CN116782950A - Assessment of risk of colorectal adenoma recurrence by non-invasive methods - Google Patents

Abstract

The present invention provides non-invasive methods for assessing the likelihood of colorectal adenoma recurrence in patients who have previously undergone surgery for colorectal cancer or adenoma resection, as well as methods for reducing the risk of colorectal adenoma recurrence in these patients. . Kits for such methods are also available.

Description

Assessing the risk of colorectal adenoma recurrence through non-invasive methods

Related applications

This application claims priority from U.S. Provisional Patent Application No. 63/116,104, filed on November 19, 2020, and U.S. Provisional Patent Application No. 63/196,582, filed on June 3, 2021, the contents of each of which are based on This document is incorporated by reference in its entirety for all purposes.

Background technique

Colorectal cancer (CRC) is the third most common cancer and the third leading cause of cancer death worldwide. Despite ongoing efforts to control the incidence of new CRC, the incidence and mortality of CRC are rapidly increasing and maintaining an upward trend in Asian countries. The global persistence of CRC requires a paradigm shift in its management strategies from clinical treatment to preclinical prevention. Early detection of colorectal adenomas (precancerous growths in the form of polyps or cysts) and appropriate treatment to prevent progression is critical for colorectal cancer. Therefore, there is an urgent need for new, reliable methods to assess the likelihood of colorectal adenoma recurrence in patients, especially those who have previously undergone polypectomy.

Microbial markers have previously been shown to be useful in the noninvasive diagnosis of colorectal cancer and adenomas. However, it is unclear whether such biomarkers can be used to predict adenoma recurrence in patients after resection of advanced adenomas. The present inventors studied microbial markers, namely Fusobacterium nucleatum (Fn), Bacteroides clarus (Bc), in the feces of patients before and after colonoscopy in which colorectal adenomas were removed. , Clostridium hathewayi (Ch), Roseburia intestinalis (Ri), and Lachnoclostridium marker m3. Significant increases in m3, Fn, and Ch were detected as well as a positive correlation between the composite score of the 4Bac (Fn, m3, Bc, and Ch) group and recurrence. Therefore, these fecal microbial markers can be used for non-invasive prediction of colorectal adenoma recurrence risk and diagnosis of adenoma recurrence. This finding therefore provides a new, important, and improved means for early detection of colorectal adenomas and effective prevention of colorectal cancer without the need for invasive measures.

Contents of the invention

The present inventors have discovered a correlation between the recurrence of colorectal adenomas and elevated levels of certain bacterial species in patients who have previously undergone polypectomy. Accordingly, a first aspect of the invention provides a method for assessing a patient's risk of experiencing recurrent colorectal adenomas following previous surgery to remove colorectal cancer or adenomas. The method includes the following steps: (a) obtaining Lacchnoclostridium species (m3), Fusobacterium nucleatum (Fn) carrying genetic marker m3 from a first stool sample collected from the individual prior to resection of the colorectal cancer or adenoma, Baseline levels of one or more of the four bacterial species Clostridium hathawayuis (Ch) and Bacteroides clarusii (Bc); (b) collected from individuals after resection of colorectal cancer or adenoma Obtain follow-up levels of one or more of Fn, m3, Bc, and Ch in a second stool sample; (c) Based on any of the four bacterial species Fn, m3, Bc, and Ch by the method specified in Table 1 calculating a composite score for one or more of the baseline and follow-up levels; and (d) detecting that the value from step (c) is higher than the standard control value, identifying the individual as having an increased risk of colorectal adenoma recurrence. On the other hand, when the follow-up value is no higher than the standard control value, the individual is considered not to have an increased risk of colorectal adenoma recurrence. Standard control values are based on one (Fn or m3 or Ch), two (m3 and Fn, or m3 and Ch), three (Fn, m3 and Ch), or four bacterial species Fn, m3, Bc and Ch ) is a composite score calculated at baseline and follow-up levels from a control group of individuals who have not experienced colorectal adenoma recurrence (e.g., approximately 3 to 10 years after removal of colorectal cancer or adenoma) of. In some cases, the claimed method for evaluating a patient experiencing recurrent colorectal adenoma following prior surgery to remove a colorectal cancer or adenoma is performed according to the steps described above by determining the level of one or more bacterial markers. In the method of risk, the bacterial marker is determined based on the level of one or more of the nucleotide sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22.

Similarly, another method for assessing an individual's risk of colorectal adenoma recurrence after colorectal cancer or adenoma resection is provided. The method includes the following steps:

(a) The following values are obtained from stool samples collected from individuals following resection of colorectal cancer or adenomas:

or

(2) A composite score of the levels of m3 and Ch of the two bacterial species, calculated by I2+β5*m3+β6*Ch; or

(3) Comprehensive score of the levels of Fn, m3 and Ch of the three bacterial species, which is calculated by

I3+β7*Fn+β8*m3+β9*Ch; or

(4) Comprehensive score of the levels of Fn, m3, Bc and Ch of the four bacterial species, which is calculated by the following

I1+β1*Fn+β2*m3+β3*Bc+β4*Ch; and

(b) Detecting a value from step (a) that is higher than a standard control value identifies an individual as having an increased risk of colorectal adenoma recurrence. Standard control values are values for the same category (i.e., Fn levels) or m3 or Ch; or based on two (m3 and Ch), three (Fn, m3, and Ch), or four bacterial species (Fn, m3, Bc, and A composite score calculated from the level of Ch), which is established from a control group of individuals who have not experienced colorectal adenoma recurrence (e.g., approximately 3 to 10 years after removal of colorectal cancer or adenoma). In some cases, the claimed method for evaluating a patient experiencing recurrent colorectal adenoma following prior surgery to remove a colorectal cancer or adenoma is performed according to the steps described above by determining the level of one or more bacterial markers. In the method of risk, the bacterial marker is determined based on the level of one or more of the nucleotide sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22.

In some embodiments of either method, the individual has a colorectal adenoma, such as a polyp or cyst removed by polypectomy, or a CRC removed by surgery. In some embodiments, the combined score for the levels of any two, three or four of the four bacterial species Fn, m3, Bc and Ch is calculated by the method specified in Table 1. In some embodiments, the genome of m3 includes the nucleotide sequence of SEQ ID NO: 19. In some embodiments, the genome of Ch includes the nucleotide sequence of SEQ ID NO:20. In some embodiments, the genome of Fn includes the nucleotide sequence of SEQ ID NO:21. In some embodiments, the genome of Bc includes the nucleotide sequence of SEQ ID NO:22. In some embodiments of either method, each of steps (a) and/or (b) includes obtaining DNA, RNA, or protein unique to at least one of the bacterial species Fn, m3, Bc, and Ch. level. In some embodiments of either method, each of steps (a) and/or (b) includes polymerase chain reaction (PCR), such as quantitative polymerase chain reaction (qPCR) or reverse transcription-polymerization Enzyme chain reaction (RT-PCR) for determination of bacterial species levels. In some embodiments of either method, a post-resection fecal sample is collected from the individual about 1 to about 5 years after the initial resection of the colorectal cancer or adenoma, e.g., about one year after the resection of the colorectal cancer or adenoma. Fecal samples were collected from individuals. In some embodiments of either method, the method further includes, after determining in step (b) or (c) that the individual is at increased risk for colorectal adenoma recurrence, performing periodic (eg, annual) colonoscopies to The step of monitoring the individual or administering to the individual an inhibitor that inhibits or eliminates one or more of the bacterial species Fn, m3, Bc and Ch in the individual, particularly in the gastrointestinal tract. In some embodiments of either method, the inhibitor is a small inhibitory RNA or antisense oligonucleotide that specifically targets at least one gene of one or more of the bacterial species Fn, m3, Bc, and Ch , or an expression cassette directing the expression of inhibitory RNA, or a viral vector including an expression cassette.

In a second aspect, the present invention provides a kit for assessing an individual's risk of colorectal adenoma recurrence after colorectal cancer or adenoma resection, comprising: (1) a first container containing an assay for determining a reagent for determining the level of bacterial species Fn; and (2) a second container containing a reagent for determining the level of bacterial species m3. In some embodiments, the kit further includes a third container containing one or more reagents for determining the level of bacterial species Bc. In some embodiments, the kit further includes a third container containing one or more reagents for determining the level of bacterial species Ch.

In some embodiments, the reagents in each container are reagents for polymerase chain reaction (PCR), eg, qPCR or RT-PCR. In some embodiments, the reagents in each container are reagents for detecting proteins unique to bacterial species such as Fn, m3, Bc, or Ch.

In a third aspect, the present invention provides a method for reducing the risk of colorectal adenoma recurrence in an individual following removal of colorectal cancer or adenoma. Methods include administering to a subject an effective amount of an inhibitor that inhibits or eliminates one or more of the bacterial species Fn, m3, and Ch in the subject, particularly in the gastrointestinal tract.

In some embodiments, the inhibitor is a small inhibitory RNA or antisense oligonucleotide that specifically targets at least one gene of one or more of the bacterial species Fn, m3, and Ch, or a guide inhibitory RNA An expression cassette for expression, or a viral vector including an expression cassette. In some embodiments, the expression cassette is included within the viral particle. In some embodiments, the method further includes, following the step of administering, the step of determining the levels of one or more bacterial species Fn, m3, and Ch in the feces of the individual. In some embodiments, the step of administering is performed within about one year after removal of the colorectal cancer or adenoma. In some embodiments, the administering step is performed multiple times (eg, once a year) over a period of time from about one year to about five or ten years after the initial resection of the colorectal cancer or adenoma. In some embodiments, the target gene is within the nucleotide sequence of SEQ ID NO: 19 (e.g., in the m3 genome), or within the nucleotide sequence of SEQ ID NO: 20 (e.g., in the Ch genome) , or within the nucleotide sequence of SEQ ID NO: 21 (e.g., in the Fn genome).

