WO2020178297A1

WO2020178297A1 - Anal bacterial biomarkers for the diagnosis of anal precancerous lesions

Info

Publication number: WO2020178297A1
Application number: PCT/EP2020/055589
Authority: WO
Inventors: Sergio SERRANO VILLAR
Original assignee: Fundación Para La Investigación Biomédica Del Hospital Ramón Y Cajal
Priority date: 2019-03-04
Filing date: 2020-03-03
Publication date: 2020-09-10

Abstract

In the present invention, we have proven that the diagnostic value of an anal cytology for the diagnosis of biopsy-proven HSIL (bHSIL) can be improved by measuring bacterial biomarkers derived from anal cytologies. For such purpose we have conducted a cross-sectional prospective study performed in HIV+ and HIV-MSM referred to a high-resolution anoscopy clinic. The primary outcome was the presence of bHSIL at the inclusion or during the previous year. We analysed mucosal microbiota to search for biomarkers predictive of the presence of bHSIL. We obtained anal mucosa specimens with an anal cytobrush. The V3-V4 region of the 16S rRNA gene was sequenced using the Illumina platform. We selected the biomarkers based on their linear discriminant analysis (LDA) scores and AUC-ROC in logistic regression models.

Description

Anal Bacterial Biomarkers for the Diagnosis of Anal Precancerous Lesions

Technical field

The present invention relates to the field of cancer diagnosis. Specifically, it relates to an improved method for the screening, diagnosis and/or monitoring of precancerous anal lesions consisting of high-degree squamous intraepithelial lesions (HSIL) or anal cancer in a human subject. In particular, said method provides an increase of specificity due to a reduction of false positive results in an anal cytology using a signature based on bacterial markers obtained from anal sampling. It further relates to the use of said method in the selection of subjects for conducting an exploratory test or for the treatment with an anti-cancer therapy.

State of the art

Albeit uncommon in the general population, there is increasing awareness of marked (40 to 130-fold) increased risk of anal cancer in people living with HIV, especially among men who have sex with men (MSM). With the changing scenario of comorbidities in the HIV-infected population, given the success of modern antiretroviral therapy (ART), anal cancer has emerged as a leading neoplasia across different cohorts in Western countries. In 2014, 29,992 people were newly diagnosed with HIV in the EU/EEA. The majority of cases (77%) were men. This was largely driven by HIV transmission between MSM, which accounted for 42% of all HIV diagnoses. Hence, the incidence of anal cancer is likely to increase in years to come. Emerging data suggest that, even other populations not typically considered at increased risk of anal cancer, might be at high risk of human papillomaviruses (HPV) oncogenic transformation. For example, the prevalence of anal cancer and high grade squamous intraepithelial lesion (HSIL) have been found dramatically high in HIV-infected women without previous history of anogenital HPV disease and HPV prevalence appears to increase following renal transplantation.

There is a vivid debate whether groups at high-risk for anal cancer should be screened for precancerous anal lesions. Subjects at higher risk include HIV-infected MSM, HIV-infected persons with a history of anogenital condylomata and HIV-infected women with abnormal vulvar or cervical histology and other types of immune suppression [2018 Guidelines of the European AIDS Clinical Society. Available at: http://www.eacsociety.org/files/2015_eacsguidelines_8.0- english_revised-20151104.pdf; Aberg JA, Gallant JE, Ghanem KG, Emmanuel P, Zingman BS, Horberg MA. Primary Care Guidelines for the Management of Persons Infected With HIV: 2013 Update by the HIV Medicine Association of the Infectious Diseases Society of America. Clin. Infect. Dis. 2014; 58:e1-e34] Anal cytology is performed annually and, in case of an abnormal cytology, the patient is referred for high-resolution anoscopy (HRA) for biopsy of acetowhite lugol-negative lesions in the anal epithelium, suggestive of HSIL. This approach is based on the capacity of the anal cytology to rule out the presence of HSIL. While this technique is highly sensitive, the specificity is very poor (95% and 58%, respectively) [Serrano-Villar S, Hernandez- Novoa B, de Benito A, et al. Screening for Precancerous Anal Lesions with P16/Ki67 Dual Stain Cytology. In: Conference on Retroviruses and Opportunistic Infections (CROI). February 13-16, 2017. Seattle. 2017], leading to an excess number of invasive procedures, including unnecessary HRA and anal biopsies, avoidable patient discomfort and adverse events, which poses a major hurdle for the wide implementation of this screening strategy and undermine the prevention of anal cancer.

Therefore, current cytology-based screening strategy is challenged since it requires widely use of HRA, which is overwhelming for health resources in most settings, which explains its poor implementation. New diagnostic procedures are warranted to overcome the limitations, low specificity, of the anal cytology-based screening in susceptible populations.

Brief description of the figures

Figure 1. Bacterial biomarkers of bHSIL using the LefSe discovery tool.

Figure 2. Diagnostic values of anal cytology alone, the combination of 4 bacterial biomarkers alone or the combination of anal cytology + the 4 bacterial biomarkers.

Description

Definitions

The terms "subject", or "individual·" are used herein interchangeably to refer to all the animals classified as mammals and includes but is not limited to domestic and farm animals, primates and humans, for example, human beings, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents. Preferably, the subject is a male or female human being of any age or race.

The term“sensitivity” as used herein refers to the proportion of subjects who have the target condition (reference standard positive) and give positive test results (TP/ (TP + FN)). It shows how good the test is at detecting a disease. Sensitivity ("sens") may be within the range of 0 (0%) < sens < 1 (100%) and ideally, the number of false negatives equaling zero or close to equaling zero and sensitivity equaling one (100%) or close to equaling one (100%). The term“specificity” as used herein refers to the proportion of subjects without the target condition (reference standard negative) and give negative test results (TN/ (TN + FP)). It shows how good the test is at identifying normal (negative) condition. Specificity ("spec") may be within the range of 0 (0%) < spec < 1 (100%) and ideally, the number of false positives equaling zero or close to equaling zero and specificity equaling one (100%) or close to equaling one (100%).

The term“accuracy” as used herein refers to the proportion of true results, either true positive or true negative, in a population. It measures the degree of veracity of a screening test on a condition, i.e., how correct is the determination and exclusion of a given condition (TN + TP)/(TN+TP+FN+FP). Accuracy ("acc") may be within the range of 0 (0%) < acc < 1 (100%) and ideally, the number of false positives and false negative equaling zero or close to equaling zero and accuracy equaling one (100%) or close to equaling one (100%).

The term“Receiver Operating Characteristic (ROC) curves” as used herein refers to a graphical plot that illustrates the performance of a binary classifier system as its discrimination threshold is varied. The curve is created by plotting the true positive rate against the false positive rate at various threshold settings. The true positive rate is also known as sensitivity. The false positive rate is calculated as 1 - specificity. The ROC curve is thus a way of graphically displaying the true positive rate versus the false positive rate (sensitivity vs (1 -specificity)) across a range of cut-offs and of selecting the optimal cut-off for clinical use. Accuracy expressed as the area under the ROC curve (AUC) provides a useful parameter for comparing test performance. An AUC approaching 1 indicates that the test is highly sensitive as well as highly specific whereas an AUC approaching 0.5 indicates that the test is neither sensitive nor specific.

The term "probe" as used herein refers to synthetic or biologically produced nucleic acids, between 10 and 285 base pairs in length which contains specific nucleotide sequences that allow specific and preferential hybridization under predetermined conditions to target nucleic acid sequences, and optionally contains a moiety for detection or for enhancing assay performance. A minimum of ten nucleotides is generally necessary in order to statistically obtain specificity and to form stable hybridization products, and a maximum of 285 nucleotides generally represents an upper limit for length in which reaction parameters can be easily adjusted to determine mismatched sequences and preferential hybridization. Probes may optionally contain certain constituents that contribute to their proper or optimal functioning under certain assay conditions. For example, probes may be modified to improve their resistance to nuclease degradation (e.g., by end capping), to carry detection ligands (e.g., fluorescein), to carry ligands for purification or enrichment purposes (e.g. biotin) or to facilitate their capture onto a solid support (e.g., poly-deoxyadenosine "tails"). The term "primers" as used herein refers to oligonucleotides that can be used in an amplification method, such as a polymerase chain reaction ("PCR"), to amplify a nucleotide sequence. Primers are designed based on the polynucleotide sequence of a particular target sequence, e.g., one specific 16S rDNA sequence.

The term“specific" as used herein in connection with a nucleotide sequence means that a nucleotide sequence will hybridize to/amplify a predetermined target sequence and will not substantially hybridize to/amplify a non-target sequence under the assay conditions, generally stringent conditions are used.

The term "hybridization" as used herein refers to a process by which, under predetermined reaction conditions, two partially or completely complementary strands of nucleic acid are allowed to come together in an antiparallel fashion to form a double-stranded nucleic acid with specific and stable hydrogen bonds, following explicit rules pertaining to which nucleic acid bases may pair with one another.

The term "substantial hybridization" means that the amount of hybridization observed will be such that one observing the results would consider the result positive with respect to hybridization data in positive and negative controls. Data which is considered "background noise" is not substantial hybridization.

The term "stringent hybridization conditions" means approximately 35 °C to 65 °C in a salt solution of approximately 0.9 molar NaCI. Stringency may also be governed by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and the temperature of hybridization. Generally, as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. As a rule, the stringency of the conditions under which hybridization is to take place will dictate certain characteristics of the preferred probes to be employed.

