyloma acuminatum, or human papillomavirus. The other aspects, the cancer may be associated with basal cell carcinoma, extramammary Paget's disease, cloacogenic carcinoma, or malignant melanoma. In a particular embodiment of the method of the invention the cancer is locally advanced rectal cancer (LARC). The term “locally advanced rectal cancer” or its acronym “LARC”, as used herein refers to rectal tumors that by clinical or radiological assessment grow through the rectal wall to an extent that complete removal by surgery alone is considered impossible. Using TNM classification, a cancer staging system for the classification of malignant tumours, the extent of cancer in a patient's body is described. LARC is typically staged as a T3 or T4 tumor, and involves the mesorectal compartment or infiltrates adjacent pelvic structures respectively.

In the context of the present invention the term “patient” refers to a person or an individual of any gender, race or age, who has been diagnosed with cancer and is suffering from said ailment.

Step (i)

The first step of the method of the invention refers to the process of determining the presence and/or abundance of the microorganisms in a sample of the patient.

The term “sample” in the context of the present invention, refers to a small quantity or isolated part which is representative of the whole, i.e., whose characteristics are identical to the whole from which the sample is taken. In a particular embodiment of the method of the invention the sample is a biological fluid or a tissue sample. The term “biological fluid” as used herein refers to any liquid or semi-solid which can be isolated from a subject. In a particular embodiment the biological fluid is selected from a group consisting of sputum, oropharyngeal washings, bronchoalveolar washings, blood, pleural fluid, amniotic fluid, plasma, feces, stools and any combination thereof. In a particular embodiment of the method of the invention the sample the sample is a stool sample. The term “tissue sample” in the context of the present invention refers to any tissue which is representative of the cancer with which the patient was diagnosed. In a particular embodiment of the method of the invention the sample is a tissue sample, preferably a tumor sample. The term "tumor" as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The term “tumor” is not limited to any stage, grade, histomorphological feature, invasiveness, aggressivity or malignancy of an affected tissue or cell aggregation. In particular stage 0 cancer, stage I cancer, stage II cancer, stage III cancer, stage IV cancer, grade I cancer, grade II cancer, grade III cancer, malignant cancer, primary carcinomas, and all other types of cancers, malignancies etc. are included. The term “tumor sample” as used herein, refers to a sample obtained from a patient. The tumor sample can be obtained from the patient by routine measures known to the person skilled in the art, i.e., biopsy taken by aspiration or punctuation, excision or by any other surgical method leading to biopsy or resected cellular material. In a particular embodiment of the method of the invention, the tumor sample is a fixed, wax-embedded tissue sample, such as a formalin-fixed, paraffin-embedded tissue sample. In term “tumor sample” is also meant to include samples obtained from a tumor where no surgical excision of the tumor has been performed. In a particular embodiment of the method of the invention the tumor sample is a sample from a tumor which has been surgically removed or is a sample from a tumor biopsy obtained without the surgical excision of the tumor.

The sample to be use in the method of the invention is obtained from a patient who, as previously state, is suffering from cancer, and wherein said patient may have already undergone one or more courses of treatment for said cancer. In a particular embodiment of the method of the invention the sample has been obtained after the patient has been treated with radiation therapy and/or with chemotherapy.

As used herein, the terms "treat", "treatment" and "treating" refer to the reduction or amelioration of the progression, severity and/or duration of a condition, disorder or disease, or the amelioration of one or more symptoms (preferably, one or more discernible symptoms) of a condition, disorder or disease. The terms "treat", "treatment" and "treating" also refer to the amelioration of at least one measurable physical parameter of a condition, disorder or disease not necessarily discernible by the patient. Furthermore, "treat", "treatment" and "treating" refer also to the inhibition of the progression of a condition, disorder or disease, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. "Treat", "treatment" and "treating" may refer, too, to the reduction or stabilization of the condition, disorder or disease. The term “therapy” refers to the treatments applied to prevent and/or treat a condition.

In the present context the terms “radiation therapy” or "radiotherapy" refer to the treatment of cancer cells with ionizing radiation in order to control or kill malignant cells. It is meant to include, for example, fractionated radiation therapy, non-fractionated radiation therapy and super-fractionated radiation therapy, as well as a combination of radiation and chemotherapy. The type of radiation may further include ionizing (y) radiation, particle radiation, low energy transfer (LET), high energy transfer (HET), X-ray radiation, UV radiation, infrared radiation, visible light, photosensitizing radiation, etc. Radiation therapy is very often combined with chemotherapy. The term “chemotherapy” as used herein refers to the treatment of cancer with anti-cancer agents, i.e., an agent that at least partially inhibits the development or progression of a cancer, including inhibiting in whole or in part symptoms associated with the cancer even if only for the short term. Several anticancer agents, without limitation, can be categorized as DNA damaging agents and these include topoisomerase inhibitors (e.g., etoposide, ramptothecin, topotecan, teniposide, mitoxantrone), DNA alkylating agents (e.g., cisplatin, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chorambucil, busulfan, thiotepa, carmustine, lomustine, carboplatin, dacarbazine, procarbazine), DNA strand break inducing agents (e.g., bleomycin, doxorubicin, daunorubicin, idarubicin, mitomycin C), anti-microtubule agents (e.g., vincristine, vinblastine), anti-metabolic agents (e.g., cytarabine, methotrexate, hydroxyurea, 5-fluorouracil, floxuridine, 6-thioguanine, 6-mercaptopurine, fludarabine, pentostatin, chlorodeoxyadenosine), anthracyclines, vinca alkaloids, or epipodophyllotoxins.

In a particular embodiment of the method of the invention the patient has been treated with neoadjuvant chemoradiotherapy.

The term “neoadjuvant therapy” as used herein refers to the administration of a therapeutic agent before/prior to the main treatment for the disease. The expression “neoadjuvant chemoradiotherapy” as used herein refers to the simultaneous treatment of cancer with short-course ionizing radiation (SORT), long-course chemoradiotherapy (LCCRT), or both (chemo)radiotherapy and systemic doses of chemotherapy (Total neoadjuvant Treatment or TNT) prior to the main treatment, which in the case of rectal cancer, is mostly surgical resection of the tumor. In a particular embodiment of the method of the invention the concurrent neoadjuvant chemoradiotheraphy is fluoropyrimidine-based concurrent neoadjuvant therapy.

The term "fluoropyrimidine" is used herein to refer to an antimetabolite chemotherapy, including, without limitation, capecitabine, floxuridine, and fluorouracil (5-Fll). In a particular embodiment of the method of the invention the fluoropyrimidine is selected from capecitabine, floxuridine and fluorouracil (5-Fll).

It will be understood by the expert in the art that independently of the type of sample used in the method of the invention, said sample allows to determine the content of at least one type of microorganism. The expression “content of at least one type of microorganism” as used herein refers to the amount, absolute or relative, of a specific microorganism class, family, genus, species or variety in relation to all the other microorganisms present in the microenvironment being sampled or the microbiome. The type of microorganism can be determined by any known technique to the expert in the field. The term “microbiome”, also known as “microbiota” as used herein refers to the entire microbial population associated with a site, organ or tissue.