In a fourth aspect, the invention provides a kit for reducing the risk of colorectal adenoma recurrence, comprising: (1) a first container containing a method for determining one or more bacterial species Fn, m3 and one or more reagents at levels of Ch; and (2) a second container containing a composition that includes an effective amount of an inhibitor that inhibits or eliminates the bacterial species Fn, m3, and Ch one or more of them. In some embodiments, the first container includes PCR reagents for determining the levels of DNA or RNA of one or more of the bacterial species Fn, m3, and Ch, such as primers or probes for PCR, such as qPCR or RT-PCR. In some embodiments, the inhibitor is a small inhibitory RNA or antisense oligonucleotide that specifically targets at least one gene of one or more of the bacterial species Fn, m3, and Ch, or a guide inhibitory RNA An expression cassette for expression, or a viral vector including an expression cassette.

Description of drawings

Figure 1. Changes in fecal bacterial markers in subjects after polypectomy compared with patients with advanced adenoma at baseline. (Figure 1, A) Subject recruitment strategy and sample/subject categories in this study. (Figure 1, B) Comparison of subjects who developed recurrent adenomas (R) and no recurrence (no-R) in cohort III with paired baseline and follow-up stools diagnosed late at index colonoscopy Levels of four fecal bacterial markers between baseline stool samples collected before adenoma and follow-up stool samples before surveillance colonoscopy. (Fig. 1, C) Comparison of the levels of four fecal bacterial markers between baseline and follow-up stool samples from all subjects recruited in this study. (Fig. 1, D) In stool samples from patients with baseline adenomas (D1) and recurrent adenomas (D2), m3, Fn, and Ch levels did not differ between patients with proximal and distal lesions. Fn, Fusobacterium nucleatum; m3, Lachnoclostridium marker m3; Ch, Clostridium hathawayii; Bc, Bacteroides clarusii.

Figure 2. Comparison of baseline (Figure 2, A) and follow-up (FU) (Figure 2, B) levels of four bacterial markers and their composite score 4Bac between patients with and without recurrent adenomas. Only patients with baseline and FU stools were included here to evaluate the effect of baseline and FU marker levels in predicting recurrent adenomas. no-R, no recurrence; R, recurrence; Fn, Fusobacterium nucleatum; m3, Lacchnoclostridium marker m3; Ch, Clostridium hathawayii; Bc, Bacteroides clarusii; 4Bac: Fn, Comprehensive score of m3, Ch and Bc.

Figure 3. Diagnostic performance of bacterial markers in predicting recurrent adenomas during follow-up (FU) surveillance. (A of Figure 3) Receiver operating characteristic (ROC) curve analysis and diagnostic performance of Fusobacterium nucleatum (Fn), Lachnoclostridium marker m3, Clostridium hathawayii (Ch), and comprehensive score 4Bac for distinguishing presence and absence Patients with recurrent adenomas. CI, confidence interval; PPV, positive predictive value; NPV, negative predictive value. (Fig. 3, B) Performance of logistic regression models involving different sets of markers in distinguishing patients with relapse from those without relapse. AUROC, area under ROC; no-R, no recurrence; R, recurrence.

Figure 4. Changes in bacterial markers at follow-up (FU) versus baseline predict adenoma recurrence. (Figure 4, A) Four bacterial markers and their combined score 4Bac showed no significant differences between baseline and FU stool in patients without recurrence. Significant increases in m3 and 4Bac were detected in FU stool compared with baseline stool in relapsed patients. (Fig. 4, B) Performance of a logistic regression model involving changes in FU stool versus baseline stool in distinguishing patients with relapse from those without relapse. The model combining changes in Fn, m3 and Ch showed the best performance. Fn, Fusobacterium nucleatum; m3, Lachnoclostridium marker m3; Ch, Clostridium hathawayii; Bc, Bacteroides clarusii; 4Bac: Fn, m3, Ch, and Bc previously designed for diagnosis of CRC and adenomas Overall rating. no-R, no recurrence; R, recurrence.

Figure 5. Validation of a new logistic regression model involving levels of Fusobacterium nucleatum (Fn), the Lachnoclostridium marker m3, and Clostridium hathawayii (Ch) in follow-up stools for the diagnosis of recurrent adenomas. Figure 5 Diagnostic performance of A new model (A1) and FIT (A2) with reference to colonoscopy and histology results. Figure 5 B Comparison of the comprehensive scores of patients with recurrent adenoma and subjects without recurrence and ROC curve analysis of the comprehensive scores. R, recurrence; no-R, no recurrence.

definition

In this disclosure, the terms "colorectal cancer (CRC)" and "colon cancer" have the same meaning and refer to cancer of the large intestine (colon), the lower part of the human digestive system, although rectal cancer is generally more specifically Cancer of the last few inches of the colon or rectum. "Colorectal cancer cells" are colon epithelial cells that have characteristics of colon cancer and encompass precancerous cells that are in the early stages of transforming into cancer cells or are prone to transforming into cancer cells. Such cells can exhibit one or more phenotypic traits characteristic of cancerous cells.

As used herein, the term "colorectal adenoma" refers to a precancerous growth, or precursor to CRC, in the form of a polyp or cyst that, if left untreated (usually by colonoscopy such as polypectomy or by surgery resection) can progress to CRC.

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single- or double-stranded form and polymers thereof. Unless specifically limited, the term encompasses nucleic acids including known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless stated otherwise, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and Complementary sequences as well as clearly stated sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixed base and/or a deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, and the mRNA encoded by a gene.

The term "gene" refers to the DNA segment involved in the production of a polypeptide chain; it includes the regions preceding and following the coding region (leader and tail) involved in the transcription/translation of the gene product and the regulation of transcription/translation, as well as the individual coding segments ( intervening sequences (introns) between exons).

In this application, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the term encompasses amino acid chains of any length, including the full-length protein of interest, in which the amino acid residues are linked by covalent peptide bonds.

The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. For the purposes of this application, amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., carbon bound to hydrogen, carboxyl, amino, and R groups, such as homoserine, norleucine, Methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones but retain the same basic chemical structure as the naturally occurring amino acid. For the purposes of this application, an amino acid mimetic refers to a compound that has a structure that differs from the usual chemical structure of an amino acid, but functions in a manner similar to naturally occurring amino acids.

Amino acids may include those with non-naturally occurring D-chirality, as disclosed in WO01/12654, which may improve the stability (eg, half-life), bioavailability of polypeptides including one or more such D-amino acids. degree and other characteristics. In some cases, one or more, and possibly all, amino acids of a therapeutic polypeptide possess D-chirality.

Amino acids may be represented herein by the commonly known three-letter symbols, or by the single-letter symbols recommended by the IUPAC-IUB Committee on Biochemical Nomenclature. Likewise, nucleotides can be represented by their commonly accepted single-letter codes.

As used herein, the term "gene expression" is used to refer to the transcription of DNA to form an RNA molecule encoding a specific protein, or the translation of a protein encoded by a polynucleotide sequence. In other words, the term "gene expression level" in this disclosure encompasses both the mRNA level and the protein level encoded by the target gene.

An "expression cassette" is a recombinantly or synthetically produced nucleic acid construct having a specified set of nucleic acid elements that permit the transcription of a specific polynucleotide sequence in a host cell, e.g., the transcription of an inhibitory RNA (e.g., such as a miRNA or siRNA) or antisense RNA targeting specific preselected sequences (e.g., Fusobacterium nucleatum (Fn), Bacteroides clarusii (Bc), Clostridium hathawayii (Ch), or Lachnoclostridium species carrying the marker m3 genomic sequence (m3 ) section). The expression cassette may be part of a plasmid, viral genome or nucleic acid fragment. In other words, the expression cassette can be transferred or delivered as part of and/or in the form of a bacterial plasmid or viral vector or virus-like particle. Typically, an expression cassette includes a polynucleotide to be transcribed operably linked to a promoter. In this context, "operably linked" means that two or more genetic elements (such as a polynucleotide coding sequence and a promoter) are positioned to permit the elements to perform their appropriate biological function (such as a promoter directing transcription of the coding sequence). ) relative position. Other elements that may be present in the expression cassette include elements that enhance transcription (eg, enhancers) and terminate transcription (eg, terminators), as well as elements that confer certain binding affinity or antigenicity to the recombinant protein produced by the expression cassette.

As used in this application, "increase" or "decrease" refers to a detectable positive or negative change in quantity compared to a comparative control, such as an established standard control or cutoff value. or baseline value. An increase is a positive change that is typically at least 10%, or at least 20%, or 50%, or 100% of the control value, and can be as high as at least 2 times, or at least 5 times, or even 10 times the control value. Likewise, a decrease is typically a negative change of at least 10%, or at least 20%, 30%, or 50%, or even up to at least 80% or 90% of the control value. Other terms indicating a change or difference in quantity compared to a baseline, such as "more than," "less than," "higher than," "less than," "greater than," and "less than," are used herein as Used in the same manner as above. In contrast, the terms "substantially the same" or "substantially no change" indicate a minimal or no change in quantity compared to a standard control value, typically within ±10% of the standard control, or ±5% , within ±2%, ±1%, or even with smaller changes compared to the standard control.