The term“therapeutically effective amount” as used herein refers to an amount that is effective, upon single or multiple dose administration to a subject (such as a human patient) in the prophylactic or therapeutic treatment of a disease, disorder or pathological condition.

Description As already indicated, there is a need for new diagnostic procedures to overcome the limitations, low specificity, of the anal cytology-based screening in susceptible populations. In this sense, the current screening strategy for anal cancer is based on the detection of high-degree squamous intraepithelial lesions (HSIL), a cancer precursor, using anal cytology followed by high resolution anoscopy and anal biopsies in case of positive cytologic results. While this approach is highly sensitive, the specificity is poor, leading to an excess number of invasive procedures, explaining the poor implementation of this screening strategy. Emerging evidence supports that epithelial-adherent bacteria amplify HPV-associated cancers and contribute to impairment of mucosal immunity from the early stages of HIV-infection.

In the present invention, we have proven that the diagnostic value of an anal cytology for the diagnosis of biopsy-proven HSIL (bHSIL) can be improved by measuring bacterial biomarkers derived from anal cytologies. For such purpose we have conducted a cross-sectional prospective study performed in HIV+ and HIV- MSM referred to a high-resolution anoscopy clinic. The primary outcome was the presence of bHSIL at the inclusion or during the previous year. We analysed mucosal microbiota to search for biomarkers predictive of the presence of bHSIL. We obtained anal mucosa specimens with an anal cytobrush. The V3-V4 region of the 16S rRNA gene was sequenced using the lllumina platform. We selected the biomarkers based on their linear discriminant analysis (LDA) scores and AUC-ROC in logistic regression models (as shown in the examples).

In the study, we included 118 HIV+ and 33 HIV- MSM: 47 had bHSIL during the previous year and 12 at the moment of the inclusion. Linear discriminant analysis (LDA) effect size (LEfSe) revealed 40 biomarkers in mucosa. After exploring the predictive value of the 15 taxa with greater LDA scores, we selected four taxa in anal samples based on the high specificity or high AUC-ROC values. On these basis, we concluded that each 25% increase in the abundance of the Ruminococcaceae NK4A214 group and Alloprevotella genus were associated with a 17% (p=0.041) and 8% (p=0.016) increased risk of bHSIL, respectively. The absence of Prevotella melanonigenica and Ruminococcaceae UCG-014 were also predictive of bHSIL (OR 6.1 , P=0.018 and OR 3.2, P=0.026, respectively). From 35 (94%) of false positive cytologic results, the combination of these four biomarkers reclassified to true negative 33 (94%), significantly improving the predictive performance of anal cytology alone to AUC 0.805.

Thus a first aspect of the invention, refers to a method for increasing the specificity or reducing the false positive rate of an anal cytology for the diagnosis and/or monitoring of precancerous anal lesions consisting of high-degree squamous intraepithelial lesions (HSIL) or anal cancer in a human subject, wherein the method comprises: optionally conducting an anal cytology in a sample isolated from the human subject, and further determining the presence or absence and/or quantifying in an anorectal epithelium sample isolated from said human subject, at least one of following bacterial markers or any combination thereof:

Ruminococcaceae NK4A214 group, preferably by using SEQ ID NO 1 as a reference sequence for said detection (of the presence or absence) and/or quantification,

- Alloprevotella genus, preferably by using SEQ ID NO 2 as a reference sequence for said detection (of the presence or absence) and/or quantification,

Prevotella melanonigenica, preferably by using SEQ ID NO 3 as a reference sequence for said detection (of the presence or absence) and/or quantification, Ruminococcaceae UCG-014, preferably by using SEQ ID NO 4 as a reference sequence for said detection (of the presence or absence) and/or quantification, Ruminococcaceae UCG-002 genus, preferably by using SEQ ID NO 5 as a reference sequence for said detection (of the presence or absence) and/or quantification,

Sneathia genus, sanguinegens species, preferably by using SEQ ID NO 6 as a reference sequence for said detection (of the presence or absence) and/or quantification,

Christensenellaceae_R-7_group, preferably by using SEQ ID NO 7 as a reference sequence for said detection (of the presence or absence) and/or quantification,

Prevotella_9 genus, preferably by using SEQ ID NO 8 as a reference sequence for said detection (of the presence or absence) and/or quantification,

Tyzzerella_4 genus, preferably by using SEQ ID NO 9 as a reference sequence for said detection (of the presence or absence) and/or quantification, Rikeneiiaceae_RC9_gut_group genus, preferably by using SEQ ID NO 10 as a reference sequence for said detection (of the presence or absence) and/or quantification,

Bifidobacterium genus, pseudocatenulatum IPLA36007 species, preferably by using SEQ ID NO 11 as a reference sequence for said detection (of the presence or absence) and/or quantification,

- Acidami nococcus genus, timonensis species, preferably by using SEQ ID NO 12 as a reference sequence for said detection (of the presence or absence) and/or quantification,

Treponema_2 genus, succinifaciens species, preferably by using SEQ ID NO 13 as a reference sequence for said detection (of the presence or absence) and/or quantification, Fusobacterium genus, nucleatum species, preferably by using SEQ ID NO 14 as a reference sequence for said detection (of the presence or absence) and/or quantification,

Sutterella genus, preferably by using SEQ ID NO 15 as a reference sequence for said detection (of the presence or absence) and/or quantification,

Megasphaera genus, elsdenii species, preferably by using SEQ ID NO 16 as a reference sequence for said detection (of the presence or absence) and/or quantification,

S5-A 14a genus, preferably by using SEQ ID NO 17 as a reference sequence for said detection (of the presence or absence) and/or quantification,

Dialister genus, propionicifaciens species, preferably by using SEQ ID NO 18 as a reference sequence for said detection (of the presence or absence) and/or quantification,

Prevotella_2 genus, preferably by using SEQ ID NO 19 as a reference sequence for said detection (of the presence or absence) and/or quantification,

Bacteroides genus, eggerthii species, preferably by using SEQ ID NO 20 as a reference sequence for said detection (of the presence or absence) and/or quantification,

Ruminococcus_1 genus, preferably by using SEQ ID NO 21 as a reference sequence for said detection (of the presence or absence) and/or quantification, Undibacterium genus, pigrum species, preferably by using SEQ ID NO 22 as a reference sequence for said detection (of the presence or absence) and/or quantification,

- Actinotignum genus, timonense species, preferably by using SEQ ID NO 23 as a reference sequence for said detection (of the presence or absence) and/or quantification,

Subdoligranulum genus, vahabile species, preferably by using SEQ ID NO 24 as a reference sequence for said detection (of the presence or absence) and/or quantification, and/or

Reyranella genus, massiliensis 521 species, preferably by using SEQ ID NO 25 as a reference sequence for said detection (of the presence or absence) and/or quantification, wherein a combined score obtained from the bacterial markers determined, aids classifying an anal cytology positive subject as presenting an increased or reduced risk of having precancerous anal lesions consisting of high-degree squamous intraepithelial lesions (HSIL) or anal cancer, by increasing the specificity of the anal cytology. It is herein noted that the phylogeny of each of the bacteria identified above is detailed in table 1 , which is sited in the examples of the present specification. It is herein further noted that reference sequences for the 16S RNA gene identification of each of the above bacterial markers, are detailed below:

Ruminococcaceae NK4A214 group:

SEQ ID NO 1.

TGGGGAATATTGGGCAATGGGCGAAAGCCTGACCCAGCAACGCCGCGTGAAGGAAGAAG GTCTTCGGATT GT AAACTTCTTTT AT CAGGG ACGAAG AACGT G ACGGT ACCT GAT G AAT AA GCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGG ATTTACTGGGTGTAAAGGGCGCGTAGGCGGGGATACAAGTCAGATGTGAAATCTATGGGC TT AACCCAT AAACTGCATTT G AAACT GTATCT CTT G AGT GTCGG AG AGGT AGACGGAATT C CT AGT GT AGCGGT GAAATGCGT AGAT ATT AGGAGGAACACCAGTGGCGAAGGCGGTCT AC TGGACGATAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACA

Alloprevotella genus

SEQ ID NO 2

T GAGGAAT ATTGGTCAAT GGACGCAAGTCT GAACCAGCCAAGT AGCGT GCAGGAAGACGG CCCT CTGGGTT GT AAACTGCTTTT AGTTGGGAAT AAAGTGCACCACGT GT GGT GTTTT GT A TGTACCATCAGAAAAAGGACCGGCTAATTCCGTGCCAGCAGCCGCGGTAATACGGAAGGT CCAGGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGCAGGCGGACCTTTAAGTCAGCT GT GAAAT ACGGCGGCTCAACCGTCGAACTGCAGTT GAT ACTGGAGGTCTT GAGTGCACAC AGGGATACTGGAATTCATGGTGTAGCGGTGAAATGCTCAGATATCATGAAGAACTCCGATC GCGAAGGCAGGTATCCGGGGTGCAACTGACGCTGAGGCTCGAAAGTGCGGGTATCAAAC A