As used herein, the term "microorganism" refers to cellular microorganisms, including bacteria, fungi, and archaea, and includes both individual organisms and populations including any number of organisms. In the context of the present description, the “at least one type of microorganism” refers to microorganisms which belong to a Fusobacterium cluster-associated microbiota and/or to the Enterobacteriaceae family, to the Anaerostipes genus, to the Ruminococcaceae genus, to the Negativibacillus genus, to the Blautia genus and the Clostridium genus.

The term “Fusobacterium" as used herein refers to a genus of anaerobic, Gramnegative, non-sporeforming bacteria belonging to Gracilicutes. Individual cells are slender, rod-shaped bacilli with pointed ends. Strains of Fusobacterium cause several human diseases, including periodontal diseases, Lemierre's syndrome, and topical skin ulcers. The Fusobacterium genus is composed of 23 species, namely F. animalis, F. canifelinum, F. equinum, F. gastrosuis, F. gonidiaformans, "F. hwasookii", "F. massiliense", F. mortiferum, F. navi forme, F. necrogenes, F. necrophorum, F. nucleatum, F. perfoetens, F. periodonticum, F. polymorphum, "F. pseudoperiodonticum", "Ca. F. pullicola", F. russii, F. simiae, F. ulcerans, F. varium, F. vincentii, and/or F. watanabei. In a particular embodiment of the method of the invention the species of the Fusobacterium genus are selected from Fusobacterium nucleatum and Fusobacterium necrophorum.

The expression “Fusobacterium cluster-associated microbiota” as used herein refers to microorganisms or groups or microorganisms which, belonging to another taxonomic classification, are closely associated with the presence of Fusobacterium. In the context of the present invention the Fusobacterium cluster-associated microbiota comprises microorganisms belonging to the Fusobacterium genus, the Porphyromonas genus, the Parvimonas genus, the Campylobacter genus and the Gemella genus.

The term “Porphyromonas genus” as used herein refers to a Gram-negative, non-spore- forming, obligatory anaerobic and non-motile genus from the family of Porphyromonadaceae. This genus has been found to be part of the salivary microbiome. In a particular embodiment of the method of the invention the microorganism of the Porphyromonas genus belongs to a species selected from P. asaccharolytica, P. bennonis, P. cangingivalis, P. canoris, P. catoniae, P. circumdentaria, P. crevioricanis, P. endodontalis, P. gingivalis, P. gingivicanis, P. gulae, P. levii, P. macacae, P. pasteri, P. pogonae, P. somerae and/or P. uenonis. In a particular embodiment of the method of the invention the microorganism of the Porphyromonas genus is P. gingivalis.

The term “Parvimonas" as used herein refers to a genus which contains only one species, namely, Parvimonas micra, characterized by being a Gram-positive anaerobic coccus which is frequently isolated from dental plaque in patients with chronic periodontitis. It is a common constituent of mixed anaerobic infections such as intra-abdominal abscess.

The term “Campylobacter genus” as used herein refers to Gram-negative bacteria, which typically appears comma or s-shaped and are motile. Some Campylobacter species can infect humans, sometimes causing campylobacteriosis, a diarrhea disease in humans. In a particular embodiment of the method of the invention the microorganism of the Campylobacter genus belongs to a species selected from a group consisting of: C. avium, C. butzleri, C. Canadensis, C. cinaedi, C. coli, C. concisus, C. corcagiensis, C. cryaerophilus, C. cuniculorum, C. curvus, C. fennelliae, C. fetus, C. gracilis, C. helveticus, C. hepaticus, C. hominis, C. hyoilei, C. hyointestinalis, C. insulaenigrae, C. jejuni, C. lanienae, C. lari, C. mucosalis, C. mustelae, C. nitrofigilis, C. peloridis, C. pylori, C. rectus, C. showae, C. sputorum, C. subantarcticus, C. upsaliensis, C. ureolyticus and/or C. volucris. In a particular embodiment of the method of the invention the microorganism of the Campylobacter genus is C. jejuni.

The term “Gemella genus” as used herein refers to Gram-positive bacteria that thrive best at high partial pressure of CO2. Gemella bacteria are primarily found in the mucous membranes of humans and other animals, particularly in the oral cavity and upper digestive tract. In a particular embodiment of the method of the invention the microorganism of the Gemella genus belongs to a species selected from a group consisting G. asaccharolytic, G. bergeri, G. cuniculi, G. haemolysans, G. morbillorum, G. palaticanis, G. parahaemolysans, G. sanguinis and/or G. taiwanensis. In a particular embodiment of the method of the invention the microorganism of the Gemella genus is G. morbillorum.

The term “Enterobacteriaceae family” as used herein refers to a large family of Gramnegative bacteria, wherein its members are characterized by being mostly bacilli (rod-shaped), and are typically 1-5 pm in length. Enterobacteriaceae typically appear as medium to largesized grey colonies on blood agar, although some can express pigments. Most have many flagella used for mobility, but a few genera are nonmotile. Most members of Enterobacteriaceae have peritrichous, type I fimbriae involved in the adhesion of the bacterial cells to their hosts. Enterobacteriaceae includes, along with many harmless symbionts, many of the more familiar pathogens, such as Salmonella, Escherichia coli, Klebsiella, and Shigella. Other disease-causing bacteria in this family include Enterobacter and Citrobacter. Members of the Enterobacteriaceae can be trivially referred to as enterobacteria or "enteric bacteria”, as several members live in the intestines of animals.

The term “Anaerostipes genus” as used herein refers to a Gram positive and anaerobic bacterial genus from the family of Lachnospiraceae. Anaerostipes occurs in the human gut and are further characterized by producing butyric acid. In a particular embodiment of the method of the invention the microorganism of the Anaerostipes genus belongs to a species selected from the group consisting of: A. butyraticus, A. caccae, A. hadrus and/or A. rhamnosivorans.

The term “Ruminococcus genus”, as used herein refers to a genus of bacteria in the class Clostridia, characterized by being anaerobic, Gram-positive gut microbes. One or more species in this genus are found in significant numbers in the human gut microbiota. In a particular embodiment of the method of the invention the microorganism of the Ruminococcus genus belongs to a species selected from a group consisting of: Ruminococcus albus, Ruminococcus bromii, Ruminococcus callidus and/or Ruminococcus flavefaciens.

The term “Negativibacillus genus” as used herein refers to a genus containing one unique species, Negativibacillus massiliensis, characterized by being a non-motile, sporeforming, Gram-stain negative, rod-shaped, strict anaerobic bacterium. In a particular embodiment of the method of the invention the microorganism of the Negativibacillus genus belongs to the species Negativibacillus massiliensis.

The term “Blautia genus” as used herein refers to anaerobic bacteria with probiotic characteristics that occur widely in the feces and intestines of mammals. In a particular embodiment of the method of the invention the microorganism of the Blautia genus belongs to the species selected from a group consisting of: B. acetigignens, B. ammoniilytica, B. argi, B. caecimuris, B. celeris, B. coccoides, B. faecicola, B. faecis, B. glucerasea, B. hansenii, B. hominis, B. hydrogenotrophica, B. intestinalis, B. liquoris, B. luti, B. obeum, B. product, B. schinkii, B. stercoris and/or B. wexlerae.