As used herein, the term "inhibiting/inhibition" refers to the inhibition of a target biological process such as RNA transcription, protein expression, cell proliferation, cell signaling, cell proliferation, tumorigenicity, metastatic potential, and disease/condition. any detectable negative effects of recurrence). Typically, inhibition is reflected as the level of relevant bacteria during the target process of administration of the inhibitor (e.g., such as Fn, Bc, Ch, or m3; or the development of colorectal adenomas) when compared to control values without administration of the inhibitor. rate) by at least 10%, 20%, 30%, 40% or 50%.

As used herein, "primer" refers to a unique polynucleotide sequence that can be used in an amplification method, such as the polymerase chain reaction (PCR), based on a sequence corresponding to a gene of interest, e.g., from a related bacterial species. , oligonucleotides that amplify nucleotide sequences from polynucleotide sequences such as gene markers m1704941 (from Fn), m370640 (from Bc) and m2736705 (from Ch), see, for example, WO2018/036503). For example, "primers" can be used in reverse transcription-polymerase chain reaction (RT-PCR) and quantitative polymerase chain reaction (qPCR) to quantify gene expression. Typically, at least one PCR primer used to amplify a polynucleotide sequence is sequence specific for the polynucleotide sequence. The exact length of the primer depends on a variety of factors, including temperature, primer source, and method used. For example, for diagnostic and prognostic applications, oligonucleotide primers typically contain at least 10, or 15, or 20, or 25 or more nucleotides, although they may contain fewer nucleosides, depending on the complexity of the target sequence. acid or more nucleotides. The factors involved in determining appropriate primer lengths are well known to those skilled in the art. Primers used in specific embodiments are shown in the Examples of the present disclosure, in which their specific applications are illustrated. In this disclosure, the term "primer pair" refers to a pair of primers that hybridize to opposite strands of a target DNA molecule, or to a region of target DNA flanking the nucleotide sequence to be amplified. In this disclosure, the term "primer site" refers to a region of target DNA or other nucleic acid to which a primer hybridizes.

As used in this application, the term "amount" or "level" refers to the amount of a bacterial species of interest present in a sample, for example, Fn, Bc, Ch or m3. Such amounts may be expressed as absolute values, i.e., the total amount of Fn, Bc, Ch, or m3 in the sample, or as relative values, i.e., as a percentage of Fn, Bc, Ch, or m3 among all bacteria in the sample.

As used in this application, the term "treating" describes actions that achieve elimination, alleviation, relief, reversal, or prevention or delay of the onset or recurrence of any symptoms of a related condition. In other words, "treating" a condition encompasses both therapeutic and preventive interventions for that condition.

As used herein, the term "effective amount" refers to an amount of a given substance sufficient to produce the desired effect. For example, an effective amount of an inhibitor of a particular bacterial species, such as Fn, Bc, Ch, or m3, is one that results in a reduction (including to undetectable levels) of inhibitor. As another example, an effective amount of an inhibitor is an amount that, when administered to a patient, is capable of achieving a detectable level of reduction in the risk of colorectal adenoma recurrence in the patient. The amount sufficient to achieve the desired effect in a therapeutic context is defined as a "therapeutically effective dose." The dosage range will vary depending on the nature of the therapeutic agent administered, as well as other factors such as the route of administration and the severity of the patient's condition.

The term "antimicrobial agent" refers to any substance capable of inhibiting, inhibiting, eliminating or preventing the growth or proliferation of bacterial species such as Fn, Bc, Ch or m3 respectively. Known agents with antimicrobial activity include various antibiotics that generally inhibit the proliferation of a broad spectrum of bacterial species as well as agents that can inhibit the proliferation of specific bacterial species such as antisense oligonucleotides, small inhibitory RNAs, and the like.

"Percent relative abundance", when used in the context of describing a specific bacterial species (e.g., Fn, Bc, Ch, or m3) in relation to all bacterial species present in the same environment, respectively, refers to the bacterial species' relative abundance among all bacterial species, respectively. A relative amount within a species, expressed as a percentage. For example, the percent relative abundance of a specific bacterial species can be determined by comparing the amount of DNA or RNA specific to that species in a given sample (e.g., by quantitative polymerase chain reaction including reverse transcription (RT)-PCR (PCR) determination) is determined by comparison with the amount of all bacterial DNA in the same sample (e.g., determined by quantitative PCR including RT-PCR and sequencing based on 16S rRNA sequence).

"Absolute abundance", when used in the context of describing the presence of a specific bacterial species (e.g., Fn, Bc, Ch, or m3) in a sample (e.g., a fecal sample collected from a test subject), refers to the presence of The amount of DNA from each bacterial species out of the total amount of DNA in the sample. For example, the absolute abundance of a bacterium can be determined by comparing the amount of DNA specific to that bacterial species in a given sample (e.g., determined by quantitative PCR including RT-PCR) to the amount of all DNA in the same sample. Compare to determine.

As used herein, the "total bacterial load" of a sample refers to the amount of all bacterial DNA relative to the amount of all DNA in the sample, respectively. For example, the absolute abundance of bacteria can be determined by comparing the amount of bacterial-specific DNA (eg, 16S rRNA determined by quantitative RT-PCR) in a given sample to the amount of all DNA in the same sample.

"Inhibitors", "activators" and "modulators" of relevant bacterial species (such as Fn, Bc, Ch or m3) refer to inhibitory, activating or regulatory molecules identified using in vitro and in vivo assays, respectively. molecules because of their ability to positively or negatively regulate bacterial proliferation or survival. The term "modulator" includes inhibitors and activators. For example, an inhibitor may partially or completely block binding, reduce, prevent, delay activation, inactivate, desensitize or downregulate the levels of associated proteins by inhibiting downstream effects, such as the growth or survival of colorectal cancer cells, or amount of reagent. In some cases, inhibitors bind directly or indirectly to targeted DNA or RNA such as antisense molecules or microRNA. Inhibitors are used herein synonymously with inactivators and antagonists. For example, an activator is an agent that may stimulate, increase, promote, enhance activation, sensitize or upregulate the level or amount of a relevant protein by promoting downstream effects, such as the growth or survival of colorectal cancer cells. Inhibitors, activators and modulators can be large molecules, such as polynucleotides, polypeptides including antibodies and antibody fragments, or they can be small molecules, including carbohydrate-containing molecules, siRNA, RNA aptamers, and the like.

As used herein, "standard control" refers to an average level corresponding to a preselected bacterial species found in a specific type of sample (e.g., a stool sample) obtained from an individual who has not had a recurrence of colorectal adenoma or based on The value of the composite score is calculated from the average level of multiple bacterial species found in the sample types collected from these individuals. For example, to examine patients who have undergone earlier surgery for colorectal cancer or adenoma resection, a "standard control" value is established to provide a cutoff value for whether the examined patient is at increased risk for colorectal adenoma recurrence. To properly establish a "standard control," a sufficient number of relapse-free individuals (e.g., at least 10, 12, 15, 20, 24, or more individuals) must be included in the control group to provide the sample To determine the average level of one or more preselected bacterial species, or a composite score calculated based on the levels of multiple bacterial species that represent the risk of colorectal adenoma recurrence.

As used herein, the term "about" means a range of values encompassing +/-10% of a predetermined value. For example, "about 10" means 9 to 11.

In this disclosure, the term "or" is generally used in its sense including "and/or" unless the content clearly dictates otherwise.

Detailed ways

I.Introduction

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Patients after polypectomy often undergo follow-up colonoscopies to detect adenoma recurrence at recommended intervals. On the other hand, the association between gut microbiota composition and adenoma recurrence after polypectomy has not been studied. A new bacterial marker m3 was previously reported to be useful in the noninvasive diagnosis of colorectal adenomas and cancers. In the present disclosure, the inventors identify novel fecal microbiome markers to predict the risk of colorectal adenoma recurrence after polypectomy.

This study included 104 patients from the Polyp Surveillance Study from 2009 to 2019. Stool samples were collected before index colonoscopy (baseline) and surveillance colonoscopy (>6 months after polypectomy). Recurrence was defined as the detection of new adenomas on colonoscopy after polypectomy. Four candidate markers including Fusobacterium nucleatum (Fn), Lacchnoclostridium marker (m3), Clostridium hathawayii (Ch), and Bacteroides clarusii were detected at baseline and follow-up by quantitative polymerase chain reaction (qPCR) Quantification in fecal samples. A fecal immunochemical test (FIT) was performed on follow-up stools.

Each of the bacterial markers Fn, m3, and Ch detected in follow-up stool samples differed significantly between subjects with adenoma recurrence and those without recurrence (n = 104; all P < 0.05). A logistic regression model combining Fn, m3 and Ch showed an area under the receiver operating characteristic curve (AUROC) of 0.741 (P<0.0001) [sensitivity=81.3%; specificity for predicting adenoma recurrence=55.4%]. Adding FIT to bacterial markers did not improve diagnostic accuracy. Among subjects with paired baseline and follow-up stool samples (n=43), m3 (P=0.006) and Fn (P=0.07) levels were higher in subjects who developed recurrent adenomas. A logistic regression model incorporating changes in fecal Fn, m3, and Ch levels at follow-up compared with baseline samples showed an AUROC for predicting risk of adenoma recurrence of 0.950 (P<0.0001) [sensitivity = 90.0%; specificity = 87.0%]. Therefore, this study identifies a novel set of fecal microbiome markers for predicting adenoma recurrence after polypectomy. These noninvasive markers may improve colonoscopy surveillance programs.

II.General approach

The practice of the invention utilizes conventional techniques in the art of molecular biology. Basic textbooks disclosing general methods for use in this invention include: Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed., 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (eds. Ausubel et al., 1994).