Prevotella melanonigenica

SEQ ID NO 3

TGAGGAATATTGGTCAATGGACGGAAGTCTGAACCAGCCAAGTAGCGTGCAGGATGACGG CCCT ATGGGTT GT AAACTGCTTTT GT ATGGGGAT AAAGTT AGGGACGT GTCCCT ATTT GCA GGTACCATACGAATAAGGACCGGCTAATTCCGTGCCAGCAGCCGCGGTAATACGGAAGGT CCAGGCGTT ATCCGGATTT ATTGGGTTT AAAGGGAGCGT AGGCTGGAGATT AAGT GT GTT G T GAAAT GT AGACGCTCAACGTCT GAATTGCAGCGCAT ACTGGTTTCCTT GAGT ACGCACAA CGTTGGCGGAATTCGTCGT GT AGCGGT GAAATGCTT AGAT AT GACGAAGAACTCCGATT G CGAAGGCAGCTGACGGGAGCGCAACTGACGCTTAAGCTCGAAGGTGCGGGTATCAAACA Ruminococcaceae UCG-014

SEQ ID NO 4

TCGGGAAT ATT GCACAATGGAGGAAACTCT GAT GCAGT GACACCGCGT AT AGGAAGAAGG CCTT AGGGTT GT AAGCT ATT GTCGT GT GAGAAGAAAAT GACT ATCACAGGAGGAAGCCCT G GCT AAAT AT GT GCCAGCAGCCGCGGT AAT ACAT AT GGGGCGAGCGTT ATCCGGATTT ATT G GGTGTAAAGGGTTCGTAGGCGGGAAAGTAAGTCAGTATGTGAAATCCCTCGGCTCAACCG AGGAACTGCAATT GAAACT ATTTTTCTT GAGT ACTGGAGGGGAAAGTGGAATTCTT AGT GT A GCGGTGAAATGCGTAGATATTAGGAAGAACACCAGTGGCGAAGGCGACTTTCTGGACAGT AACT GACGCT GAGGAACGAAAGT GTGGGGAGCAAACA

Ruminococcaceae UCG-002 genus

SEQ ID NO 5

TGGGGAATATTGGGCAATGGGCGCAAGCCTGACCCAGCAACGCCGCGTGAAGGAAGAAG GCTTTCGGGTT GT AAACTTCTTTT GTCAGGGACGAGT AGAAGACGGT ACCT GACGAAT AAG CCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAT TTACTGGGTGTAAAGGGCGTGTAGCCGGGAGGGCAAGTCAGATGTGAAATCCACGGGCT CAACTCGT G AACTGCATTT GAAACT ACT CTT CTT GAGT AT CGG AGAGGCAATCGG AATTCC T AGT GT AGCGGT GAAATGCGT AGAT ATT AGGAGGAACACCAGT GGCGAAGGCGGATTGCT GGACGACAACTGACGGTGAGGCGCGAAAGCGTGGGGAGCAAACA

Sanguinegens species

SEQ ID NO 6

TGGGGAAT ATT GGACAATGGAGGCAACTCT GATCCAGCAATTCT GT GT GT GT GAAGAAGGT TTT AGGACT GT AAAACACTTTT AGT AGGGAAGAAAGAAAT GACGGT ACCT ACAGAAGAAGC GACGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGTCGCGAGCGTTATCCGGAAT TATTGGGCTTAAAGGGCATCTAGGCGGTTAAACAAGTTGAAGGTGAAAACCTGTGGCTCAA CCAT AGGCTTGCCT ACAAAACT GT AT AACT AGAGT ACT GGAAAGGTGGGTGGAACT ACACG AGT AGAGGT GAAATTCGT AGAT AT GT GT AGGAAT GCCGAT GAT GAAGAT AACTCACTGGAC AGCAACTGACGCTGAAGTGCGAAAGCTAGGGGAGCAAACA

Christensenellaceae R-7 group

SEQ ID NO 7

TGGGGAATATTGGGCAATGGGCGCAAGCCTGACCCAGCAACGCCGCGTGAGGGAAGAAG GTTTTCGGATT GT AAACCTCT GTCGCAGGGGACGAAGGAAGT GACGGT ACCCT GT AAGAA AGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCGAGCGTTGTCCG GAATTACTGGGCGTAAAGGGAGCGTAGGCGGTCGATTAAGTTAGATGTGAAACCCCCGGG CTT AACTT GGGGACTGCATCT AAT ACTGGTT GACTT AGAGT ACAGGAGAGGGAAGCGGAAT TCCT AGT GT AGCGGT GAAATGCGT AGAT ATT AGGAGGAACACCAGTGGCGAAGGCGGCTT TCTGGACT GACACT GACGCT GAGGCTCGAAAGCGTGGGGAGCAAACA

Prevotella 9 genus

SEQ ID NO 8

T GAGGAAT ATTGGTCAATGGACGAGAGTCT GAACCAGCCAAGT AGCGTGCAGGAAGACGG CCCT ATGGGTT GT AAACTGCTTTT AT AAGGGAAT AAAGT GAGAGTCGT GACTCTTTTTGCAT GTACCTTATGAATAAGGACCGGCTAATTCCGTGCCAGCAGCCGCGGTAATACGGAAGGTC CGGGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGCCGGAGATTAAGCGTGTTG T GAAAT GT AGATGCTCAACATCTGCACTGCAGCGCGAACTGGTTTCCTT GAGT ACGCACAA AGTGGGCGGAATTCGT GGT GT AGCGGT GAAATGCTT AGAT ATCACGAAGAACTCCGATT G CGAAGGCAGCTCACTGGAGCGCAACT GACGCT GAAGCTCGAAAGTGCGGGT ATCGAACA

Tyzzerella 4 genus

SEQ ID NO 9

TGGGGAAT ATTGCACAATGGGGGAAACCCT GAT GCAGCGACGCCGCGT GAGCGAAGAAG

T ATTTCGGT AT GT AAAGCTCT ATCAGCAGGGAAGAAAT AGGACGGT ACCT GACT AAGAAGC

ACCGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGGTGCAAGCGTTATCCGGATT

TACTGGGTGTAAAGGGAGCGTAGACGGTTGTGTAAGTCTGATGTGAAAGCCCGGGGCTCA

ACCCCGGGACT GCATTGGAAACT AT GT AACT AGAGT GTCGGAGAGGT AAGCGGAATTCCT

AGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTG

GACGATCACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAACA

Rikenellaceae RC9 gut group genus

SEQ ID NO 10

TGAGGAATATTGGTCAATGGGCGGAAGCCTGAACCAGCCATGCCGCGTGAAGGACTAAGG CCCT ACGGGTCGT AAACTTCTTT AGACGCAGAGCAAT AAGGTCCACGCGTGGACCGAT GA GAGTATGCGTAGAATAAGCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGGGGA TGCGAGCGTTATCCGGATTCATTGGGTTTAAAGGGTGCGTAGGCGGCCGAGTAAGTCAGC GGT GAAAGACCGGGGCTCAACCCTGGAAGTGCCGTT GAT ACT GTTTGGCTGGAAT GATCC CGCCGCGGGAGGAAT GAGT GGT GT AGCGGT GAAATGCAT AGAT ATCACTCAGAACACCGA TTGCGAAGGCATCTCGCGAGGGGTCAATTGACGCTGAGGCACGAAAGCGTGGGGATCGA

ACA pseudocatenulatum IPLA36007 species

SEQ ID NO 1 1

TGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGCGGGATGACG

GCCTTCGGGTT GT AAACCGCTTTT GATCGGG AGCAAGCCTTCGGGT GAGT GT ACCTTTCG

AATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAT

CCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCA

TCGCTTAACGGTGGATCTGCGCCGGGTACGGGCGGGCTGGAGTGCGGTAGGGGAGACT

GGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGC

AGGTCTCTGGGCCGTTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACA timonensis species

SEQ ID NO 12

TGGGGAATCTTCCGCAATGGACGAAAGTCT GACGGAGCAACGCCGCGT GAGT GAT GAAG GTCTTCGGATT GT AAAACT CT GTT GTCAGGGACG AAT GT ACT GAT CT AT AAT ACATTTCGGT ATTGACGGTACCTGACGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACG TAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCATGTAGGCGGGCTTTTAAG TCCGACGTGAAAATGCGGGGCTTAACCCCGTATGGCGTTGGATACTGGGAGTCTTGAGTG CAGGAGAGGAAAGGGGAATTCCCAGT GT AGCGGT GAAATGCGT AGAT ATTGGGAGGAACA CCAGTGGCGAAGGCGCCTTTCTGGACTGTGTCTGACGCTGAGATGCGAAAGCCAGGGTA GCAAACG succinifaciens species

SEQ ID NO 13

CTAAGAATATTCCGCAATGGGGGGAACCCTGACGGAGCGACGCCGCGTGGGCGAGGAAG

GCCGGAAGGTTGTAAAGCCCTTTTGCGCGCGAGGAATAAGGGGAGGAGGGAATGCCTTC

CCGGTGACTGTAGCGCGCGAATAAGCGCCGGCTAATTACGTGCCAGCAGCCGCGGTAAC

ACGTAAGGCGCGAGCGTTGTTCGGAATCATTGGGCGTAAAGGGCGTGTAGGCGGCCCTG

CAAGCCTGGCGTGAAATCCCGGGGCCCAACCCCGGAACCGCGCTGGGAACTGCTGGGCT

TGAGCCGCTGTGGCGCAGCCGGAATTCCAGGTGTAGGGGTGAAATCTGTAGATATCTGGA

AGAACACCGATGGCGAAGGCAGGCTGCGAGCGGACGGCTGACGCTGAGGCGCGAAGGC

GCGGGGAGCGAACA nucleatum species

SEQ ID NO 14

TGGGGAAT ATT GGACAATGGACCGAGAGTCT GATCCAGCAATTCT GT GTGCACGATGAAG TTTTTCGGAAT GT AAAGT GCTTTCAGTT GGGAAGAAAT AAAT GACGGT ACCAACAGAAGAA GTGACGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGTCACGAGCGTTATCCGGA TTTATTGGGCGTAAAGCGCGTCTAGGTGGTTATGTAAGTCTGATGTGAAAATGCAGGGCTC AACTCT GT ATT GCGTTGGAAACT GT AT AACT AGAGT ACTGGAGAGGT AAGCGGAACT ACAA GT GT AGAGGT GAAATTCGT AGAT ATTTGT AGGAATGCCGATGGGGAAGCCAGCTT ACTGG ACAGAT ACT GACGCT AAAGCGCGAAAGCGTGGGT AGCAAACA