The term “Clostridium genus” as used herein refers to bacterium characterize by being an anaerobic, non-motile, gram-positive that reproduces by sporulation. This genus includes several significant human pathogens, including the causative agents of botulism and tetanus. In a particular embodiment of the method of the invention the microorganisms of the Clostridium genus are selected from a group consisting of: Clostridium botulinum, Clostridium innoccum, Clostridium perfringens, Clostridium tetani, Clostridium difficile, Clostridium perfringens, Clostridium ramnosum, Clostridium sordelii, Clostridium septicum, Clostridium tertiumm, Clostridium histolyticum, Clostridium paraputrific and Clostridium sordellii.

As will be of the understanding of the expert in the field that step i) of the method of the invention comprises the determination of the content at least one type of microorganism, but that two or more at possible. In a particular embodiment of the method of the invention step i) comprises the determining the content of two, three, four, five, six, seven, eight, nine or ten types of microorganisms, wherein each type belongs to the Fusobacterium cluster-associated microbiota and/or the Enterobacteriaceae family, the Anaerostipes genus, the Ruminococcaceae genus, the Negativibacillus genus, the Blautia genus and the Clostridium genus.

The determination of the content of a microorganism in a sample can be accomplished by several methods known to the expert in the field, such as, without limitation, 16S rRNA sequencing, quantitative PCR (qPCR), in situ hybridization and shotgun metagenome sequencing. In a particular embodiment of the method of the invention the content of at least one type of microorganism is determined by 16S rRNA sequencing, quantitative PCR (qPCR) and/or in situ hybridization.

The term "16S rRNA" or "16S" refers to sequence derived by characterizing the nucleotides that comprise the 16S ribosomal RNA gene(s). The bacterial 16S rDNA is approximately 1500 nucleotides in full length and can be referred to fragments ranging from a few nucleotides to full length of the 16S rDNA.16S rDNA is used in reconstructing the evolutionary relationships and sequence similarity of one bacterial isolate to another using phylogenetic approaches.16S sequences are used for phylogenetic reconstruction as they are in general highly conserved, but contain specific hypervariable regions that harbor sufficient nucleotide diversity to differentiate genera and species of most bacteria. The term "V1-V9 regions" of the 16S rRNA refers to the first through ninth hypervariable regions of the 16S rRNA gene that are used for genetic typing of bacterial samples. These regions in bacteria are defined by nucleotides 69-99, 137-242, 433-497, 576-682, 822-879, 986-1043, 1117- 1173, 1243-1294 and 1435-1465 respectively using numbering based on the E. coli system of nomenclature. As used herein, the term "16S rRNA sequencing" refers to the sequencing of 16S ribosomal RNA (rRNA) gene sequences by using primers such as universal primers and/or species-specific primers to identify the bacteria present in a sample. 16S rRNA genes contain both highly conserved sites and hypervariable regions that can provide species- specific signature sequences useful for identification of bacteria. Such universal primers are well known in the art.

In a particular embodiment of the method of the invention the content in the at least one type of microorganism is determined as the relative content of the 16S rRNA gene of the microorganism with respect to the total content of 16S RNA genes of the microorganisms present in the sample.

The expression “relative content of the 16S RNA gene” as used herein refers to the proportional amount of the number of nucleotide sequences, obtained by any method which leads to the production of repeated nucleotide sequences, which are attributed or inferred to belong to a determined 16S RNA gene of a certain bacteria genus or species in relation to all the nucleotide sequences which are attributed or inferred to belong to other 16S RNA genes which belong to other bacteria genus or species. In order to do such attribution or inferring, all the nucleotide sequences, which can be produced during the sequencing are compared and search in public databases, such as NCBI GenBank, which contain the nucleotide sequence for the 16S rRNA gene of most microorganisms. Secondary database that collects only 16S rRNA sequences are widely used as well. Examples of such databases are EzBioCloud, Ribosomal Database Project, SILVA, and GreenGenes.

In a particular embodiment of the method of the invention the relative content of the 16S RNA gene is determined by measuring the number of amplicons of the whole 16S RNA gene or a fragment thereof. In another particular embodiment of the method of the invention the relative content of the 16S RNA gene is determined by measuring the number of amplicons of a fragment of the 16S RNA gene, wherein said fragment has a length of at least 20 bp, at least 30 bp, at least 40 bp, at least 50 bp, at least 60 bp, at least 70 bp, at least 80 bp, at least 90 bp, at least 100 bp, at least 100 bp, at least 150 bp, at least 200 bp, at least 250 bp, at least 300 bp, at least 350 bp, at least 400 bp, at least 450 bp, at least 500 bp.

“Amplicons” as used herein refer to replicated DNA (or ribonucleic acid - RNA) strands that are formed by polymerase chain reaction (PCR), ligase chain reactions (LCR), or other DNA duplication methods, where the strands are copies of a target region of a genome Amplicons for sequencing have a length typically in the range between 100 bp and 500 bp. In a particular embodiment of the method of the invention the relative content of the 16S RNA gene is determined by measuring the number of amplicons from at least one region of the variable regions V1 , V2, V3, V4, V5, V6, V7, V8 and V9.

In a particular embodiment of the method of the invention the relative content of the 16S RNA gene is determined by measuring the number of amplicons from at least two regions of the variable regions selected from the group consisting of: V1-V2, V1-V3, V1-V4, V1-V5, VIVO, V1-V7, V1-V8, V1-V9, V2-V3, V2-V4, V2-V5, V2-V6, V2-V7, V2-V8, V2-V9, V3-V4, V3-V5, V3-V6, V3-V7, V3-V8, V3-V9, V4-V5, V4-V6, V4-V7, V4-V8, V4-V9, V5-V6, V5-V7, V5-V8, V5- V9, V6-V7, V6-V8, V6-V9, V7-V8, V7-V9 and V8-V9.

In a particular embodiment of the method of the invention the relative content of the 16S RNA gene is determined by measuring the number of amplicons from the V3-V4 regions in said genes.

The content of at least one type of microorganism can be determined by 16S rRNA sequencing as exemplified in the Example section.

In a particular embodiment of the method of the invention the content of at least one type of microorganism is determined by quantitative PCR (qPCR).

The term “qPCR”, “quantitative polymerase chain reaction”, “real-time polymerase chain reaction” or “real-time PCR” as used herein, indicates a polymerase chain reaction performed to monitor amplification of a target polynucleotide during the PCR (in real time). qPCR can be used to detect a target polynucleotide quantitatively (quantitative real-time PCR) or semi- quantitatively (above/below a certain amount of target polynucleotide) (semi-quantitative realtime PCR). Typically, qPCR monitoring is performed through use of non-specific fluorescent dyes that intercalate with any double-stranded DNA or sequence-specific polynucleotide probes consisting of oligonucleotides that are labelled with a fluorescent reporter, which permits detection only after hybridization of the probe with its complementary sequence. An example of the use of qPCR for the quantification of the content of at least one type of microorganisms is exemplified by Jian et al. (2020, PLOS ONE 15(1): e0227285).

In a particular embodiment of the method of the invention the content of at least one type of microorganism is determined by in situ hybridization.