For nucleic acids, the size is given in kilobase pairs (kb) or base pairs (bp). Nucleic acid sizes are estimates obtained from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, the size is given in kilodaltons (kDa) or number of amino acid residues. Protein size was estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.

For example, using the automated synthesizer described in Van Devanter et al., Nucleic Acids Res. 12:6159-6168 (1984), the solid phase subcontractor was first described in Beaucage and Caruthers, Tetrahedron Lett. 22:1859-1862 (1981). The phosphoramidite triester method can chemically synthesize oligonucleotides that are not commercially available. Oligonucleotides are performed using any art-recognized strategy, such as native acrylamide gel electrophoresis or anion-exchange high-performance liquid chromatography (HPLC) as described in Pearson and Reanier, J. Chrom. 255:137-149 (1983) Purification of Acid.

The target sequence used in the present invention can be verified, for example, to be unique to the target bacterial species using methods well known in the relevant research fields, such as the chain termination method for double-stranded templates in Wallace et al., Gene 16:21-26 (1981) DNA or RNA polynucleotide sequences, as well as synthetic oligonucleotides (e.g., primers).

III. Obtaining Samples and Analyzing Bacterial DNA or RNA

The present invention relates to the determination of the level or amount of signature DNA or RNA of one or more bacterial species found in human fecal samples as a means of assessing the risk of recurrence of colorectal adenomas. Therefore, a first step in practicing the present invention is to obtain a fecal sample from a test subject and extract DNA or RNA from said sample.

A. Obtaining and Preparing Fecal Samples

Using the methods of the present invention, fecal samples are obtained from persons who are to be tested or monitored for recurrent colorectal adenomas. Individual stool sample collection can be easily accomplished in the clinic or at the patient's home. Appropriate amounts of feces are collected and can be stored according to standard procedures prior to further preparation. Bacterial DNA or RNA found in stool samples of patients according to the present invention can be analyzed using established techniques. Methods for preparing stool samples for nucleic acid extraction are well known to those skilled in the art. See, e.g., Yu et al., Gut. 2015-09-25pii:gutjnl-2015-309800.doi:10.1136/gutjnl-2015-309800.

B. Extraction and Quantification of DNA and RNA

Methods for extracting DNA from biological samples are well known and routinely practiced in the field of molecular biology (eg, described in Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3rd Edition, 2001). RNA contamination should be eliminated to avoid interference with DNA analysis.

Likewise, there are many methods for extracting mRNA from biological samples. General procedures for mRNA preparation can be followed, see, for example, Sambrook and Russell, supra; various commercially available reagents or kits, such as Trizol reagent (Invitrogen, Carlsbad, CA), Oligotex Direct mRNA Kit (Qiagen, Valencia , CA), RNeasy Mini Kit (Qiagen, Hilden, Germany) and Series 9600 ^TM (Promega, Madison, WI), can also be used to obtain mRNA from biological samples from test subjects. Combinations of more than one of these methods can also be used. It is necessary to eliminate all contaminating DNA from the RNA preparation. Therefore, samples should be handled carefully, treated thoroughly with DNase, and appropriate negative controls should be used during amplification and quantification steps.

1. PCR-based quantitative determination of DNA or RNA levels

When DNA or mRNA is extracted from a sample, the amount of predetermined bacterial DNA or RNA (such as 16s rDNA or RNA encoded by bacterial genes unique to the bacterial species) can be quantified. Preferred methods for determining DNA or RNA levels are amplification-based methods, such as by polymerase chain reaction (PCR), including reverse transcription-polymerase chain reaction (RT-PCR) for RNA quantitative analysis.

When bacterial DNA is amplified directly, bacterial RNA must first be reverse transcribed. Before the amplification step, a DNA copy of the target RNA (cDNA) must be synthesized. This process is achieved by reverse transcription, which can be performed as a separate step, or in a homogeneous reverse transcription-polymerase chain reaction (RT-PCR), a method used to amplify RNA. A modified polymerase chain reaction. Methods suitable for amplifying ribonucleic acids by PCR are described in: Romero and Rotbart, Diagnostic Molecular Biology: Principles and Applications pp. 401-406; Persing et al., eds., Mayo Foundation, Rochester, MN, 1993; Egger et al., J. Clin. Microbiol. 33:1442-1447, 1995; and U.S. Patent No. 5,075,212.

General methods of PCR are well known in the art and are therefore not described in detail herein. For a review of PCR methods, protocols, and principles of designing primers, see, for example, Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, Inc. N.Y., 1990. PCR reagents and protocols are also available from commercial suppliers such as Roche Molecular Systems.

Most commonly, PCR is performed in an automated process using thermostable enzymes. During this process, the temperature of the reaction mixture is automatically cycled through the denaturation zone, primer annealing zone, and extension reaction zone. Instruments specifically adapted for this purpose are commercially available.

Although PCR amplification of target bacterial DNA or RNA is typically used in the practice of the present invention, those skilled in the art will recognize that amplification of these DNA or RNA species in a sample can be accomplished by any known method, such as ligase chains Each of these methods can provide adequate amplification, including LCR, transcription-mediated amplification, and semi-conservative sequence replication or nucleic acid sequence-based amplification (NASBA). Recently developed branched DNA technology can also be used to quantitatively determine the amount of DNA or mRNA in a sample. For a review of branched DNA signal amplification for direct quantification of nucleic acid sequences in clinical samples, see Nolte, Adv. Clin. Chem. 33:201-235, 1998.

2. Other quantitative methods

Target bacterial DNA or RNA can also be detected using other standard techniques well known to those skilled in the art. Although an amplification step is typically performed before the detection step, amplification is not necessarily required in the method of the invention. For example, DNA or RNA can be identified by size fractionation (eg, gel electrophoresis) with or without prior amplification steps. After running the sample in an agarose or polyacrylamide gel and labeling it with ethidium bromide according to well-known techniques (see, e.g., Sambrook and Russell, supra), the presence of a band of the same size as the standard control indicates the target The amount of target DNA or RNA is then compared to the control based on the intensity of the band. Alternatively, oligonucleotide probes specific for target bacterial DNA or RNA can be used to detect the presence of such DNA or RNA and, based on the intensity of the signal provided by the probe, indicate the bacterium by comparison to a standard control Amount of DNA or RNA.

Sequence-specific probe hybridization is a well-known method for detecting specific nucleic acids, including nucleic acids from other species. Under sufficiently stringent hybridization conditions, the probe specifically hybridizes only to substantially complementary sequences. The stringency of hybridization conditions can be relaxed to allow for varying amounts of sequence mismatches.

There are many hybridization modes known in the art, including but not limited to: liquid phase, solid phase or mixed phase hybridization analyses. The following articles provide reviews of various modes of hybridization analysis: Singer et al., Biotechniques 4:230, 1986; Haase et al., Methods in Virology, pp. 189-226, 1984; Wilkinson, In situHybridization, edited by Wilkinson, IRL Press , Oxford University Press, Oxford; and Hames and Higgins, eds., Nucleic Acid Hybridization: A Practical Approach, IRL Press, 1987.

Hybridized complexes are detected according to well-known techniques. Nucleic acid probes capable of specifically hybridizing to a target nucleic acid (ie, bacterial 16s rDNA) can be labeled by any of several methods commonly used to detect the presence of hybridizing nucleic acids. A commonly used detection method is autoradiography using probes labeled with ^3H , ^125I , ^35S , ^14C or ^32P . The choice of radioisotope depends on the choice of parameters for the study due to ease of synthesis, stability, and half-life of the selected isotope. Other labels include compounds (eg, biotin and digoxigenin) that are conjugated to antiligands or antibodies labeled with fluorophores, chemiluminescent reagents, and enzymes. Alternatively, probes can be directly conjugated to labels such as fluorophores, chemiluminescent reagents and enzymes. The choice of label depends on the required sensitivity, ease of conjugation to the probe, stability needs, and available instrumentation.

Probes and primers required for practicing the present invention can be synthesized and labeled using well-known techniques. For example, using an automated synthesizer as described in Needham-Van Devanter et al., Nucleic Acids Res. 12:6159-6168, 1984, solid phase as first described in Beaucage and Caruthers, Tetrahedron Lett. 22:1859-1862, 1981 The phosphoramidite triester method allows the chemical synthesis of oligonucleotides used as probes and primers. Purification of oligonucleotides can be performed by native acrylamide gel electrophoresis, or by anion exchange HPLC as described in Pearson and Reanier, J. Chrom. 255:137-149, 1983.

IV. Quantification of Bacterial Proteins

A. Preparing samples for bacterial protein detection

The presence of relevant bacterial species in a sample can also be determined quantitatively by analyzing one or more proteins unique to the bacteria. Fecal samples from subjects are used in the practice of the present invention and can be obtained and processed for analysis according to known methods or as described in previous sections.

B. Determination of bacterial protein levels

A variety of immunological assays can be used to detect proteins, such as those that indicate bacterial identity. In some embodiments, a sandwich assay can be performed by capturing the target protein from the test sample using an antibody with specific binding affinity for the protein. It can then be detected using labeled antibodies with specific binding affinity for the protein. Such immunological analyzes can be performed using microfluidic devices such as microarray protein chips. Proteins of interest (eg, proteins unique to a bacterial species) can also be detected by gel electrophoresis (such as two-dimensional gel electrophoresis) and Western blot analysis using specific antibodies. Alternatively, the target protein can be detected by standard immunohistological techniques using appropriate antibodies. Monoclonal and polyclonal antibodies, including antibody fragments with desired binding specificities, can be used for specific detection of target proteins. Antibodies and binding fragments thereof with specific binding affinity for a specific protein can be generated by known techniques.