Sutterella genus

SEQ ID NO 15

TGGGGAATTTTGGACAATGGGGGAAACCCTGATCCAGCCATGCCGCGTGCGGGAAGAAG GCCTTCGGGTT GT AAACCGCTTTT GTCAGGGACGAAAAGTCGT ATTT AAAT AAAGT ACGAT GCT GACGGT ACCT GAAGAAT AAGCACCGGCT AACT ACGTGCCAGCAGCCGCGGT AAT ACG TAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTGGATAAG ACAGAT GT GAAATCCCCGGGCTT AACCT GGGAACCGCATTT GT GACT GTCT GACT GGAGT AT GTCAGAGGGGGGTGGAATTCCACGT GT AGCAGT GAAATGCGT AGAT AT GTGGAAGAAC ACCGATGGCGAAGGCAGCCCCCTGGGGCAAAACTGACGCTCATGCACGAAAGCGTGGGG AGCAAACA elsdenii species

SEQ ID NO 16

TGGGGAATCTTCCGCAATGGACGAAAGTCT GACGGAGCAACGCCGCGT GAACGAT GACG GCCTTCGGGTT GT AAAGTTCT GTT AT ACGGGACGAATGGCGT AACGGTCAAT ACCCGTT GC GAGT GACGGT ACCGT AAGAGAAAGCCACGGCT AACT ACGT GCCAGCAGCCGCGGT AAT AC GTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCGCGCAGGCGGCGTCGTA AGTCGGTCTTAAAAGTGCGGGGCTTAACCCCGTGAGGGGACCGAAACTGCGATGCTAGAG T ATCGGAGAGGAAAGCGGAATTCCT AGT GT AGCGGT GAAATGCGT AGAT ATT AGGAGGAA CACCAGTGGCGAAAGCGGCTTTCTGGACGACAACTGACGCTGAGGCGCGAAAGCCAGGG GAGCAAACG

S5-A14a genus

SEQ ID NO 17 TGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCAACGCCGCGTGAACGAAGAAG GCCTTCGGGTCGT AAAGTTCT GTCCAT AGGGAAGAAACAAAT GACGGT ACCT ATGGAGGA AGCCCCGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGGGGCAAGCGTTATCCG GAATTATTGGGCGTAAAGAGTGCGTAGGTGGTGGCTTAAGCGCAGGGTTTAAGGCAATGG CTT AACT ATT GTTCGCCTT GCGAACT GGGTCACTT GAGT ACAGGAGAGGAAAGCGGAATTC CTAGTGTAGCGGTGAAATGCATAGATATTAGGAGGAACACCGGTGGCGAAGGCGGCTTTC TGGACTGCAACT GACACT GAGGCACGAAAGCGTGGGT AGCAAACA propionicifaciens species

SEQ ID NO 18

TGGGGAATCTTCCGCAATGGACGAAAGTCT GACGGAGCAACGCCGCGT GAGT GAAGACG GCCTTCGGGTT GT AAAGCTCT GT GATCCGGGACGAAAGAGCCT GAGGTT AAT AGCCT AAG GAAGTGACGGTACCGGAAAAGCAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAAT ACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGCTTCC T AAGTCCATCTT AAAAGTGCGGGGCTT AACCCCGT GATGGGAT GGAAACT GGGAAGCT GG AGT ATCGGAGAGGAAAGTGGAATTCCT AGT GT AGCGGT GAAAT GCGT AGAGATT AGGAAG AACACCGGTGGCGAAGGCGACTTTCTGGACGAAAACTGACGCTGAGGCGCGAAAGCGTG GGGAGCAAACA

Prevotella 2 genus

SEQ ID NO 19

T GAGGAAT ATTGGTCAATGGGCGAGAGCCT GAACCAGCCAAGT AGCGTGCAGGAT GACG

GCCCTATGGGTTGTAAACTGCTTTTATGCGGGGATAAAGTGAGCCACGAGTGGCTTATTGC

AGGTACCGCATGAATAAGGACCGGCTAATTCCGTGCCAGCAGCCGCGGTAATACGGAAGG

TCCGGGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGCCGTCTGGTAAGCGTGT

TGTGAAATGTCGGGGCTCAACCTGGGCATTGCAGCGCGAACTGTCAGACTTGAGTGCGCG

GGAAGT AGGCGGAATTCGTCGT GT AGCGGT GAAATGCTT AGAT ATGACGAAGAACTCCGA

TTGCGAAGGCAGCCTGCTGTAGCGCAACTGACGCTGAAGCTCGAAAGCGTGGGTATCGAA

CA eggerthii species

SEQ ID NO 20

T GAGGAAT ATTGGTCAAT GGACGAGAGTCT GAACCAGCCAAGT AGCGT GAAGGAT GACT G CCCTATGGGTTGT AAACTT CTTTT AT ACGGG AAT AAAGT GG AGT AT GC AT ACTCCTTT GTAT GTACCGTATGAATAAGGATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATC CGAGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGCGGGTGCTTAAGTCAGTTG T GAAAGTTT GCGGCTCAACCGT AAAATTGCAGTT GAT ACTGGGT ACCTT GAGT GCAGCAT A GGT AGGCGGAATTCGT GGT GT AGCGGT GAAATGCTT AGAT ATCACGAAGAACTCCGATT G CGAAGGCAGCTT ACTGGACT GT AACT GACGCT GATGCTCGAAAGT GT GGGT ATCAAACA

Ruminococcus 1 genus

SEQ ID NO 21

TGGGGAAT ATT GCACAATGGAGGAAACTCT GAT GCAGCGATGCCGCGT GAGGGAAGAAG GTTTT AGGATT GT AAACCTCT GTCTTCAGGGACGAAAAAAAAGACGGT ACCT GAGGAGGAA GCTCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGAGCGAGCGTTGTCCGG AATTACTGGGTGTAAAGGGAGCGTAGGCGGGATCGCAAGTCAGATGTGAAAACTATGGGC TT AACCCAT AAACTGCATTT GAAACT GT GGTTCTT GAGT GAAGT AGAGGT AAGCGGAATTC CT AGT GT AGCGGT GAAAT GCGT AGAT ATT AGGAGGAACATCAGT GGCGAAGGCGGCTT AC TGGGCTTT AACT GACGCT GAGGCTCGAAAGCGTGGGGAGCAAACA pigrum species

SEQ ID NO 22

TGGGGAATTTTGGACAATGGGGGCAACCCTGATCCAGCAATGCCGCGTGAGTGAAGAAGG

CCCTCGGGTTGTAAAGCTCTTTTGTCAGGGAAGAAACGGTCTACTCTAATACAGTGGGCTA

ATGACGGTACCTGAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGT

AGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTTATAAGA

CAGAGGT GAAATCCCCGGGCTCAACCTGGGAACTGCCTTT GT GACT GT AAGGCT AGAGT G

TGTCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGATATGTGGAGGAATA

CCGATGGCGAAGGCAGCCCCCTGGGATAACACTGACGCTCATGCACGAAAGCGTGGGGA

GCAAACA timonense species

SEQ ID NO 23

TGGGGGATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGGGGGATGAAG

GCTTTCGGGTTGTAAACCTCTTTCGCTATCGAACAAGGCCCGCTTAGGGTGGGTTGAGGG

TAGGTGGAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCG

CGAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTTGTAGGCGGCTTGTTGCGCCTGCTG

TGAAAACGCGGGGCTTAACTTCGCGCGTGCAGTGGGTACGGGCAGGCTTGAGTGTGGTA

GGGGT GACTGGAATTCCAGGT GT AGCGGTGGAAT GCGCAGAT ATCT GGAGGAACACCGAT GGCGAAGGCAGGTCACTGGGCCATTACTGACGCTGAGGAGCGAAAGCATGGGTAGCGAA

CA variabile species

SEQ ID NO 24

TGGGGGATATTGCACAATGGGGGAAACCCTGATGCAGCGACGCCGCGTGGAGGAAGAAG

GTTTTCGGATT GT AAACTCCT GTCGTT AGGGACGAT AAT GACGGT ACCT AACAAGAAAGCA

CCGGCTAACTACGTGCCAGCAGCCGCGGTAAAACGTAGGGTGCAAGCGTTGTCCGGAATT

ACTGGGTGTAAAGGGAGCGCAGGCGGACCGGCAAGTTGGAAGTGAAAACTATGGGCTCA

ACCT AT AAATTGCTTTCAAAACTGCTGGCCTT GAGT AGT GCAGAGGT AGGT GGAATTCCCG

GTGTAGCGGTGGAATGCGTAGATATCGGGAGGAACACCAGTGGCGAAGGCGACCTACTG

GGCACCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCAAACA massiliensis 521 species

SEQ ID NO 25

TGGGGAAT ATTGGACAAT GGGGGCAACCCT GATCCAGCCATGCCGCGT GAGT GAT GAAG

GCCTTCGGGTTGTAAAGCTCTTTTGGCGGGGACGATGATGACGGTACCCGCAGAATAAGC

CCCGGCTAACTTCGTGCCAGCAGCCGCGGTAAGACGAAGGGGGCTAGCGTTGTTCGGAA

TTACTGGGCGTAAAGCGCGTGTAGGCGGTTATCCAAGTCGGGTGTGAAAGCCTTGAGCTC

AACTCAAGAAAT GCACTCGGT ACTGGGT GACT AGAGGACCGGAGAGGAT AGTGGAATTCC

CAGT GT AGT GGT GAAAT ACGT AGAGATTGGGAAGAACACCAGT GGCGAAGGCGGCTATCT

GGACGGTTTCTGACGCTAAGACGCGAAAGCGTGGGGAGCAAACA

In a preferred embodiment of the first aspect of the invention, said determination of the presence or absence and/or quantification in an anorectal epithelium sample isolated from said human subject of the one or more bacterial markers, increases the specificity of an anal cytology to at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or to at least 99%.