The term "in situ hybridization" refers to specific binding of a nucleic acid to a target nucleic acid in its normal place in a sample, such as on metaphase or interphase chromosomes. The terms "hybridizing" and "binding" are used interchangeably to mean specific binding between a nucleic acid probe and its complementary sequence. In situ hybridization technique is mostly done with a fluorescent or chromogenic label in the in-situ hybridization probe such as a “fluorescence in situ hybridization” or “chromogenic in situ hybridization”. The term “fluorescence in situ hybridization (FISH)” refers to a cytogenetic technique developed by biomedical researchers in the early 1980s that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes/genomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence complementarity. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific RNA targets (mRNA, IncRNA and miRNA) in cells, circulating tumor cells, and tissue samples. DNA and RNA probes may be also labelled with a chromogen which can be visualized using a conventional optic microscope. Examples of the use of in situ hybridization for the visualization of selected bacteria within the tissue context are exemplified by Serna et al (2020, Annals of Oncology, (10):1366-1375) and Tomkovich et al (2019, J Clin lnvest.;129(4):1699-1712).

The term "chromogenic in situ hybridization" or "CISH" refers to a type of ISH with a chromogenic label. ISH, FISH and CISH methods are well known to those skilled in the art (see, for example, Stoler, Clinics in Laboratory Medicine 10(l):215-236 (1990); In situ hybridization. A practical approach, Wilkinson, ed., IRL Press, Oxford (1992); Schwarzacher and Heslop-Harrison, Practical in situ hybridization, BIOS Scientific Publishers Ltd, Oxford (2000)).

The determination of the content of a microorganism in a sample can also be accomplished by determining the present of products derived from said microorganisms, which are byproducts of theirs metabolism and cellular growth. In a particular embodiment of the method of the invention the content of at least one type of microorganism is determined by metabolomics, transcriptomics, lipidomics and/or proteomics.

The term “metabolomics” as used herein refers to the scientific study of chemical processes involving metabolites, the small molecule substrates, intermediates, and products of cell metabolism. Specifically, metabolomics is the "systematic study of the unique chemical fingerprints that specific cellular processes leave behind", the study of their small-molecule metabolite profiles. The metabolome represents the complete set of metabolites in a biological cell, tissue, organ, or organism, which are the end products of cellular processes. The several techniques which are used to study metabolics ultimately allow to determine the constituents of the microbiome which is the target of the study.

The term “transcriptomics” as used herein refers to the quantitative science that encompasses the assignment of a list of strings ("reads") to the object ("transcripts" in the genome), i.e., the assignment of RNAs to the genes from which they have originated.

The term “Lipidomics” as used herein refers to the large-scale study of pathways and networks of cellular lipids in biological systems. The word "lipidome" is used to describe the complete lipid profile within a cell, tissue, organism, or ecosystem and is a subset of the "metabolome".

The term “proteomics” as used herein refers to the large-scale study of proteins from cells, tissues, organisms, or ecosystems.

Step (ii) of the method of the invention relates to the comparison of the content of at least one type of microorganism to a reference value.

The term “reference value”, as used herein, relates to a predetermined criterion used as a reference for evaluating the values or data obtained from the samples collected from a subject. This “reference value” may also be referred as “cut-off value” or “threshold value”. The predetermined criteria used as a reference to evaluate the values or data obtained from samples collected from a subject. The reference value can be an absolute value, a relative value, a value that has an upper or lower limit, a range of values, a mean value, a median value, a mean value, or a value compared to a particular control or baseline value. In a preferred method of the invention the reference value is the content of the microorganisms belonging to a Fusobacterium cluster-associated microbiota, wherein said Fusobacterium cluster-associated microbiota is selected is formed by microorganisms of the Porphyromonas genus, the Parvimonas genus, the Campylobacter genus and the Gemella genus and/or the content of at least one type of microorganism belonging to the Enterobacteriaceae family, to the Anaerostipes genus, the Ruminococcus genus, to the Negativibacillus genus, to the Blautia genus and the Clostridium genus in a sample from a patient suffering from cancer and in which the cancer has not progressed.

The expression “cancer has not progressed” refers to the cancer not advancing in the normal progression of the disease, i.e., the cancer has not grown, got worse or metastasized. The reference value of the method of the invention can be determined in a similar way to the content of at least one microorganism as described above.

The method of the invention allows to predict the progression of cancer in a cancer patient if there is an increased content in said at least one type of microorganisms belonging to the Fusobacterium cluster-associated microbiota with respect to the reference value and/or wherein a decreased content of at least one type of microorganism belonging to the Enterobacteriaceae family, to the Anaerostipes genus, to the Ruminococcus genus, to the Negativibacillus genus, to the Blautia genus or the Clostridium genus is indicative of an increased risk of progression of the cancer in said patient.

The expression “increased content in a least one type of microorganism” as used herein refer to the rise, elevation or augment of a type of microorganism in a statistically significant amounts in relation to a reference value. It will be understood that an increased content in the microorganism with respect to a reference value can also be referred to as being “present” in the sample. In a particular embodiment of the method of the invention the increase in at least one type of microorganism is of at least 0.1%, at least 1 %, at least 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more than 100% increase in relation to the reference value. In another particular embodiment of the method of the invention the increase in at least one type of microorganism is of at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times or more than 10 times in relation to the reference value. In another particular embodiment of the method of the invention the increase in at least one type of microorganism is of at least a 10- fold increase, at least about 20-fold increase, at least about 50-fold increase, at least about 100-fold increase, at least about 1000-fold increase, or more than 1000-fold increase in relation to the reference value.

The expression “decrease content in a least one type of microorganism” as used herein refer to the reduction, lowering or diminution of a type of microorganism in a statistically significant amounts in relation to a reference value. It will be understood that a decreased content in the microorganism with respect to a reference value can also be referred to as being “absent” in the sample. In a particular embodiment of the method of the invention the decrease in at least one type of microorganism is of at least 0.1 %, at least 1%, at least 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more than 100% decrease in relation to the reference value. In another particular embodiment of the method of the invention the decrease in at least one type of microorganism is of at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times or more than 10 times in relation to the reference value. In another particular embodiment of the method of the invention the decrease in at least one type of microorganism is of at least a 10-fold decrease, at least about 20-fold decrease, at least about 50-fold decrease, at least about 100-fold decrease, at least about 1000-fold decrease, or more than 1000-fold decrease in relation to the reference value.

The increased content in said at least one type of microorganisms belonging to the Fusobacterium cluster-associated microbiota and/or the decreased content of at least one type of microorganism belonging to the Enterobacteriaceae family, to the Anaerostipes genus, to the Ruminococcus genus, to the Negativibacillus genus, to the Blautia genus or the Clostridium genus is indicative of an increased risk of progression of the cancer in said patient.

As used herein, the expression "increased risk of progression of the cancer" refers to a higher probability, statistically significant, of said cancer progressing to a more advanced stage of the disease in relation to the stage in which the cancer is currently at. As will be understood by those skilled in the art, the prediction, although preferred to be, need not be correct for 100% of the subjects to be evaluated. The term, however, requires that a statistically significant portion of subjects can be identified as having an increased probability of having a given outcome. Whether a subject is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney test, etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90% at least 95%. The p-values are, preferably 0.05, 0.02, 0.01 or lower.