In practicing the present invention, other methods may also be used to determine the levels of marker proteins. For example, several techniques based on mass spectrometry measurements have been developed to rapidly and accurately quantify target proteins (even in large samples). These methods involve high-precision instrumentation such as triple Q instruments using multiple reaction monitoring (MRM) technology, matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI TOF/TOF), using selective ion detection (SIM) mode ion trap instrument, and electrospray ionization (ESI)-based QTOP mass spectrometer. See, eg, Pan et al., J Proteome Res. 2009 Feb;8(2):787–797.

V. Surveillance and Treatment of Recurrent Colorectal Adenomas

By illustrating the correlation between the enrichment of certain bacterial species in the human intestine and the increased risk of recurrence of colorectal adenomas, the present invention provides a preventive measure for the prophylactic treatment of those at increased risk of developing colorectal adenomas again. Patients who have had colorectal cancer or adenomas removed previously: The first step is regular surveillance, such as scheduling annual colonoscopies, so that newly developing colon polyps and cysts can be detected and removed immediately. The second measure is to treat the patient with one or more inhibitors to inhibit the relevant bacterial species and reduce their presence/levels in the patient's gut.

Inhibitors of relevant bacterial species can be of virtually any chemical and structural nature: they can be polypeptides (e.g. antibodies, antibody fragments, aptamers), polynucleotides (e.g. antisense DNA/RNA, small inhibitory RNA or microRNA) and small molecules. As long as they have a confirmed inhibitory effect on the target bacteria (e.g., inhibiting bacterial proliferation or inducing bacterial cell death), such inhibitors can be used to inhibit the development of recurrent adenomas in the patient's intestines, and thus can be used to inhibit or prevent Recurrence of colorectal adenomas.

Furthermore, after detecting the enrichment of certain bacterial species in the gut of patients after previous surgical resection for colorectal cancer or polypectomy, the inventors show that this is associated with an increased likelihood of recurrence of colorectal adenomas, one can identify patients There is an increased risk of developing adenomas again in the future. As a result of this determination, patients may be required to undergo subsequent monitoring and treatment or preventive/monitoring measures that may prevent, eliminate, ameliorate, reduce severity and/or frequency, or delay the recurrence of colorectal adenomas. For example, doctors may prescribe medications and non-drug treatments, such as lifestyle changes (e.g., losing 5% or more of body weight, adopting a healthier lifestyle, including following a high-fiber/low-salt diet and maintaining higher physical activity, such as walking for at least 150 minutes per week, and undergoing more frequent regular screenings/examinations, such as colonoscopies every 1-2 years instead of every 5 years).

A. Modulators of relevant bacterial species

Inhibition of bacterial species can be achieved through the use of inhibitor nucleic acids (such as siRNA, microRNA, microRNA (mini RNA), lncRNA, antisense oligonucleotides, aptamers) that target specific bacterial genes. Such nucleic acids may be single-stranded nucleic acids, such as mRNA, or double-stranded nucleic acids, such as DNA, which can be converted under appropriate conditions to the inhibitory active form of the target bacterial RNA.

In one embodiment, the nucleic acid encoding the inhibitor is provided in the form of an expression cassette, typically produced recombinantly, with a promoter operably linked to a polynucleotide sequence encoding the inhibitor. In some cases, the promoter is one that specifically directs expression in the bacterial cell of choice. Administration of such nucleic acids can inhibit target bacterial gene expression, and thus the bacterial population. Since almost all known bacteria have been completely sequenced and the information has been stored in databases, suitable inhibitor nucleic acids can be designed based on the sequence information.

When bacterial cultures are exposed to candidate compounds, inhibitors of the relevant bacterial species can be identified in the assay and the compound's effect on the culture analyzed. For example, inhibitors may be observed to exhibit inhibitory or inhibitory effects on bacterial cultures, resulting in reduced growth and/or increased bacterial cell death. An inhibitory effect is detected when a negative effect on the bacterial culture is established in the test group. Preferably, the negative effect is at least a 10% reduction; more preferably, the reduction in cell proliferation is at least 20%, 50%, 75%, 80% or higher.

As mentioned above, these bacterial inhibitors can have different chemical and structural characteristics. For example, an inhibitor can be any small or large molecule that affects only the growth or survival of a specific bacterial species. Basically any compound can be tested as a potential inhibitor. Such inhibitors can be identified by screening combinatorial libraries containing large numbers of potentially effective compounds. As described herein, such combinatorial chemical libraries can be screened in one or more assays to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. Thus, the identified compounds may serve as conventional "lead compounds" or may themselves be used as potential or actual therapeutic agents.

The preparation and screening of combinatorial chemical libraries is well known to those skilled in the art. Such combinatorial chemical libraries include, but are not limited to: peptide libraries (see, eg, U.S. Patent No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature 354: 84-88 (1991)), and carbohydrate libraries (see, eg, Liang et al., Science, 274:1520-1522 (1996) and U.S. Patent No. 5,593,853). Other chemistries used to generate chemically diverse libraries may also be used. Such chemicals include, but are not limited to: peptoids (PCT Publication WO 91/19735), encoded peptides (PCT Publication WO 93/20242), random bio-oligomers (PCT Publication WO 92/00091), benzodiazepines (U.S. Patent No. 5,288,514), Diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylenes Vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), non-peptide peptide mimetics with a β-D-glucose backbone (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), similar organic synthesis of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al. Human, Science 261:1303 (1993)) and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see, Ausubel, Berger and Sambrook, all supra ), peptide nucleic acid libraries (see, e.g., U.S. Patent No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature BioteChnology, 14(3):309-314 (1996) and PCT/US96/10287), small organic Molecular libraries (see, e.g., Benzodiazepines, Baum C&EN, Jan. 18, p. 33 (1993)); Isoprenoids, U.S. Patent No. 5,569,588; Thiazolidinones and Meta-thiazindinones , U.S. Patent No. 5,549,974; pyrrolidines, U.S. Patent Nos. 5,525,735 and 5,519,134; morpholine compounds, U.S. Patent No. 5,506,337; and benzodiazepines, U.S. Patent No. 5,288,514).

B. Pharmaceutical compositions

1. Preparation

Inhibitors of relevant bacterial species can be used in the preparation of pharmaceutical compositions or medicaments. The pharmaceutical composition or medicament can be administered to a subject to treat recurrent colorectal adenomas, especially for prophylaxis.

The compounds used in the treatment methods of the present invention can be used to prepare pharmaceutical compositions or medicaments including an effective amount of the compounds, combined or mixed with excipients or carriers suitable for use.

Exemplary pharmaceutical compositions for such therapeutic uses include: (i) polynucleosides encoding inhibitors as described herein (e.g., siRNA, microRNA, microRNA, lncRNA, antisense oligonucleotides) an expression cassette for the acid sequence, and (ii) a pharmaceutically acceptable excipient or carrier. The terms "pharmaceutically acceptable" and "physiologically acceptable" may be used synonymously herein. For use in the treatment methods as described herein, a therapeutically effective dose of the expression cassette can be provided.

Inhibitors can be administered via liposomes, which serve to target the conjugate to specific tissues, as well as increase the half-life of the composition. Liposomes include emulsions, foaming agents, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, etc. In these formulations, the inhibitor is to be delivered as part of a liposome, either alone or in combination with molecules capable of binding, for example, receptors that are ubiquitous in target cells or other therapeutic or immunogenic compositions. Thus, liposomes filled with the desired inhibitors of the present invention can be directed to a treatment site (eg, the colon), where the liposomes then deliver the selected inhibitor composition. Liposomes used in the present invention are formed from standard vesicle-forming lipids, which typically include neutral and negatively charged phospholipids and sterols (such as cholesterol). The following factors are often considered to guide lipid selection: e.g., liposome size, acid instability and stability of the liposomes in the blood stream. Various methods for preparing liposomes are available and are described, for example, in Szoka et al. (1980) Ann. Rev. Biophys. Bioeng. 9:467, U.S. Patent Nos. 4,235,871, 4,501,728, and 4,837,028.

Pharmaceutical compositions or medicaments for use in the present invention may be prepared by standard techniques using one or more physiologically acceptable carriers or excipients. Suitable pharmaceutical carriers are described herein and in "Remington's Pharmaceutical Sciences" by E. W. Martin. The compounds and agents of the present invention and their physiologically acceptable salts and solvates may be formulated for administration by any suitable route, including inhalation, topical, nasal, or oral administration. , parenteral or rectal administration.

Typical formulations for topical or topical administration include creams, ointments, sprays, lotions and plasters. However, the pharmaceutical compositions may be formulated for any type of administration, local and systemic, for example, intradermal injection, subcutaneous injection, intravenous injection, intramuscular injection, intranasal injection using a syringe or other device. Intracerebral injection, intratracheal injection, intraarterial injection, intraperitoneal injection, intravesical injection, intrapleural injection, intracoronary injection or intratumoral injection. Formulations for administration by inhalation (eg, aerosol) or oral, rectal, or vaginal administration are also contemplated.

2. Route of administration

Suitable formulations for topical application, for example to the skin and eyes, are preferably aqueous solutions, ointments, creams or gels well known in the art. Such preparations may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

Suitable formulations for transdermal administration include an effective amount of a modulator of the invention in association with a carrier. Preferred carriers include absorbable, pharmaceutically acceptable solvents to facilitate penetration through the skin of the host. For example, a transdermal device takes the form of a bandage that includes a support member, a reservoir containing a compound and optionally a carrier, and optionally a rate control barrier to perform the treatment at a controlled and predetermined rate over an extended period of time. Means for delivering the compound to the skin of the host, and for securing the device to the skin. Matrix transdermal formulations may also be used.