In a preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, said one or more bacterial markers is Ruminococcaceae NK4A214 group, wherein an increase in the abundance of Ruminococcaceae NK4A214 group in the isolated sample in comparison to a reference sample from a healthy population of individuals, is associated with an increased risk of bHSIL and to an increased in the specificity of an anal cytology when both methods are combined. In another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, said one or more bacterial markers is Alloprevotella genus, wherein an increase in the abundance of Alloprevotella genus in the isolated sample in comparison to a reference sample from a healthy population of individuals, is associated with an increased risk of bHSIL and to an increased in the specificity of an anal cytology when both methods are combined.

In another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, said one or more bacterial markers is Prevotella melanonigenica, wherein the absence of Prevotella melanonigenica in the isolated sample is associated with an increased risk of bHSIL and to an increased in the specificity of an anal cytology when both methods are combined.

In another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, said one or more bacterial markers is Ruminococcaceae UCG-014, wherein the absence of Ruminococcaceae UCG-014 in the isolated sample is associated with an increased risk of bHSIL and to an increased in the specificity of an anal cytology when both methods are combined.

In another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, said one or more bacterial markers are selected from the combination of markers from the list consisting of Ruminococcaceae NK4A214 group, Alloprevotella genus, Prevotella melanonigenica, and Ruminococcaceae UCG-014 (see figure 2).

In yet another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, the anorectal epithelium sample is collected or isolated from cytobrushes with and without spatulas, cytopicks, cotton, Dacron, rayon, or nylon-flocked (NF)-swabs.

A second aspect of the present invention, refers to a method for the diagnosis and/or monitoring of precancerous anal lesions consisting of high-degree squamous intraepithelial lesions (HSIL) or anal cancer in a human subject, wherein the method comprises: a) conducting an anal cytology in a sample isolated from a human subject; b) detecting the presence or absence and/or quantifying at least one of the bacterial markers identified in any of the embodiments of the first aspect of the invention in an anorectal epithelium sample isolated from said human subject, preferably collected or isolated from cytobrushes with and without spatulas, cytopicks, cotton, Dacron, rayon, or nylon-flocked (NF)-swabs; c) calculating a combined score from the bacterial markers (levels and/or presence or absence) determined in b); and d) classifying an anal cytology positive subject as presenting an increased risk of having precancerous anal lesions consisting of high-degree squamous intraepithelial lesions (HSIL) or anal cancer, according to the combined score obtained in c).

In a preferred embodiment of the second aspect of the invention, in step d) said method comprises comparing the combined score in the subject’s sample with a reference combined score; wherein a difference of the combined score in the subject’s sample with regard to said reference combined score is indicative of precancerous anal lesions consisting of high-degree squamous intraepithelial lesions (HSIL) or anal cancer.

In another preferred embodiment of the second aspect of the invention or of any of its preferred embodiments, in step b) the bacterial markers identified are selected from the list comprising or consisting of Ruminococcaceae NK4A214 group, Alloprevotella genus, Prevotella melanonigenica, and Ruminococcaceae UCG-014, or any combination thereof.

The combined score of step (c) is a value obtained according to a given mathematical algorithm wherein the quantification values of each of the bacterial markers determined in step b) are variables of said mathematical algorithm. For instance, said combined score may be calculated as the sum of the products of standardized beta coefficients obtained in a regression analysis for each marker and wherein the bacterial marker values are used as variables.

Typically, in step d) said method comprises comparing the combined score in the subject sample with a reference combined score; and a significant difference of the combined score in the subject sample with regard to said reference combined score is indicative of the disease or of the pre-cancerous state.

The term“reference combined score” as used herein is a reference value obtained according to a given mathematical algorithm wherein reference expression values of each of the bacterial markers used in the method of the invention are variables of said mathematical algorithm.

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 reference value or reference level can be an absolute value, a relative value, a value that has an upper or a lower limit, a range of values, an average value, a median value, a mean value, a tertile value, or a value as compared to a particular control or baseline value. In a particular embodiment, optionally in combination with one or more of the embodiments or features described above or below, said reference value is the mean value or the tertile value.

The reference value according to the methods of the invention can be obtained from one or more subjects not having the disease (i.e., healthy control subjects), or from the same subject that was diagnosed as having anal cancer or a pre-cancerous state thereof but at an earlier time point.

In the methods of the invention, the combined score (or bacterial marker amount) is considered “decreased” when said combined score (or bacterial marker amount) is lower than a reference combined score (or a reference value). Preferably, the combined score is considered to be lower than a reference combined score (or a reference value) when it is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, or more lower than the reference combined score (or a reference value).

Likewise, in the context of the methods of the invention, the combined score (or bacterial marker amount) is considered“increased” when said combined score is higher than a reference combined score (or a reference value). Preferably, the combined score (or bacterial marker amount) is considered to be higher than a reference combined score (or a reference value) when it is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, or more higher than a reference combined score (or a reference value).

Alternatively or in addition, subjects having more than about 1.1 ,12, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 fold levels deviation (i.e., increase or decrease) than the reference combined score (or reference value) as described herein.

The term "diagnosis”, as used in any of the aspects of the present invention, refers both to the process of attempting to determine and/or identify a possible disease in a subject, i.e. the diagnostic procedure, and to the opinion reached by this process, i.e. the diagnostic opinion. As such, it can also be regarded as an attempt at classifying an individual's condition into separate and distinct categories that allow medical decisions about treatment and prognosis to be made. It is to be understood that the method, in a preferred embodiment, is a method carried out in vitro, i.e. not practiced on the human or animal body. In particular, the diagnosis to determine precancerous anal lesions or anal cancer patients, may relate to the capacity to identify and classify said patients.

The methods of the invention may also be used for the differential diagnosis between patients having precancerous anal lesions and anal cancer patients.

The most effective and economic measure to reduce anal cancer incidence and mortality are anal cancer risk screening and monitoring tests. Screening tests are grouped into those that primarily detect cancer early; and those that can detect cancer early and also can detect precancerous anal lesions, thus providing a greater potential for prevention.

The term “screening” is understood herein as the examination or testing of a group of asymptomatic individuals pertaining to the general population, or of a group of individuals having one or more risk factors (e.g., a subject with intermediate or high risk of developing a disease), with the objective of discriminating healthy individuals from those who have or are suspected of having a disease. A method of screening is generally used for the“early detection” of a disease. The expression“early detection” refers to detection before the presence of clinical signs.

The goal of cancer screening is to reduce mortality through early detection and treatment thus enabling a reduction in incidence of advanced disease which generally has a worse prognosis. The method of the screening of the invention may be conducted in individuals that do not present signs and/or symptoms of anal cancer (referred herein as“asymptomatic individuals”). It may also be carried out in subjects with or without personal or family history of anal cancer, or other risk factors.

Therefore, in a futher preferred embodiment of the first or second aspects of the present invention, said subject is a subject suspected of having precancerous anal lesions consisting of high-degree squamous intraepithelial lesions (HSIL) or anal cancer.

In another preferred embodiment of the first or second aspects of the present invention, said subject is an asymptomatic subject.

On the other hand, it is noted that determination of the bacterial markers of any of the methods of the present invention, may be conducted by any molecular biology method. Molecular biology methods for measuring quantities of target nucleic acid sequences are well known in the art. These methods include but are not limited to end point PCR, competitive PCR, quantitative PCR (qPCR), PCR-pyrosequencing, PCR-ELISA, DNA microarrays, nucleic acid sequencing, such as next generation sequencing methods, in situ hybridization assays such as dot-blot or Fluorescence In Situ Hybridization assay (FISH), mass spectrometry, branched DNA (Nolte, Adv. Clin. Chem. 1998,33:201-235) and to multiplex versions of said methods (see for instance, Andoh et al. , Current Pharmaceutical Design, 2009; 15,2066-2073) and the next generation of any of the techniques listed and combinations thereof, all of which are within the scope of the present invention.

Diverse next-generation sequencing methods have been described and are well known to a person skilled in the art. These include for instance sequencing by synthesis with cyclic reversible termination approaches (e.g., Illumina, SEQLL, Qiagen), sequencing by synthesis with single-nucleotide addition approaches (e.g., Roche-454, Thermo Fisher-Ion Torrent), sequencing by ligation (e.g., Thermo Fisher 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), see for instance Goodwin S, et al., Nat Rev Genet. 2016, 17(6):333-51).