In a particular embodiment of the method of the invention the risk of progression in said patient is increase by at least at least 0.1 %, at least 1 %, at least 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more than 100% increase. In another particular embodiment of the method of the invention the risk of progression in said patient is increase by at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times or more than 10 times. In another particular embodiment of the method of the invention the risk of progression in said patient is increase by at least a 10-fold increase, at least about 20-fold increase, at least about 50-fold increase, at least about 100-fold increase, at least about 1000-fold increase, or more than 1000-fold increase.

The risk of progression of the method of the invention can also be determined as the risk of relapse after neoadjuvant chemoradiotherapy.

In the present invention the term “risk of relapse” refers to the probability of a subject of redeveloping cancer after a disease-free period. In a particular embodiment of the method of the invention the risk of relapse in said patient is increase by at least at least 0.1 %, at least 1 %, at least 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more than 100% increase. In another particular embodiment of the method of the invention the risk of relapse in said patient is increase by at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times or more than 10 times. In another particular embodiment of the method of the invention the risk of relapse in said patient is increase by at least a 10-fold increase, at least about 20-fold increase, at least about 50-fold increase, at least about 100-fold increase, at least about 1000-fold increase, or more than 1000-fold increase.

In the present context the term “relapse” refers to the reappearance of a disease, i.e. cancer, after a period, normally said period being post-treatment, where it could not be detected. The reappearance of the disease or cancer, can be local, i.e. a “local relapse” wherein the cancer returns in a same local or place it first started, can be a “distant relapse” referring to the fact that the cancer can back in another part of the body, some distance from where it started.

In another particular embodiment of the method of the invention the relapse is local or distant relapse.

Further methods of the invention

The method of the invention finds use in other methods related to the treating or preventing cancer.

All previous definitions and embodiments relating to previous aspects are equally applicable to the aforementioned aspects and their embodiments.

Another aspect of the present invention relates to a method for selecting a personalized therapy for a patient suffering from cancer, from now onwards the method II of the invention, wherein the method comprises determining the risk of progression of the cancer by the method of the invention and selecting a therapy adequate for the treatment of cancer in those patients in which the cancer is predicted to progress.

A further aspect of the present invention relates to a method for preventing the progression of cancer of a patient or for preventing the relapse of cancer in patient, from now onwards the method III of the invention, said method comprising determining the risk of progression of the cancer by the method of the invention and treating the patient with a therapy adequate for the cancer which is predicted to progress or to relapse.

In the context of the present invention the term “personalized therapy” refers to the set of interventions for treatment of the cancer of the patient which is adapted to the genetic substratum of the patient, the molecular profile of the disease and the microorganism profile of the patient as determined by the method of the invention.

In a particular embodiment of the method II and of the method III of the invention the therapy adequate for the treatment of cancer is surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy and any combination thereof. In another particular embodiment of the method II and of the method III of the invention the therapy adequate for the treatment of cancer is antibiotics, prebiotics, probiotics, bacteria consortia, postbiotics, faecal microbiota transplant and any combination thereof, wherein the therapy is designed to decrease content in a least one type of the microorganisms of the Fusobacterium cluster-associated microbiota or to the increased content in a least one type of beneficial microorganism. In a particular embodiment of the method II or the method II of the invention the decrease in at least one type of microorganisms of the Fusobacterium cluster-associated microbiota is of at least 0.1 %, at least 1%, at least 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more than 100% decrease in relation to a reference value obtained before the therapy. In a particular embodiment of the method II or method III of the invention the increase in at least one type of beneficial microorganism is of at least 0.1%, at least 1 %, at least 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more than 100% increase in relation to a reference value obtained before the therapy.

The term "targeted therapy", as used herein, relates to the application to a patient of a chemical substance known to block growth of cancer cells by interfering with specific molecules known to be necessary for tumorigenesis or cancer or cancer cell growth. The term "immunotherapy" as used herein relates to the treatment of cancer by modulation of the immune response of a subject. Said modulation may be inducing, enhancing, or suppressing said immune response, e.g. by administration of at least one cytokine, and/or of at least one antibody specifically recognizing cancer cells.

As used herein, the term “antibiotic” is defined as a compound having either a bactericidal or bacteriostatic effect upon bacteria contacted by the compound, in specific a bactericidal or bacteriostatic effect upon the Fusobacterium cluster-associated microbiota. As used herein, the term “bactericidal” is defined to mean having a destructive killing action upon bacteria. As used herein, the term “bacteriostatic” is defined to mean having an inhibiting action upon the growth of bacteria. Non-limiting exemplary antibiotics include those classified as aminoglycosides, beta-lactams, quinolones or fluoroquinolones, macrolides, sulfonamides, sulfamethaxozoles, tetracyclines, streptogramins, oxazolidinones (such as linezolid), clindamycins, lincomycins, rifamycins, glycopeptides, polymxins, lipo-peptide antibiotics. The term also includes antimicrobial agents isolated from natural sources or chemically synthesized. The term “antibiotic” also refers to antimicrobial agents for use in human therapy. Exemplary antibiotics include: tetracyclines, fluoroquinolones, chloramphenicol, penicillins, cephalosporins, puromycin, nalidixic acid, and rifampin. Additional specific examples of antibiotics include clindamycin, carbenicillin, cefoperazone, cefamandole, sulfonamides, quinolones, oxazolidinones, carbapenems, aminoglycosides, erythromycin, tetracycline and sulbactam. The term also includes pharmacologically acceptable sodium salts, pharmacologically acceptable calcium salts, pharmacologically acceptable potassium salts, lipid formulations, derivatives and/or analogs of the above.

The term “probiotic” as used herein refers to a microorganism or group of microorganisms that provides health benefits, or that is non-pathogenic, to a subject when consumed, ingested, or otherwise administered to a subject, for example, a reduction in the likelihood of relapse following cancer treatment. As used herein, the term probiotic can be used to describe, for example, probiotic bacteria and can include the bacteria described herein as well as other bacteria.

The term “prebiotic” in the present context refers to a substance that promotes the growth, proliferation and/or survival of one or more bacteria or yeast. As used herein, the term prebiotic can be used to describe, for example, a nutritional supplement including plant fiber, or one or more of poorly-absorbed complex carbohydrates, oligosaccharides, inulin-type fructans or arabinoxylans. In the context of the present invention the term “postbiotic” refers to a substance derived from a probiotic organism. As used herein, the term postbiotic can be used to describe, for example, a protein expressed by one or more bacteria, a metabolic product of one or more bacteria, or media from a culture of one or more strains of bacteria.

The term “bacteria consortia” or “microbial consortium” or “microbial community” are used interchangeably herein and refer to two or more bacterial or microbial groups living symbiotically, wherein at least one of said microorganisms are a probiotic, or the community is itself probiotic or leads to the production of postbiotics.

The term “faecal microbiota transplant”, “FMT”, also known as a “stool transplant”, is the process of transferring fecal bacteria and other microbes from a healthy individual into another individual.

The expression “reduce or kill the microorganisms of the Fusobacterium cluster- associated microbiota” as used herein refers to the process of lowering the relative or absolute number of microorganisms detected by the method of the invention

The term “beneficial microorganism” as used herein refers to a probiotic.

In a particular embodiment of the method II and of the method III of the invention the chemotherapy is adjuvant chemotherapy.