For oral administration, the pharmaceutical composition or medicament may take the form of, for example, tablets or capsules prepared in conventional manner using pharmaceutically acceptable excipients. Preferred are tablets and gelatin capsules comprising: the active ingredient (i.e. inhibitor or activator), and (a) a diluent or filler, for example, lactose, glucose, sucrose, mannitol, sorbitol, cellulose (for example, ethyl cellulose, microcrystalline cellulose), sugar gum, pectin, polyacrylate and/or calcium hydrogen phosphate, calcium sulfate, (b) lubricant, for example, silica, talc, stearic acid, hard Magnesium fatty acid or calcium stearate salt, metallic stearate, colloidal silicon dioxide, hydrogenated vegetable oil, corn starch, sodium benzoate, sodium acetate and/or polyethylene glycol; for tablets, may also include (c) Binders, for example, magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone and/or hydroxypropylcellulose; if desired, may also include ( d) disintegrants, for example, starch (for example, potato starch or sodium starch), glycolates, agar, alginic acid or its sodium salt, or foaming mixtures, (e) wetting agents, for example, laurel sodium sulfate, and/or (f) absorbents, colorants, flavors and sweeteners.

Tablets may also be film-coated or enteric-coated according to methods known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as dry products for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared in a conventional manner using pharmaceutically acceptable additives such as suspending agents such as sorbitol syrup, cellulose derivatives or hydrogenated edible fats; emulsifiers such as lecithin or gum arabic; a non-aqueous medium, such as almond oil, oil esters, ethanol, or graded vegetable oils; and a preservative, such as methyl or propyl paraben, or sorbic acid. The formulations may also contain buffer salts, flavouring, coloring and/or sweetening agents, as appropriate. Where desired, formulations for oral administration may be suitably formulated to achieve controlled release of the active compound.

The compounds and agents of the present invention may be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. Formulations for injection may be presented in unit dose form, for example, in ampoules or multi-dose containers with an added preservative. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and preferably suppositories prepared from fatty emulsions or suspensions. The compositions may be sterile and/or contain adjuvants such as preservatives, stabilizers, wetting or emulsifying agents, dissolution promoters, salts to adjust the osmotic pressure, and/or buffers. Alternatively, the active ingredient may be in powder form for constitution with a suitable medium (for example, sterile pyrogen-free water) before use. In addition, they may contain other substances of therapeutic value. The composition is prepared according to conventional mixing, granulating or coating methods, respectively, and contains about 0.1% to 75%, preferably about 1% to 50% of the active ingredient.

For administration by inhalation, the active ingredient may be conveniently delivered as an aerosol spray from a pressurized pack or nebulizer using suitable propellants, such as dichlorodifluoromethane, trichlorofluoromethane, Dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve to deliver a metered amount. For example, gelatin capsules or cartridges for use in an inhaler or insufflator may be prepared containing a powder mixture of the compound and a suitable powder base (eg, lactose or starch).

The conditioning agents may also be formulated in rectal compositions, eg, suppositories or retention enemas, eg, containing conventional suppository bases, eg, cocoa butter or other glycerides.

In addition, the active ingredients can be formulated as depot preparations. Such long-acting formulations may be administered by implantation (eg, subcutaneously or intramuscularly) or intramuscular injection. Thus, for example, the active ingredients may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or may be formulated as sparingly soluble derivatives, for example, as a sparingly soluble salt.

In some cases, a pharmaceutical composition or medicament of the invention includes: (i) an effective amount of a compound as described herein that inhibits a population of one or more related bacterial species identified herein, and ( ii) Another therapeutic agent. When used with a compound of the present invention, such therapeutic agents may be used alone, sequentially, or with one or more other such therapeutic agents (e.g., a first therapeutic agent, a second therapeutic agent, and an antibacterial inhibitor of the present invention). agent) used in combination. Administration may be by the same or different routes of administration or may be administered in the same pharmaceutical formulation.

3.Dose

The subject may be administered a pharmaceutical composition or medicament in a therapeutically effective dose capable of preventing, treating, or controlling the recurrence of a colorectal adenoma as described herein. The pharmaceutical composition or drug is administered to the subject in an amount sufficient to elicit an effective therapeutic response in the subject.

The dosage of active agent administered will depend on the individual's weight, age, individual condition, surface area or volume of the area to be treated, and the form of administration. Dosage size will also be determined by the presence, nature, and extent of any adverse reactions associated with administration of a particular compound in a particular subject. For example, each type of inhibitor or nucleic acid encoding an inhibitor may have a unique dosage. A unit dose for oral administration to a mammal of about 50 to 70 kg may contain about 5 to 500 mg of active ingredient. Generally, the dosage of the active compounds of the present invention is a dosage sufficient to achieve the desired effect. The optimal dosing regimen can be calculated from measurements of drug accumulation in the subject. Typically, dosages may be administered once or more daily, weekly, or monthly. Those skilled in the art can readily determine optimal dosages, administration methods and repetition rates.

To achieve the desired therapeutic effect, the compound or agent may be administered over multiple days at a therapeutically effective daily dose. Accordingly, therapeutically effective administration of a compound to treat a relevant condition or disease described herein in a subject requires periodic (eg, daily) administration lasting from three days to two weeks or more. Typically, the agent is administered for at least three consecutive days, usually at least five consecutive days, more usually at least ten consecutive days, and sometimes for 20, 30, 40 or more consecutive days. Although continuous daily dosing is the preferred route to achieving a therapeutically effective dose, therapeutically beneficial effects can be achieved without daily administration of the agent, so long as dosing is repeated frequently enough to maintain a therapeutically effective concentration of the agent in the subject. For example, the agent may be administered every other day, every third day, or once a week if a higher dosage range is used and tolerated by the subject.

The optimal dose, toxicity, or therapeutic efficacy of the compound or agent may vary depending on the relative potency of the individual compound or agent and may be determined by standard pharmaceutical methods in cell culture or experimental animals, for example, by determining LD ₅₀ (the dose that causes death in 50% of the population) and ED ₅₀ (the dose that is therapeutically effective in 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index and can be expressed as the ratio LD ₅₀ /ED ₅₀ . Agents that exhibit a large therapeutic index are preferred. Although agents exhibiting toxic side effects can be used, consideration should be given to designing delivery systems that target such agents to the site of affected tissue, thereby minimizing potential damage to normal cells and thus reducing side effects.

For example, data obtained from cell culture analyzes and animal studies can be used to develop dosage ranges for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that includes the _ED50 but has little or no toxicity. The dosage may vary within this range depending on the dosage form used and route of administration. For any agent used in the methods of the invention, the therapeutically effective dose can first be estimated from cell culture assays. Doses can be formulated in animal models to achieve a range of circulating plasma concentrations that includes the _IC50 (the concentration of the agent that achieves half the maximal inhibition of symptoms) as determined in cell culture. This information can be used to more precisely determine available doses for humans. For example, levels in plasma can be determined by high performance liquid chromatography (HPLC). Generally, the dosage equivalent of the agent will be about 1 ng/kg to 100 mg/kg for a typical subject.

Exemplary dosages of inhibitors or nucleic acids encoding inhibitors described herein are provided. When administered IV, the dose of nucleic acid encoding the inhibitor (such as an expression vector) may be 0.1-0.5 mg (eg, 5-30 mg/kg). Small organic compound inhibitors may be administered orally at 5-1000 mg or intravenously infused at 10-500 mg/ml. Peptide inhibitors may be administered by intravenous injection or infusion in the following amounts: 50-500 mg/ml (over 120 minutes); 1-500 mg/kg (over 60 minutes); or 1-100 mg/kg (bolus) , 5 times a week. Inhibitors may be administered at 10-500 mg subcutaneously; 0.1-500 mg/kg intravenously twice daily, or approximately 50 mg once weekly, or 25 mg twice weekly.

The pharmaceutical compositions of the present invention may be administered alone, or they may be administered in combination with at least one other therapeutic compound. Exemplary beneficial therapeutic compounds include systemic and local anti-inflammatory drugs, analgesics, antihistamines, anesthetic compounds, and the like. Other therapeutic compounds can be administered simultaneously with the main active ingredient, or even in the same composition. Other therapeutic compounds may also be administered separately, in separate compositions, or in dosage forms separate from the main active ingredient. Some doses of the main ingredient can be administered simultaneously with other therapeutic compounds, while other doses can be administered alone, depending on the specific findings of the intestinal bacterial population and the characteristics of the individual.

The dosage of the pharmaceutical composition of the present invention can be adjusted during treatment based on various factors, including the distribution of the patient's intestinal bacterial population and physiological response to the treatment regimen. Those skilled in the art are often involved in the adjustment of such treatment regimens.