In some embodiments, said molecular biology quantification methods are based on sequence specific amplification. Such an amplification-based assay comprises an amplification step which comprises contacting a sample (preferably an isolated DNA sample) with two or more amplification oligonucleotides specific for a target sequence in a target nucleic acid to produce an amplified product if the target nucleic sequence is present in the sample. 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), which includes quantitative PCR.

One particularly preferred quantification method is quantitative PCR (qPCR), also known as real-time PCR. It relates to a type of PCR that amplifies and simultaneously quantifies a target DNA molecule. Its key feature is that the amplified DNA is detected as the reaction progresses in real time. Different instruments are available, such as ABI Prism 7700 SDS, GeneAmp 5700 SDS, ABI Prism 7900 HT SDS from Applied Biosystems; iCycler iQ from Bio-Rad; Smart Cycler from Cepheid; Rotor-Gene from Corbett Research; LightCycler from Roche Molecular Biochemicals and Mx4000 Multiplex from Stratagene. The qPCR process enables accurate quantification of the PCR product in real-time by measuring PCR product accumulation very early in the exponential phase of the reaction, thus reducing bias in the quantification linked to the PCR amplification efficiency occurring in end-point PCR. Real-time PCR is well known in the art and is thus not described in detail herein. Technology overview and protocols for qPCR are available for instance from the above-mentioned vendors, e.g., http://www.sigmaaldrich.com/technical-documents/protocols/biology/sybr-green-qpcr.html or http://www.sigmaaldrich.com/life-science/molecular-biology/pcr/quantitative-pcr/qpcr-technical- guide.html. For a review of qPCR methods see Wong ML y Medrano JF, Biotechniques 2005, 39(1):75-85. In a particular embodiment, the quantification method is a multiplex qPCR.

Different detecting chemistries are available for qPCR. All of them can be used with the above- mentioned qPCR instruments. The term “detection chemistry” refers to a method to report amplification of specific PCR product in real-time PCR. These detecting chemistries may be classified into two main groups; the first group comprises double-stranded DNA intercalating molecules, such as SYBR Green I and EvaGreen, whereas the second includes fluorophore- labeled oligonucleotides. The latter, in turn, has been divided into three subgroups according to the type of fluorescent molecules used in the PCR reaction: (i) primer-probes (Scorpions, Amplifluor®, LUX™, Cyclicons, Angler®); (ii) probes; hydrolysis (TaqMan, MGB-TaqMan, Snake assay) and hybridization (Hybprobe or FRET, Molecular Beacons, HyBeacon™, MGB- Pleiades, MGB-Eclipse, ResonSense®, Yin-Yang or displacing); and (iii) analogues of nucleic acids (PNA, LNA®, ZNA™, non-natural bases: Plexor™ primer, Tiny-Molecular Beacon) see E. Navarro eta I..Clinica Chimica Acta, Volume 439, 15 January 2015, Pages 231-250.

Said probes may be dual-labeled oligonucleotides, such as hydrolysis probes or molecular beacons. The 5’ end of the oligonucleotide is typically labelled with a fluorescent reporter molecule while the 3’ end is labeled with a quencher molecule. The sequence of the probe is specific for a region of interest in the amplified target molecule. In a more preferred embodiment, said probe is a hydrolysis probe which is designed so that the length of the sequence places the 5’ fluorophore and the 3’ quencher in close enough proximity so as to suppress fluorescence. Several reporter molecules and quenchers for use in qPCR probes are well known in the art.

Generally, for the quantification of nucleotide sequences specific oligonucleotides, such as probes and / or primers, are used. The term "a primer and / or a probe" specifically includes "primers and / or probes". Both expressions are used interchangeably herein and encompass for example a primer; a probe; a primer and a probe; a pair of primers; and a pair of primers and a probe. Design and validation of primers and probes is well known in the art. For the design of primers and probes in quantitative real-time PCR methods, see for instance Rodriguez A et al. (Methods Mol Biol., 2015, 1275:31-56). Preferred primers and/or probes which may be used in the methods of the invention are described herein below under the kits of the invention.

Preferably, oligonucleotides useful in the methods of the 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, oligonucleotides specifically hybridizing with the target or reference sequence are about 19 to about 21 nucleotides in length. In a particular embodiment, said oligonucleotides have been modified for detection purposes or to enhance assay performance as described herein.

These oligonucleotides may be ribonucleotides or deoxyribonucleotides. In particular embodiments, the oligonucleotides may have at least one chemical modification. For instance, suitable oligonucleotides may be comprised of one or more“conformationally constrained” or bicyclic sugar nucleoside modifications, for example,“locked nucleic acids.” “Locked nucleic acids” (LNAs) are modified ribonucleotides that contain an extra bridge between the 2’ and 4’ carbons of the ribose sugar moiety resulting in a“locked” conformation that confers enhanced thermal stability to oligonucleotides containing the LNAs. In other embodiments, the oligonucleotides may comprise peptide nucleic acids (PNAs), which contain a peptide-based backbone rather than a sugar-phosphate backbone. Other chemical modifications that the oligonucleotides may contain include, but are not limited to, sugar modifications, such as 2’-0- alkyl (e.g. 2’-0-methyl, 2’-0-methoxyethyl), 2’-fluoro, and 4’ thio modifications, and backbone modifications, such as one or more phosphorothioate, morpholino, or phosphonocarboxylate linkages. For instance, these oligonucleotides, particularly those of shorter lengths (e.g., less than 15 nucleotides) can comprise one or more affinity enhancing modifications, such as, but not limited to, LNAs, bicyclic nucleosides, phosphonoformates, 2’ O-alkyl and the like. In some embodiments, the oligonucleotides may be chemically modified, for instance to improve their resistance to nuclease degradation (e.g., by end capping), to carry detection ligands (e.g., fluorescein) or to facilitate their capture onto a solid support (e.g., poly-deoxyadenosine "tails").

The term“quantification levels” might be the concentration (DNA amount per unit of volume), the DNA amount per number of cells, the cycle threshold value (Ct value) or any mathematical transformation thereof. In a preferred embodiment, the quantification of said bacterial sequences is performed by qPCR and the quantification levels are expressed as the Ct value. The Ct (cycle threshold) value is defined as the number of qPCR cycles required for the fluorescent signal to cross the threshold. Ct levels are 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).

Quantification of the abundance of a target nucleic acid sequence (e.g. SEQ ID NO: 1) in a sample of the invention might be absolute or relative. Relative quantification is based on one or more internal reference genes, i.e., 16S rRNA genes from reference strains, such as determination of total bacteria (Eubacteria) using universal primers and expressing the abundance of the target nucleic acid sequence as a percentage of Eubacteria (e.g. SEQ ID NO: 1/Eubacteria ratio). Absolute quantification gives the exact number of target molecules by comparison with DNA standards.

In a particular embodiment of the methods of the invention, prior to the quantification of said bacterial sequences, DNA is extracted from the sample. Several DNA extraction methods from samples are well known to the skilled person in the art.

In yet further preferred embodiments of the first or second aspects of the present invention, said molecular biology method is qPCR, preferably wherein the quantification levels are expressed as Ct value.

In yet further preferred embodiments of the first or second aspects of the present invention, said method further comprises storing the method results in a data carrier, preferably wherein said data carrier is a computer readable medium.

A third aspect of the invention refers to a computer implemented method, wherein the method is as defined as reflected in the second aspect of the invention, and the computer implements at least step c) and optionally d) of the method.

A fourth aspect of the invention refers to a data-processing apparatus comprising means for carrying out the steps of a method of the third aspect of the invention.

A fifth aspect of the invention refers to the use of a kit suitable for detecting and/or quantifying any of the bacterial species identified in the first aspect of the invention, preferably any of the bacterial markers selected from the list comprising or consisting of Ruminococcaceae NK4A214 group, Alloprevotella genus, Prevotella melanonigenica, and Ruminococcaceae UCG-014, or any combination thereof, for implementing any of the methods of the first or second aspect of the present invention.

Finally, the present invention further refers to a method of treating a subject having precancerous anal lesions consisting of high-degree squamous intraepithelial lesions (HSIL) or anal cancer, wherein said subject has been selected by a method for the screening, diagnosis and/or monitoring according to the invention, and wherein said method further comprises administering to the subject an anti-cancer therapy.

It is finally contemplated that any features described herein can optionally be combined with any of the embodiments of any method, medical use, kit and use of a kit of the invention; and any embodiment discussed in this specification can be implemented with respect to any of these. It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The use of the word "a" or "an" may mean "one," but it 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” may also refer to one or more. The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

As used in this specification and claim(s), 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 "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The term “comprises” also encompasses and expressly discloses the terms“consists of” and“consists essentially of”. As used herein, the phrase "consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. As used herein, the phrase "consisting of” excludes any element, step, or ingredient not specified in the claim except for, e.g., impurities ordinarily associated with the element or limitation.

The term "or combinations thereof” as used herein 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: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context. As used herein, words of approximation such as, without limitation, "about", "around”, “approximately” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as "about" may vary from the stated value by ±1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10%. Accordingly, the term“about” may mean the indicated value ± 5% of its value, preferably the indicated value ± 2% of its value, most preferably the term“about” means exactly the indicated value (± 0%).

The following examples serve to illustrate the present invention and should not be construed as limiting the scope thereof.

Examples

The present example shows a set of anal-associated bacterial taxa, obtained by anal cytobrush, that aids to significantly improve the accuracy of anal cytology for the diagnosis of biopsy- proven HSIL.