The term "adjuvant chemotherapy" in the present context refers to chemotherapy that is administered after a clinical intervention. Depending on the specific clinical scenario, adjuvant chemotherapy, is usually administered starting about four weeks after surgery.

As used herein, the term "prevent/preventing/prevention" refers to avoiding or delaying the manifestation of one or more symptoms or measurable markers of a disease or disorder, in the present case the cancer. The delay in the presentation of symptoms or markers is a delay in the time to manifest such symptoms or markers in an untreated subject relative to a control or a similar likelihood or susceptibility to develop the disease or disorder. Relative to a control or a symptom or marker that occurs in an untreated subject having a similar likelihood or susceptibility to progress the disease or disorder, or relative to a population based on the disease or disorder. The term "prevention" in terms of histological or statistical measures of possible symptoms or markers includes not only the complete avoidance or prevention of symptoms or markers, but also the reduced severity of any of these symptoms or markers. Or degree. By "reduced severity" is meant reducing the severity or extent of a symptom or measurable disease marker by at least 10% relative to a control or reference, for example, at least 15%, 20%, 30%, 40%, 50% 60%, 70%, 80%, 90%, 95%, 99% or even 100% (i.e. no symptoms or measurable markers).

In another particular embodiment of the method II and of the method III of the invention the relapse is local or distant relapse.

***

The invention will be described by way of the following examples, which are to be considered as merely illustrative and not limitative of the scope of the invention.

Examples

Example 1

METHODOLOGY

Study cohorts

The study cohort comprises 40 non-consecutive patients who were diagnosed with rectal cancer at Vail d’Hebron University Hospital between 2007 and 2018 and with available fresh frozen tissue of the surgically resected primary tumor for omics analysis. This cohort represents a subgroup of patients included in our recently published analysis with available Fusobacterium status by RNA-in situ hybridization, immune microenvironment, clinicopathologic, response, and follow up data (G. Serna et al, Ann Oncol. 2020, 10:1366- 1375). The treated group included patients with locally advanced rectal adenocarcinomas (LARC) who received neoadjuvant chemoradiotherapy (nCRT). The untreated group included patients with stages I to III rectal cancer who did not receive preoperative treatment given the tumor stage at diagnosis (stage I, low risk stage II upper rectal cancer), emergency surgery, or ineligibility for radiotherapy treatment (i.e. previous pelvic radiotherapy for prostate or cervix neoplasms). Relapse status was available for patients of the treated subgroup.

16S rRNA gene amplicon sequencing

The composition and structure of the tumor tissue microbial communities was assessed through bacterial DNA extraction, amplification and sequencing of the V3-V4 variable regions of the 16S rRNA gene. The Illumina Miseq sequencing 300x2 approach was used. Amplification was performed after 25 PCR cycles. A negative control of the DNA extraction was included as well as a positive Mock Community control to ensure quality control. Once the desired amplicon is confirmed in 1.5 % agarose gel electrophoresis, samples were stored at -20° C until sequencing library preparation. Illumina sequencing adapters and dual indices were attached using Nextera XT Index Kit (Illumina, Inc.) followed by the corresponding PCR amplification program as described in the MiSeq 16S rRNA Amplicon Sequencing protocol. Sequencing was performed on an Illumina MiSeqTM platform (Illumina, Inc.) according to the manufacturer's specifications to generate paired end reads of 300 bp length in each direction. The quality of MiSeq raw sequences was assessed using the FastQC software (http://www.Bioinformatics.Babraham.Ac.Uk/Projects/Fastqc/). Raw demultiplexed forward and reverse reads were processed using DADA2 R package (Callahan et al 2016), that included primer trimming, quality filtering, denoising, pair-end-merging and phylotype calling. Taxonomic assignment of phylotypes was performed using a Bayesian Classifier trained with Silva database version 132 (99% OTUs full-length sequences) (Wang et al. 2007).

Bioinformatics analysis

The bioinformatic analysis allowed us to generate a prediction of the taxonomy at the phylum, family and gender level using the reference dataset SILVA database version 132. To analyze the microbial diversity between the samples an alpha and beta diversity were used. Alpha diversity allowed us to analyze microbial diversity within the sample. In detail, to evaluate the biodiversity of the samples, alpha diversity was calculated using the Eveness index that given as the Pielou's evenness index, quantifies how equal the community is numerically, it considers the number and the abundance of phylotypes in a community. To evaluate the differences in bacterial composition between the different samples, beta diversity calculated using the Bray-Curtis metric (phylogenetic qualitative measure) was analyzed and represented through a PCoA (Principal Coordinator Analysis) 2D. In PCoA, each dot represents one sample, distances between dots represents the ecological distances between samples.

Statistical analysis

Samples were stratified in three different experimental groups according to Fusobacterium relative abundance by 16S rRNA (16S) gene sequencing (NEG, <3%; LOW, 3-10%; HIGH, >10%), treatment received (treated vs untreated), and outcome (relapse vs no relapse). Comparisons between groups were performed using the Wilcoxon-Mann-Whitney non-parametric test (for two groups comparison) and the Kruskal-Wallis non-parametric test (for three groups comparison). Beta diversity distance matrices was used to calculate principal coordinates analysis (PCoA) and to make ordination plots using R software package version 3.6.0. The significance of groups present in community structure was tested using Permanova tests. Significant thresholds were set at 0.05. Differential relative abundances of taxa between groups were tested using both by Linear discriminant analysis (LDA) effect size (LEfse) analysis to estimate the effect size of each differentially abundant feature (LDA score of 2) and by Wilcoxon-Mann-Whitney non-parametric test (significant threshold p value of 0.05).

RESULTS

Data quality check.

Negative controls were analyzed to detect contaminant amplicons. Almost every contaminant amplicon was either absent in the samples or was over two orders of magnitude less abundant than in the control. Twelve (12) samples were excluded from the analysis: 7 due to the low number of reads (cut-off of 931) and 5 due to histological criteria (3 samples were adenoma while 2 were normal tissue). After quality check, a total of 28 samples were used for the analysis (13 from the treated cohort and 15 from the untreated one). Follow up was available for all 13 patients of the treated cohort: 5 patients experienced a relapse whereas 8 patients did not.

Identification of Fusobacteri um-associated taxa

We first sought to identify the most common Fusobacterium species in rectal cancer. Fusobacterium nucleatum was the predominant species of the genus Fusobacterium observed followed by Fusobacterium necrophorum w ic appears to be enriched in 3 samples which had no concentration of Fusobacterium nucleatum (Figure 1A). To identify taxa associated with Fusobacterium, tumors were divided into 3 groups according to Fusobacterium relative abundance by 16S rRNA gene: NEGATIVE (<3%), LOW (3-10%), and HIGH (>10%). When we compared relative abundance with Fusobacterium levels by in situ hybridization performed on paired formalin fixed and paraffin embedded (FFPE) tumor samples, we found a good correlation between techniques with 30% (3 out 10), 54% (7 out of 13) and 83% (5 out of 6) of negative, low, and high samples by 16S classified as positive by RNA ISH, respectively (Figure 1 B).