VI. Kits and Devices

The invention provides compositions and kits for performing the methods described herein to determine the level or relative abundance of one or more of relevant bacterial species, such as Fn, Bc, Ch and m3, in a sample. To assess the risk of colorectal adenoma recurrence in samples, such as stool samples, obtained from human patients with previous colorectal cancer, cysts, or polyps removed by surgery or polypectomy. For example, the level or relative abundance of one or more of Fn, Bc, Ch, and m3 is determined in a stool sample collected from a patient so that the patient can be treated accordingly, e.g., if the patient is considered likely to have For recurrent colorectal adenomas, patients will receive more surveillance, such as being scheduled for colonoscopies more frequently (e.g., yearly instead of every 5 years), and being prescribed changes in diet and physical activity regimen, up to and including antimicrobial therapy to inhibit the level of relevant bacterial species, such as Fn, Bc, Ch and m3 (e.g., administration of an expression cassette, such as one contained in a viral vector, directing the expression of inhibitory RNA molecules, particularly in colorectal epithelial tissue, to inhibit the expression of one or more genes of Fn, Bc, Ch, or m3); otherwise, the patient would not receive additional monitoring but would remain on a regular surveillance schedule, such as colonoscopy every 5 years for average-risk individuals. Microscopic examination. In any case, when a patient is screened for possible recurrence of colorectal adenomas (e.g., by colonoscopy), abnormal colorectal tissue (cysts, polyps, and tumors, including adenomas and tumors) so discovered may be removed according to established procedures. cancer, etc.).

In the context of assessing the likelihood of colorectal adenoma recurrence in multiple patients who underwent colorectal cancer/polyp/cyst resection, the levels or relative values of Fn, Bc, Ch, or m3 in the first patient's postoperative stool sample Abundance or their combined score increased by a greater percentage compared to the corresponding baseline level or score in his stool sample obtained before surgery, whose CRC/polyp/cyst removal surgery was considered to have a higher chance than the second patient and those with recurrent colorectal adenomas who had a smaller or no increase in percentage from baseline to postoperative values. Therefore, the first patient is given more monitoring (e.g., scheduled colonoscopies more frequently, such as once a year instead of every 5 years) and treatment (e.g., antibacterial therapy, especially targeted therapy) than the second patient. antimicrobial therapy to one or more bacterial species (Fn, Bc, Ch, or m3), while the second patient may receive less monitoring and/or treatment, including regularly scheduled colonoscopies and/or no antimicrobial therapy at all , especially if the second patient is considered not to be at increased risk of colorectal adenoma recurrence.

In patients who are identified as being at increased risk for colorectal adenoma recurrence and are therefore given antimicrobial therapy to prevent or reduce the risk of colorectal adenoma recurrence, their stool samples are optionally further tested after antimicrobial therapy, allowing determination of the bacterial species Fn , Bc, Ch, or m3 levels, or their combination scores, to confirm risk reduction.

In some embodiments, kits for performing analysis of the level or relative abundance of at least one optional multiple bacterial species Fn, Bc, Ch, or m3 typically include reagents for performing RT-PCR or qPCT to perform Qualitative and/or quantitative determination of polynucleotide sequences, such as bacterial species-specific 16S rRNA: e.g., at least one oligonucleotide for reverse transcription and at least one set of three oligonucleotide primers for PCR amplification Unique polynucleotide sequences. In some cases, one or more oligonucleotides can be labeled with a detectable moiety. In some cases, hydrolysis probes are included in the kit to allow for immediate quantitative determination of amplification products. Typically, hydrolysis probes have a fluorescent label and a quencher. Some examples of such primers and probes are provided in the Examples section of this disclosure.

Typically, the kit also includes positive and negative controls for the specific analytical method. Additionally, the kits of the present invention may provide instructions to guide the user in analyzing samples and assessing the likelihood of colorectal adenoma recurrence in test subjects.

On the other hand, the invention may also be implemented in a device or system comprising one or more such devices capable of performing all or part of the method steps described herein. For example, in some cases, after receiving a first sample (e.g., a stool sample from a test subject prior to surgery to remove colorectal cancer/polyps/cysts, such as polypectomy) and a second sample of the same type ( For example, in a fecal sample from the same test subject after surgery (e.g., about one year, about two years, or about one to about two, three, four, or five years) after surgery, the device or system performs the following steps to: Assessing the subject's likelihood of colorectal adenoma recurrence: (a) determining the level or relative abundance of one or more bacterial species Fn, Bc, Ch, or m3 in the first sample and the second sample (e.g., The level or relative abundance of any two, three or four of these species (e.g., Fn and m3, optionally further including Ch) based on their unique 16S rRNA sequence) or calculated according to the description provided herein a comprehensive score of the degree, optionally further including a FIT score; (b) comparing the level or composite score of the first sample with the level or composite score of the second sample; (c) providing an output indicating that the test subject Are at high risk for recurrent colorectal adenomas and should therefore undergo prophylactic surveillance and/or treatment as described herein to eliminate or reduce such risk. In some cases, the device or system of the present invention performs the tasks of steps (b) and (c) after step (a) has been performed and the level or composite score from step (a) has been entered into the device.

Preferably, the device or system of the invention is partially or fully automated.

Example

The following examples are provided by way of illustration only and not limitation. Those skilled in the art will readily recognize that various non-critical parameters may be changed or modified to produce substantially the same or similar results.

Example I

introduction

Colorectal cancer (CRC) is one of the most common cancers worldwide [1]. Most CRCs start as adenomas and progress to cancer. Colonoscopic polypectomy has been used as a first-line approach to prevent CRC. Adenomatous polyps can be detected in 20% to 40% of patients undergoing screening colonoscopy, and their occurrence is associated with an increased risk of CRC. Although endoscopic resection of colorectal adenomas significantly reduces the risk of CRC, regular surveillance examinations are required because the risk of recurrence after polypectomy is 37% to 60% [2]. Recently, new biomarkers have been developed for CRC diagnosis, including fecal DNA [3], plasma DNA [4], and fecal bacterial markers [5]. The U.S. Food and Drug Administration (FDA) has approved two non-invasive tests, a multi-target fecal DNA [3] and a plasma DNA test for CRC screening [4]. However, these tests are less accurate in diagnosing precancerous lesions, especially non-advanced adenomas, because genetic or epigenetic changes in cancer cells are rarely seen in small precancerous lesions.

Altered gut microbiota composition is associated with the development and progression of adenomas and CRC [6-10]. In particular, a direct pathogenic role of the intestinal microbiota on the development of CRC has been demonstrated in germ-free animal models [11]. Specific bacterial pathogens, such as Fusobacterium nucleatum (Fn) [12-14] and Anaerobic Peptostreptococcus [15], have been proposed to contribute to colorectal tumorigenesis. It has been previously reported that fecal bacterial markers can be used as non-invasive detection of adenomas and CRC [5,7,16,17]. Quantification of candidate bacterial markers using a probe-based double-stranded quantitative polymerase chain reaction (qPCR) assay for a panel of fecal bacterial markers including Fn, Lachnoclostridium marker m3, Clostridium/Hungatella hathewayi (Ch), and Clarus Bacteroidetes (Bc), the so-called 4Bac group of Fn, m3, Ch and Bc, have shown good diagnostic performance in the detection of adenomas and CRC [5,16]. It has been hypothesized that these bacterial markers would also be effective in detecting recurrent adenomas. In this study, the inventors evaluated the utility of the 4Bac panel of Fn, m3, Ch, and Bc in predicting the risk of adenoma recurrence after colonoscopic polypectomy.

Materials and methods

research subjects

This study was included as part of a polyp surveillance study conducted between 2009 and 2019 at the Prince of Wales Hospital, a secondary and tertiary referral center for the Chinese University of Hong Kong, China (CREC reference number: 2010.198). All subjects provided a stool sample before the index colonoscopy (baseline). Subjects with adenomas discovered during the index colonoscopy underwent polypectomy and had regular colonoscopies in accordance with international polyp surveillance guidelines [2,18,19]. In surveillance colonoscopy, recurrence-free is defined as the absence of adenomas, sessile serrated polyps, hyperplastic polyps larger than 10 mm, or CRC on colonoscopy. Recurrence was defined as colonoscopy in which at least one adenoma was found. To identify new lesions, only polypectomies that occurred at least 6 months after the index colonoscopy were included. The adenoma detection rate of colonoscopy is >30%. Collect stool samples before each surveillance colonoscopy. Three groups of subjects were included - Group I: 118 patients with adenomas, for whom stool samples were collected before the index colonoscopy (baseline samples); Group II: 61 subjects, for whom stool samples were collected during the colonoscopy Fecal samples (follow-up samples); and Group III: 43 subjects, paired baseline and follow-up samples before colonoscopy) (Fig. 1, A). All lesions were confirmed by an experienced pathologist (TKF). Advanced adenomas were defined as adenomas 1 cm or larger in size, with a tubulovillous or villous component, or with high or severe dysplasia. Exclusion criteria included subjects who had taken antibiotics within the past 3 months. Informed consent was obtained from all subjects. The study was approved by the NTEC-CUHK Joint Clinical Research Ethics Committee.

Human feces sample collection

Baseline stools (n=161) were collected before index colonoscopy in Group I (n=118) and Group III (n=43), with surveillance colonoscopies 2.5 ± 1.6 years after the index colonoscopy. 104 follow-up stools were previously collected from Group II (n=61) and Group III (n=43). Forty-eight of 104 postpolypectomy patients (28 in group II and 20 in group III) had adenomas detected on follow-up colonoscopy (Fig. 1, A), 7 of which were advanced adenomas. Detailed clinical characteristics are shown in Table 4. Subjects collected fecal samples in standardized containers at home and immediately stored the samples in a -20°C refrigerator at their home. Frozen samples were then delivered to the hospital in insulated polystyrene foam containers and immediately stored at -80°C until further analysis.