Materials and methods

Anal Cytology

A Dacron^® cytobrush moistened with tap water was used to sample the anal canal, and was rinsed in a vial containing 20 ml of PreservCyt (Hologic, Inc., Marlborough, MA, United States) fixative medium. The Bethesda System (TBS 2001) criteria were used for cytology reporting [Darragh TM, Birdsong G, Luff R, Davey D. The Bethesda system for reporting cervical cytology: definitions, criteria, and explanatory notes. 2nd ed. 2001] Cytological results were classified as unsatisfactory, negative, atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL), or HSIL.

HRA and histology

Concomitant HRA with biopsy of acetowhite lugol-negative lesions or suspicious of HSIL was offered for all participants. HRA was performed using the standard procedure, including topical application of 3% acetic acid and lugol solution in the anal canal. Anal biopsies were taken from suspicious areas revealed by HRA as acetowhite lugol-negative lesions with baby-Tischler forceps. The histologic results were been classified according to the Lower Anogenital Squamous Terminology (LAST) project recommendations [18] Results were reported as negative, LSIL, HSIL, or squamous cell carcinoma. When multiple biopsies were obtained, the most severe result was used as the histological diagnosis in the analysis. Treatment with infrared coagulation was offered to patients in whom the biopsy revealed high-degree intraepithelial lesions (HSIL). Biopsies were not obtained in participants who were evaluated as having no visual abnormalities suggestive of SIL on HRA examination, who were classified as being negative for SILs.

DNA extraction from tissue samples For DNA extraction in samples derived from anal cytobrushes, we followed the MasterPureTM DNA Purification Kit protocol (lllumina Inc., Madison. Wl). Total DNA, corresponding to genomic DNA of co-extracted bacterial and human DNA, was precipitated with isopropanol and subsequently washed with ethanol 70° and quantified using Qubit fluorometry.

16S RNA gene analysis (V3-V4 regions). Biodiversity and clustering

Amplicon data from the 16S rRNA gene was analyzed following the recommendations of the metagenomic state-of-the-art pipe line QIIME v1.8[19] 16S rRNA gene reads with low quality score (<20 out of 40 quality units assigned by the 454), short read lengths (<170 nucleotides) and singletons were filter and using the USEARCH[20] pipe line. Reference-based and de novo potential chimeras were also removed from the remaining sequences applying the UCHIME algorithm [Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 2011 ; 27:2194-200] taking as template the “Gold” database. Taxonomic information of the 16S rDNA sequences were obtained by comparison using the Ribosomal Database Project-ll (RDP) [Cole JR, Wang Q, Cardenas E, et al. The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res. 2009; 37:D141-5] and the Greengenes database available in QIIME v1.8 software. The Operational Taxonomic Units (OTUs) are the representation of the different clusters of species that are sharing the same microbiome. The criteria for collapsing each of the different sequences into OTUs are given by the percentage of identity between the different sequences, normally taken the 97% of similarity as a standard practice for mapping the 16S rRNA amplicon sequences to its corresponding species. OTUs were created by Uclust [Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 2010; 26:2460-1] applying a cluster criteria of 97% similarity. The most representative sequence for each OTU was then compared against the Qiime cluster version of the Greengenes database [DeSantis TZ, Hugenholtz P, Larsen N, et al. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl. Environ. Microbiol. 2006; 72:5069-72] (database 97_otus.fasta). The annotation was accepted when the bootstrap confidence estimation value was over 0.8, stopping the assignation at the last well-identified phylogenetic level. The genus abundance table was summarized from the resulting otu_table.biom file by the script summarize_taxa_through_plots.py. Representative sequences were aligned with Pynast [Nkuize M, Mulkay JP, Adler M, et al. Response of black African patients with hepatitis C virus genotype 4 to treatment with peg-interferon and ribavirin. Acta Gastroenterol. Belg. 2013; 76:291-299] against the clustered version of the Greengenes database (database core_set_aligned.fasta. imputed), to use as input to reconstruct the phylogenetic tree using the FastTree software [Price MN, Dehal PS, Arkin AP. FastTree 2-approximately maximum- likelihood trees for large alignments. PLoS One 2010; 5:e9490] Distances analysis between samples were performed by the weighted normalized Unifrac using the beta_significance.py (~ numjters 100) from the Qiime v1.8.0 pipe line to obtain the Principal Coordinates Analysis (PCoA). The Shannon index and the richness estimator Chad were calculated to assess the OTUs and genus diversity within the community using the alpha_diversity.py script from the Qiime pipeline.

Biomarker discovery

The linear discriminative analysis (LDA) effect size (LEfSE) biomarker discovery tool [Segata N, Izard J, Waldron L, et al. Metagenomic biomarker discovery and explanation. Genome Biol. 2011 ; 12:R60] was used to identify specific taxa as biomarkers for cases and controls. We fixed an alpha(a) value <0.05. The bacterial taxa with significant differences between samples were used to build the LDA model and to estimate its effect as a discriminant feature among them. The threshold used to consider a discriminative feature for the logarithmic LDA score was set to >2. We further examined the predictive ability of the standard cytology vs. each bacterial biomarker identified in the previous step by fitting consecutive multivariate logistic regression model. Finally, we tested whether the combination of 4 bacterial biomarkers incremented the predictive value of the liquid-based cytology by using a test of equality of the ROC areas of the predictive model including the anal cytology alone against the model including the cytology and the four biomarkers with the magnitude of association with bHSIL.

Results

We included 118 HIV+ and 33 HIV- men: 47 had bHSIL during the previous year and 12 at the moment of the inclusion. Differences in alpha and bacterial diversity were significant between mucosa and feces, but non-significant in the comparison by presence of bHSIL or HIV status. Linear discriminant analysis (LDA) effect size (LEfSe) revealed 40 biomarkers in samples derived from ano-rectal cytobrushes (figure 1) able to discrimate subjects with or without bHSIL. The characterization of these key taxa, alongside with their sequences and taxonomic information is provided in table 1 below.

Then, we explored the predictive value of the 15 taxa with greater LDA scores, we selected four taxa in anal samples. For example, genus was the most enriched taxon among subjects with bHSIL (OTU115 in figure 1). The method to calculate its diagnostic value was as follow:

1. We fitted a logistic regression model, being the continuous relative abundance of OTU1 15 the predictor (Iog10 transformed) and the presence of bHSIL the outcome:

Logistic regression Number of obs 146

Wald chi2 (1) 5.43

Prob > chi2 0.0197

Log pseudolikelihood -90.54847 Pseudo R2 0.0280

I Robust

bHSIL I Coef . Std. Err. z P>|z| [95% Conf . Interval] logOTU115 |

.418069

.179345

2.33

0.020

.0665593

.7695786

cons I -.8836587 .2024986 -4.36 0.000 -1.280549 -.4867688

2. We calculated the AUC, the diagnostic ability of the model by computing the post/estimation AUC, sensitivity and specificity. We selected the first cut-off reaching at specificity of 90% (highlighted below):

Detailed report of sensitivity and specificity

Correctly

Cutpoint Sensitivity Specificity Classified LR+ LR-

( >= 0 ) 100.00% 0.00% 33.56% 1.0000

( >= .30103 ) 28.57% 83.51% 65.07% 1.7321 0.8554

( >= 2.09691 ) 18.37% 92.78% 67.81% 2.5452 0.8798

( >= 2.103.. ) 16.33% 92.78% 67.12% 2.2624 0.9018 ( >= 2.10721 ) 16.33% 93.81% 67.81% 2.6395 0.8919

( >= 2.143.. ) 16.33% 94.85% 68.49% 3.1673 0.8822

( >= 2.206.. ) 14.29% 94.85% 67.81% 2.7714 0.9037

( >= 2.338.. ) 12.24% 94.85% 67.12% 2.3755 0.9252

( >= 2.348.. ) 10.20% 94.85% 66.44% 1.9796 0.9468

( >= 2.499.. ) 8.16% 94.85% 65.75% 1.5837 0.9683

( >= 2.503.. ) 8.16% 95.88% 66.44% 1.9796 0.9579

( >= 2.639.. ) 8.16% 96.91% 67.12% 2.6395 0.9477

( >= 2.720.. ) 8.16% 97.94% 67.81% 3.9592 0.9377

( >= 2.934.. ) 8.16% 98.97% 63.49% 7.9184 0.9279

( >= 3.001.. ) 8.16% 100.00% 69.18% 0.9184

( >= 3.332.. ) 6.12% 100.00% 68.49% 0.9388

( >= 3.547.. ) 4.08% 100.00% 67.81% 0.9592

( >= 3.550.. ) 2.04% 100.00% 67.12% 0.9796

( > 3.550.. ) 0.00% 100.00% 66.44% 1.0000

3. We repeated the logistic regression model using the dichotomized variable at the selected cut-off (in this case, 1.982):

Logistic regression Number of obs 146

Wald chi2 (1) 3.38

Prob > chi2 0.0659

Log pseudolikelihood = -91.470685 Pseudo R2 0.0181

4. We fitted a third regression model for the same taxa, exploring the predictive ability of the presence or absence of this taxa:

Logistic regression Number of obs 146

Wald chi2 (1) 2.83

Prob > chi2 0.0926

Log pseudolikelihood = -91.756598 Pseudo R2 0.0151

After repeating the same steps for the 15 taxa with highest LDA scores, we selected the two taxa with the highest AUC for diagnosis for bHSIL, Ruminococcaceae NK4A214 group and Alloprevotella genus, and the two taxa with greatest association (odds ratio) with bHSIL diagnosis, when explored as presence or absence ( Prevotella melanonigenica and Ruminococcaceae UCG- 014). Each 25% increase in the abundance of the Ruminococcaceae NK4A214 group and Alloprevotella genus were associated with a 17% (p=0.041) and 8% (p=0.016) increased risk of bHSIL, respectively. This was calculated by applying the formula: (logistic regression coefficient)*log(1.25). In addition, the absence of Prevotella melanonigenica and Ruminococcaceae UCG-014 were predictive of bHSIL (OR 6.1 , P=0.018 and OR 3.2, P=0.026, respectively).