The alpha diversity analysis showed significant differences between the Fusobacterium HIGH vs NEG (p value= 0.03) and LOW vs NEG (p value= 0.01) tumors as defined by 16S gene relative abundance, with no significant differences found between the Fusobacterium HIGH and Fusobacterium LOW subgroups (p value= 0.93) (Figure 2A). The beta diversity analysis showed significant differences between the three experimental subgroups (p value = 0.001) demonstrating different ecological compositions of the tumor microbiota according to the presence or absence of Fusobacterium (Figure 2B).

Linear discriminant analysis (LDA) was performed to identify differentially abundant taxas between Fusobacterium status subgroups (Figure 3). Anaerococcus, Gemella, Peptostreptococcus, Parvimonas and Fusobacterium were shared between the Fusobacterium HIGH and LOW subgroups and differentially expressed in Fusobacterium- positive tumors (HIGH and LOW) when compared with the Fusobacterium-negative tumors. Importantly, 4 out 5 shared taxas, which are Gemella, Peptostreptococcus, Parvimonas and Fusobacterium itself, were previously reported to be frequently associated with Fusobacterium and enriched in the Fusobacterium cluster as compared to Bacteroides cluster (Dohlman et al) in ORC, thus confirming a) the methodological validity of our analysis and b) a similar and robust tumor-associated microbiota between rectal cancer and colorectal cancer in general.

Bacteria predictive of relapse after nCRT in LARC.

In our previous work (Serna el al, Annals of Oncology 2020) we showed that the persistence of Fusobacterium by RNA-ISH in surgical samples was a negative prognostic biomarker in LARC after nCRT. To investigate whether other bacteria were prognostic, we compared the microbiota of tumors from the treated cohort which relapsed or were disease- free after nCRT. Alpha diversity analysis showed a non-significant reduced diversity in tumors from patients that relapsed as compared with those who did not (p=0.17, Figure 4A). Beta diversity analysis showed that samples from patients with relapse clustered together and differently from those who did not suggesting a different ecological compositions of the tumor microbiota according to the presence or absence of relapse (p=0.024, Figure 4B).

The LDA confirmed the enrichment of Fusobacterium in tumors that relapsed and identified enrichment of other previously undescribed species such as Porphyromonas, Parvimonas, Campilobacter and Gemella. Species that were depleted in tumors that relapsed were Enterobacteriaceae, Anaerostipes, Ruminococcus, and Negativibacillus (Figure 5).

A Wilcoxon-Mann-Whitney non-parametric test was also performed to identify differentially abundant taxas between tumors with or without relapse (Table 1). This analysis confirmed the results of the LDA, showing that Fusobacterium, Porphyromonas, Parvimonas, Campilobacter and Gemella were enriched and Ruminococcus , anaerostipes, Blautia, and innocuum_group were depleted in tumors from patients who relapsed, indicating that maybe these bacteria (alone or in combination) can be used as predictors of outcome in LARC after nCRT. Tablel : Wilcoxon statistical test. Differentially abundant taxa are shown, (p value<0.05).

Interestingly, 3 out of 5 bacteria found enriched in tumors with worse prognosis were also differentially abundant in Fusobacterium-pos ive compared to Fusobacterium-negative tumors in our study and all 5 relapse-associated taxas were previously reported to be enriched in Fusobacterium-positive CRC (Dohlman et al.). These findings suggest that Fusobacterium is part of a relatively stable community of CRC tumor-associated bacteria which presence/persistence in the tumor microenvironment after nCRT is predictive of relapse. This community of bacteria used alone or in combination, together with other bacteria depleted in tumors which relapsed, may be used as biomarker providing prognostic information and guide treatment decision. To note, not all the bacteria of the Fusobacterium cluster described by Dohlman et al (which include also Solobacterium, Peptostreptococcus, Alloprevotella, Leptotrichia, Prevotella, and Dialister) were found to be predictive of relapse in our study, suggesting that the development of a prognostic molecular test must take into consideration only those taxas found to be linked with outcome.

Example 2

MATERIAL AND METHODS

Study cohorts

To determine the prevalence of the microbiota signature in colorectal cancer (CRC) and other tumor types, various published cohorts were considered for an in-silico analysis. Table 2 provides an overview of different CRC cohorts, while Table 3 summarizes cohorts related to other tumor types. These cohorts encompassed tissue data, comprising both adjacent and tumoral tissues, or exclusively tumoral tissues, often with longitudinal data. Some cohorts included paired tissue and stool data, while others exclusively featured stool data, comparing samples from cancer patients to those from healthy individuals. The fecal samples employed as controls were sourced from cohorts within the same country and, whenever possible, featuring amplification of the same 16S region to ensure the consistency of results.

Table 2: Overview of the different CRC cohorts included in the in-silico analysis. Different characteristics including sample type, number of samples, 16S region amplified and country are described.

Table 3: Summary of other tumor type cohorts included in the in-silico analysis. Different characteristics including sample type, number of samples, 16S region amplified and country are described.

Bioinformatics analysis

The bioinformatic analysis was assessed with the nfcore/ampliseq pipeline v2.6.1 (ampliseq). The pipeline was executed using Nextflow v 21.10.6 and singularity.

Nfcore performs by default different steps that comprehend: 1. The quality control analysis of raw sequences (assess using the FastQC a single sample quality control, and MultiQC ; 2. The primerand adapters trimming (process using Cutadapt); 3. The raw demultiplexed forward and reverse reads (processed using DADA2), that include quality filtering and trimming, denoising and pairend-merging; 4. DADA2 infers exact amplicon sequence variants (ASVs) from highthroughput amplicon sequencing data, replacing the coarser and less accurate OTU clustering approach; 5. Predict whether ASVs are ribosomal RNA sequences with Barrnap; 6. Taxonomic classification (available via a native implementation of the RDP naive Bayesian classifier, and species-level assignment to 16S fragments by exact matching and classify using a reference taxonomies database SILVA v138 (99 % OTUs full-length sequences).

Statistical analysis

To study the differential abundance of our bacterial cluster, we opted to enhance our analysis by calculating the generalized fold change (gFC) and assessing the variance in average fold changes. The gFC is determined by computing the mean of the log 10 values within the quartile range of 0.1 to 0.9 (0.1 , 0.15, 0.20,..., 0.85, 0.9), utilizing relative abundance data across all cohorts. For paired samples, we compute the difference for each quantile, subsequently deriving the mean of these differences. In both scenarios, a t-test was employed to compare either the resulting means of each group or the differences to the population mean. The gFC is visually represented through a forest plot, accompanied by the depiction of 95% confidence intervals. Meta-analysis is conducted using surveomp, incorporating both the logFC and the confidence intervals.

RESULTS

CRC cohorts We conducted a comprehensive metanalysis of publicly available metagenomic datasets of stool and tissue (tumor and adjacent control tissue) specimens from CRC studies to explore whether the enrichment of the Fusobacterium cluster signature could be found in independent CRC cohorts. The metanalysis conducted on tissue samples, comprising five cohorts, revealed a significant enrichment of many genera of the Fusobacterium cluster in cancers as compared to normal tissue controls, including Fusobacterium, Parvimonas, Gemella, Campylobacter and Peptostreptococcus (Figure 6A). The metanalysis of stool samples across three distinct CRC datasets also showed increased abundance of many genera of the cluster in stool from CRC patients as compared to healthy controls which was statistically significant for Fusobacterium, Parvimonas, Prevotella, Porphyromonas, and Peptostreptococcus (Figure 6B). Healthy-associated genera inversely correlated with Fusobacterium showed a trend towards depletion in CRC versus controls which was significant for Blautia, Ruminococcus torques group, Negativibacillus, and Anaerostipes in tumor tissue as compared to normal adjacent mucosa. Interestingly, the pattern identified in the tissue samples analysis aligns with the findings from the stool samples, suggesting that the analysis of the cluster may be conducted in both specimen types.