Last, to determine the diagnostic value of the combination of these biomarkers when added to the anal cytology, we fitted a logistic regression model including these four taxa and the anal cytology as predictors, and the presence or absence of bHSIL as outcomes:

From 35 (94%) of false positive cytologic results, the combination of these four biomarkers reclassified to true negative 33 (94%), significantly improving the predictive performance of anal cytology alone to AUC 0.805 (Figure 2).

We show below the observed cases of bHSIL versus the predicted observations by our model:

Logistic model for hsil combined ever

True

Total I 47 81 I 128 While following this method we selected 4 bacteria which combination was able to improve the diagnostic accuracy measured as AUC of the standard procedure (anal cytology), all the taxa listed in table 1 have diagnostic value, as were identified using the LefSe biomarker discovery tool.

For example, for Fusobacterium nucleatum, a species listed in table 1 and previously linked with colorectal cancer:

Detailed report of sensitivity and specificity

Correctly

Cutpoint Sensitivity Specificity Classified LR+ LR-

As an additional example, we report below the diagnostic values of Bacteroides egghertii abundance, also listed in table 1 :

Detailed report of sensitivity and specificity

Correctly

Cutpoint Sensitivity Specificity Classified LR+ LR-

We have, therefore, found anal-associated bacteria indicative of higher risk of precancerous anal lesions, which combination was highly specific. Thus determining that the microbiota could be exploited as a complementary diagnostic tool for anal cytology to overcome the low specificity and high rate of false positive results of the current screening strategy for anal cancer screening.

Claims

1. A method for increasing the specificity or reducing the false positive rate of an anal cytology for the diagnosis and/or monitoring of precancerous anal lesions consisting of high-degree squamous intraepithelial lesions (HSIL) or anal cancer in a human subject, wherein the method comprises: optionally conducting an anal cytology in a sample isolated from the human subject, and further determining the presence or absence and/or quantifying in an anorectal epithelium sample isolated from said human subject, at least one of following bacterial markers or any combination thereof:

Sneathia sanguinegens, preferably by using SEQ ID NO 6 as a reference sequence for said detection (of the presence or absence) and/or quantification, Christensenellaceae_R-7_group, preferably by using SEQ ID NO 7 as a reference sequence for said detection (of the presence or absence) and/or quantification,

Bifidobacterium pseudocatenulatum IPLA36007, preferably by using SEQ ID NO 11 as a reference sequence for said detection (of the presence or absence) and/or quantification, - Acida mi nococcus genus, timonensis species, preferably by using SEQ ID NO 12 as a reference sequence for said detection (of the presence or absence) and/or quantification,

Treponema_2 genus, succinifaciens species, preferably by using SEQ ID NO 13 as a reference sequence for said detection (of the presence or absence) and/or quantification,

Fusobactehum genus, nucleatum species, preferably by using SEQ ID NO 14 as a reference sequence for said detection (of the presence or absence) and/or quantification,

Megasphaera elsdenii, preferably by using SEQ ID NO 16 as a reference sequence for said detection (of the presence or absence) and/or quantification, S5-A 14a genus, preferably by using SEQ ID NO 17 as a reference sequence for said detection (of the presence or absence) and/or quantification,

Bacteroides eggerthii, preferably by using SEQ ID NO 20 as a reference sequence for said detection (of the presence or absence) and/or quantification, Ruminococcus_1 genus, preferably by using SEQ ID NO 21 as a reference sequence for said detection (of the presence or absence) and/or quantification, Undibacterium pigrum, preferably by using SEQ ID NO 22 as a reference sequence for said detection (of the presence or absence) and/or quantification,

- Actinotignum timonense, preferably by using SEQ ID NO 23 as a reference sequence for said detection (of the presence or absence) and/or quantification, Subdoligranulum variabile, preferably by using SEQ ID NO 24 as a reference sequence for said detection (of the presence or absence) and/or quantification, and/or

Reyranella massiliensis 521, preferably by using SEQ ID NO 25 as a reference sequence for said detection (of the presence or absence) and/or quantification, wherein a combined score obtained from the bacterial markers determined, aids classifying an anal cytology positive subject as presenting an increased or reduced risk of having precancerous anal lesions consisting of high-degree squamous intraepithelial lesions (HSIL) or anal cancer, by increasing the specificity of the anal cytology.

2. The method of claim 1 , wherein said determination of the presence or absence and/or quantification in an anorectal epithelium sample isolated from said human subject of the one or more bacterial markers, increases the specificity of an anal cytology to at least 85%.

3. The method of any of claims 1 or 2, wherein said one or more bacterial markers is Ruminococcaceae NK4A214 group, wherein an increase in the abundance of Ruminococcaceae NK4A214 group in the isolated sample in comparison to a reference sample from a healthy population of individuals, is associated with an increased risk of bHSIL.

4. The method of any of claims 1 to 3, wherein said one or more bacterial markers is Alloprevotella genus, wherein an increase in the abundance of Alloprevotella genus in the isolated sample in comparison to a reference sample from a healthy population of individuals, is associated with an increased risk of bHSIL.

5. The method of any of claims 1 to 4, wherein said one or more bacterial markers is Prevotella melanonigenica, wherein the absence of Prevotella melanonigenica in the isolated sample is associated with an increased risk of bHSIL.

6. The method of any of claims 1 to 5, wherein said one or more bacterial markers is Ruminococcaceae UCG-014, wherein the absence of Ruminococcaceae UCG-014 in the isolated sample is associated with an increased risk of bHSIL.

7. The method of any of claims 1 to 6, wherein said one or more bacterial markers are selected from the combination of markers from the list consisting of Ruminococcaceae NK4A214 group, Alloprevotella genus, Prevotella melanonigenica, and Ruminococcaceae UCG-014.

8. The method of any of claims 1 to 7, wherein the anorectal epithelium sample is collected or isolated from cytobrushes with and without spatulas, cytopicks, cotton, Dacron, rayon, or nylon-flocked (NF)-swabs.

9. A method for the diagnosis and/or monitoring of precancerous anal lesions consisting of high-degree squamous intraepithelial lesions (HSIL) or anal cancer in a human subject, wherein the method comprises:: a) conducting an anal cytology in a sample isolated from a human subject; b) detecting the presence or absence and/or quantifying at least one of the bacterial markers identified in any of claims 1 to 7 in an anorectal epithelium sample isolated from said human subject, preferably collected or isolated from cytobrushes with and without spatulas, cytopicks, cotton, Dacron, rayon, or nylon-flocked (NF)-swabs; c) calculating a combined score from the bacterial markers determined in b); and d) classifying an anal cytology positive subject as presenting an increased or reduced risk of having precancerous anal lesions consisting of high-degree squamous intraepithelial lesions (HSIL) or anal cancer, according to the combined score obtained in c).

10. The method according to claim 9, wherein in step d) said method comprises comparing the combined score in the subject’s sample with a reference combined score; and wherein a difference of the combined score in the subject’s sample with regard to said reference combined score is indicative of precancerous anal lesions consisting of high-degree squamous intraepithelial lesions (HSIL) or anal cancer.

11. The method according to any of claims 9 or 10, wherein in step b) the bacterial markers identified are the ones indicated in any of claims 3 to 7 or any combination thereof.

12. The method according to any of claims 1 to 11 , wherein said subject is a subject suspected of having precancerous anal lesions consisting of high-degree squamous intraepithelial lesions (HSIL) or anal cancer.

13. The method according to any of claims 1 to 12, wherein said subject is an asymptomatic subject.

14. The method according to any of claims 1 to 13, wherein determination of the bacterial markers is conducted by a molecular biology method selected from the group consisting of next generation sequencing, quantitative PCR (qPCR), PCR-pyrosequencing, PCR-ELISA, DNA microarrays, branched DNA, dot-blot, Fluorescence In Situ Hybridization assay (FISH), and multiplex versions of said methods.

15. The method according to claim 14, wherein said molecular biology method is qPCR, preferably wherein the quantification levels are expressed as Ct value.

16. A method according to any of claims 9 to 15, wherein said method further comprises storing the method results in a data carrier, preferably wherein said data carrier is a computer readable medium.

17. A computer implemented method, wherein the method is as defined in any of claims 9 to 15, and the computer implements at least step c) and optionally d) of the method.

18. A data-processing apparatus comprising means for carrying out the steps of a method of claim 17.

19. Use of a kit suitable for detecting and/or quantifying any of the bacterial species identified in any of claims 1 to 7, or any combination thereof, in a method for the screening, diagnosis and/or monitoring of precancerous anal lesions consisting of high-degree squamous intraepithelial lesions (HSIL) or anal cancer, according to any of claims 1 to 18.

20. A method of treating a subject having precancerous anal lesions consisting of high- degree squamous intraepithelial lesions (HSIL) or anal cancer, wherein said subject has been selected by a method for the screening, diagnosis and/or monitoring according to any of claims 1 to 18, and wherein said method further comprises administering to the subject an anti-cancer therapy.