Other tumor type cohorts

To assess the specificity of the Fusobacterium and its associated microbiota for CRC, we conducted a comprehensive examination of its presence across diverse cohorts from different primary origin (oral, melanoma, prostate, breast, pancreas, stomach, and ovary) utilizing publicly available datasets (Table 3).

Stool data analysis showed a variable and consistent enrichment of several genera of the Fusobacterium cluster in the different tumor types analysed. Remarkably, almost all genera within the Fusobacterium cluster were increased in stool datasets from the pancreatic cancer cohorts (Figure 7A-B). Consistent with this finding in pancreatic cancer, the analysis of the stool datasets from gastric, prostate, and ovarian cancer cohorts unveiled a similar enrichment of Fusobacterium and its associated microbiota in patients compared to control subjects (Figure 7C-E). From the breast cancer cohort, three out of ten genera of the cluster including Fusobacterium, Prevotella and Bacteroides displayed an increase in cancer patients compared to controls (Figure 7F). Inconsistent results were observed between the two distinct melanoma cohorts examined- Two genera of the cluster (Dialister and Gemella) were found enriched in patients compared to healthy control in one cohort but were not significantly different in the other one included in our analysis (Figure 7G-H). The identification of the cluster was not limited to a specific specimen type. We could identify Fusobacterium and its associated microbiota in tumor tissues of different tumors histology as well. Remarkably, the tissue dataset of the gastric cancer cohort showed an enrichment of all genera of the cluster which was significant for Fusobacterium, Parvimonas and Prevotella (Figure 8A). Fusobacterium, Prevotella, and Gemella were also enriched in tissue samples from the breast cancer cohort (Figure 8B). Conversely, only Bacteroides demonstrated a tendency towards an enrichment in the tissue dataset of patients with prostate cancer compared to control subjects (Figure 8C).

In summary, our comprehensive in silico analysis reveals the presence of the Fusobacterium cluster across all the various tumor types investigated (Melanoma, Ovarian, Breast, Pancreas, Stomach, and Prostate). This suggests that its occurrence is not exclusive to CRC. Interestingly, a minimum of 2 (in melanoma stool) and a maximum of 7 (in pancreas and prostate cancer stool) out of 10 genera of the Fusobacterium cluster were found significantly enriched in cancer patients as compared to controls.

Importantly, the identification of the Fusobacterium cluster is not limited to a particular sample type, as it can be identified in both tissue and stool samples. Notably, our investigation indicates that the analysis of stool samples not only provides a more accurate and consistent detection of the Fusobacterium cluster across various tumor types but also emerges as a less invasive alternative to traditional tissue biopsies.

Claims

1 . Method for predicting the progression of cancer in a patient, comprising the steps of i) determining in a sample of said patient the content of at least one type of microorganism belonging to a Fusobacterium cluster-associated microbiota, wherein said cluster-associated microbiota is formed by microorganisms of the Fusobacterium genus, Porphyromonas genus, the Parvimonas genus, the Campylobacter genus and the Gemella genus and/or the content of at least one type of microorganism belonging to the Enterobacteriaceae family, to the Anaerostipes genus, to the Ruminococcus genus, to the Negativibacillus genus, to the Blautia genus and the Clostridium genus and ii) comparing said content to a reference value, wherein an increased content in said at least one type of microorganisms belonging to the Fusobacterium cluster-associated microbiota with respect to the reference value and/or wherein a decreased content of at least one type of microorganism belonging to the Enterobacteriaceae family, to the Anaerostipes genus, to the Ruminococcus genus, to the Negativibacillus genus, to the Blautia genus or the Clostridium genus is indicative of an increased risk of progression of the cancer in said patient.

2. The method according to claim 1 , wherein the content in the at least one type of microorganism is determined as the relative content of the 16S rRNA gene of the microorganism with respect to the total content of 16S RNA genes of the microorganisms present in the sample.

3. The method according to claim 2 wherein the relative content of the 16S RNA gene is determined by measuring the number of amplicons of the whole length of said gene or a fragment thereof.

4. The method according to any one claim 1 to 3, wherein the reference value is the content of the microorganisms belonging to a Fusobacterium cluster-associated microbiota, wherein said Fusobacterium cluster-associated microbiota is formed by microorganisms of the Fusobacterium genus, Porphyromonas genus, the Parvimonas genus, the Campylobacter genus and the Gemella genus and/or the content of at least one type of microorganism belonging to the Enterobacteriaceae family, to the Anaerostipes genus, the Ruminococcus genus, to the Negativibacillus genus, to the Blautia genus and the Clostridium genus in a sample from a patient suffering from cancer and in which the cancer has not progressed.

5. The method according to any one of claims 1 to 4, wherein the cancer colorectal cancer.

6. The method of claim 5 wherein the cancer is locally advanced rectal cancer (LARC).

7. The method according to any one of claims 1 to 4 wherein the cancer is oral cancer, melanoma, prostate cancer, breast cancer, pancreas cancer, stomach cancer, or ovary cancer.

8. The method according to any one of claims 1 to 7 wherein the sample has been obtained after the patient has been treated with radiation therapy and/or with chemotherapy.

9. The method according to claim 8 wherein the patient has been treated with neoadjuvant chemoradiotherapy.

10. The method according to claim 9 wherein the neoadjuvant chemoradiotherapy is fluoropyrimidine-based neoadjuvant therapy.

11 . Method according to any one of claims 1 to 10 wherein the sample is a tumor sample.

12. The method according to claim 11 wherein the tumor sample is a sample from a tumor which has been surgically removed or is a sample from a tumor biopsy obtained without the surgical excision of the tumor.

13. The method according to any one of claims 1 to 10 wherein the sample is stool sample.

14. The method according to any one of claims 1 to 13 wherein the risk of progression is determined as the risk of relapse after neoadjuvant chemoradiotherapy.

15. The method according to claim 14 wherein the relapse is distant or local relapse.

16. A method for selecting a personalized therapy for a patient suffering from cancer, wherein the method comprises determining the risk of progression of the cancer by a method as defined in any one of claims 1 to 15 and selecting a therapy adequate for the treatment of cancer in those patients in which the cancer is predicted to progress.

17. A method for preventing the progression of cancer of a patient or for preventing the relapse of cancer in patient, said method comprising determining the risk of progression of the cancer by a method as defined in any one of claims 1 to 15 and treating the patient with a therapy adequate for the cancer which is predicted to progress or to relapse.

18. The method according to claim 16 or 17 wherein the therapy adequate for the treatment of cancer is surgery, radiotherapy, chemotherapy, antibiotic specific for the microorganisms of the Fusobacterium cluster-associated microbiota and any combination thereof.

19. The method according to claim 18 wherein the chemotherapy is adjuvant chemotherapy.

20. The method according to any one of claims 17 to 19 wherein the relapse is local or distant relapse.