Fecal DNA extraction and probe-based bacterial marker quantification by dual quantitative PCR (qPCR)

Stool DNA extraction was performed using the Norgen Stool DNA Isolation Kit (Norgen Biotek Corp, Ontario, Canada) following the manufacturer's instructions. Determination of DNA quality and quantity using gel electrophoresis and NanoDrop spectrophotometer. Fecal levels of four bacterial DNA markers (Fn, m3, Bc and Ch) were quantified by qPCR covering previously shown expression in CRC (Fn and Ch), adenoma and CRC (m3) patient samples as well as healthy subjects (bc ) markers enriched in the sample. In our previous studies, the targeting specificity of the primer and probe sequences for the targeting markers and 16s rDNA internal control has been verified [5,16]. Each probe carries a 5' reporter dye FAM (6-carboxyfluorescein) or VIC (4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein) and a 3' quencher dye TAMRA (6-carboxytetramethyl-rhodamine). Primers and hydrolysis probes were synthesized by Invitrogen (Carlsbad, CA). qPCR amplification was performed on the ABI QuantStudio sequence detection system as previously described, with thermal cycler parameters of 95°C for 10 minutes and (95°C for 15 seconds, 60°C for 1 minute) × 45 cycles [5,16]. Each experiment included positive and negative controls for markers (H ₂ O as template). Each sample was assayed in triplicate. The relative level of each marker was calculated by using the delta Cq method compared to the internal control (Power(2,-(Cq _target -Cq _control )) and is shown as a Log value of '*10e6+1'.

Scoring Algorithms and Critical Values

The composite score of four bacterial markers (4Bac) using a logistic regression model (4Bac score = I ₁ +β ₁ *Fn + β ₂ *m3 + β ₃ *Bc + β ₄ *Ch) was determined in a previous study [ 5]. The composite score of follow-up markers using the logistic regression model was as follows: I ₂ +β5*m3+β6*Ch, or I3+β7*Fn+β8*m3+β9*Ch (Table 2). The combined score for baseline and follow-up markers using a logistic regression model was as follows: I ₁₀ + (β _i *Fn _follow-up – β _j *Fn _baseline ) + (β _k *m3 _follow-up – β _l *m3 _baseline ) + (β _m * Ch _follow-up – β _n *Ch _baseline ) (Table 1). In the regression model, I represents the intercept, β represents the regression coefficient, and the marker represents the corresponding Cq value. The cutoff value is determined by receiver operating characteristic (ROC) analysis that maximizes the Youden index (J = sensitivity + specificity - 1) [20].

Fecal Immunochemical Test (FIT)

Quantitative OC sensor testing was performed on an automated OCsensor instrument (Eiken Chemical, Japan) following the manufacturer's instructions, using a positive cutoff value equivalent to a hemoglobin concentration of 100 ng per milliliter (ng Hb/mL).

Statistical Analysis

Values are expressed as median (interquartile range) or mean ± SD, as appropriate. Differences in bacterial levels were determined by Mann-Whitney U test or paired t-test. Continuous clinical and pathological variables were compared by t-test or one-way ANOVA. ROC curves are used to evaluate the diagnostic value of bacterial markers or models in differentiating patients with and without recurrent adenomas. Pairwise comparisons of the area under the ROC (AUROC) for each method/marker were performed using non-parametric methods [21]. All tests were performed with Graphpad Prism 5.0 (Graphpad Software Inc., San Diego, CA) or MedCalc StatisticalSoftware V.18.5 (MedCalc Software bvba, Ostend, Belgium; website: medcalc.org; 2018). P<0.05 was considered statistically significant.

result

Fecal bacterial markers increased in subjects with recurrent adenoma

Levels of four bacterial markers, Fn, Ch, m3 and Bc, were first compared in baseline stool samples from patients with advanced adenoma and in follow-up stool samples from subjects with or without recurrent adenoma after polypectomy. Fn (P < 0.05) and m3 (P < 0.0001) were significantly increased in follow-up stool samples from subjects with recurrent adenomas compared with baseline stool samples, but in follow-up samples from subjects without recurrence in Group III, these There were no significant changes in markers (Mann Whitney test). The marker Ch was decreased in follow-up samples from subjects without recurrence (P=0.066) but did not change significantly in follow-up samples from patients with adenoma recurrence in Group III compared with baseline stool samples. The marker Bc showed no changes in follow-up stool samples regardless of adenoma recurrence status compared with corresponding baseline samples (Fig. 1, B). These findings were further validated in an expanded data set involving all samples from Groups I to III. Compared with baseline stool, Fn and m3 were significantly increased in the follow-up stool of subjects with recurrent adenoma, while Ch was significantly decreased in the follow-up stool of subjects without recurrent adenoma (all P<0.05) (Figure 1, C) . Regardless of adenoma recurrence status, the marker Bc remained unchanged in follow-up stool compared with baseline stool. Importantly, fecal levels of Fn, m3, and Ch in subjects with recurrent adenomas did not show differences between patients with proximal and distal lesions (Fig. 1, D1, and Fig. 1, D2). These findings indicate that after colonoscopic resection of advanced adenomas, CRC or adenoma-enriched bacterial markers are increased in the stool of subjects with recurrent adenomas or decreased in the stools of subjects without adenoma recurrence.

Fecal bacterial marker levels differ between subjects with adenoma recurrence and those without recurrence

To assess whether fecal bacterial markers during index colonoscopy and surveillance colonoscopy are associated with colonoscopy outcomes, subjects with baseline and follow-up feces were divided into two groups of "relapse" based on surveillance colonoscopy and histology results. (n=20; 46.5%) and "no recurrence" (n=23; 53.5%). Based on the levels of bacterial markers in baseline stool samples, no significant differences were found in the levels of bacterial markers tested between subjects with adenoma recurrence at follow-up and those without recurrence (Fig. 2, A). At follow-up time points, levels of Fn (P<0.01), m3 (P<0.001), and Ch (P<0.05) were observed, as well as the 4Bac composite score (Fn, m3, Ch, and Bc) (P<0.001), was significantly higher in subjects with adenoma recurrence than in subjects without recurrence (Figure 2, B). These results suggest that quantification of bacterial markers during surveillance follow-up may help predict patients at high risk for adenoma recurrence.

Adding FIT did not improve prediction of adenoma recurrence

All follow-up stools were further included to evaluate the performance of bacterial markers in predicting adenoma recurrence at follow-up time points. ROC curve analysis showed that individual Fn, m3 and Ch markers significantly differentiated subjects with adenoma recurrence and subjects without recurrence, with AUROCs of 0.640, 0.676 and 0.597 respectively (all P<0.05), while FIT failed to distinguish adenomas. Subjects with recurrence and subjects without adenoma recurrence (AUROC=0.551, P=0.38) (Fig. 3, A). m3 performed better than Fn and Ch in predicting adenoma recurrence, with a sensitivity of 52.0% and a specificity of 80.4%. Although Ch showed a relatively small AUROC, its specificity for predicting adenoma recurrence was 100% and its sensitivity was 20.8%. In contrast, FIT showed limited sensitivity (8.3%) in detecting recurrent adenomas, with the majority detected being non-advanced adenomas.

At a specificity of 71.4%, the composite score using 4Bac performed well in distinguishing patients with recurrent adenomas from those with non-recurrent adenomas, with an AUROC of 0.701 (P=0.0001) and a sensitivity of 62.5%. However, since 4Bac was not significantly better than other single markers (P>0.05 by comparing ROC curves), different marker groups or different scoring algorithms to combine these markers ensure better prediction accuracy. Therefore, logistic regression was applied to combine bacterial markers. A logistic regression model involving m3 and Ch showed an AUROC of 0.725 (P<0.0001), a sensitivity of 62.5%, and a specificity of 80.4%. The model combining Fn, m3, and Ch showed the highest AUROC for predicting adenoma recurrence of 0.732 (P<0.0001), with a sensitivity of 81.3% and a specificity of 55.4% (Fig. 3, B). Inclusion of FIT results was not associated with improved diagnostic performance of bacterial markers.

Fecal bacterial marker panel (m3, Fn, and Ch) shows high accuracy in predicting adenoma recurrence

Monitoring changes in fecal bacterial marker levels at the time of colonoscopy were further compared to fecal samples collected before the index colonoscopy. M3 levels and composite score 4Bac were found to be significantly increased in follow-up stool samples compared with baseline samples in subjects with colonoscopy-confirmed adenoma recurrence (P<0.05 by paired test) (Fig. 4, A) . In contrast, levels of bacterial markers did not change significantly in follow-up samples from subjects who subsequently had normal colonoscopies during the surveillance period (P>0.05). Using a logistic regression model that included “change” in stool bacterial markers at follow-up compared with baseline stool, m3 alone was found to show good diagnostic performance in predicting adenoma recurrence, with an AUROC of 0.843 (P<0.0001) and a sensitivity of 85.0%, The specificity was 87.0%. Although the numbers are not significant (Figure 4, B), this result is better than the model using the 4Bac score. Combining Fn or Ch (rather than Bc) with m3 in the model further improved the diagnostic performance of m3 alone, although this was not significant. The combination of m3, Fn, and Ch performed best in predicting adenoma recurrence, with an AUROC of 0.950 (P<0.0001), a sensitivity of 90.0%, and a specificity of 87.0% (Fig. 4, B).

A fecal bacterial marker panel predicting adenoma recurrence

Tables 1 and 2 present the AUROC, sensitivity, and specificity results for various combinations from various faecal bacterial marker panels, as well as the corresponding calculation methods if more than one biomarker is used. Depending on the panel of fecal bacterial markers used, the risk of adenoma recurrence can be predicted by (1) comparing individual levels or composite scores in samples collected from baseline and follow-up or (2) using standard controls (Table 1 or Table 2) Individual-level or composite scores in samples collected at follow-up.

Table 1 Examples of fecal bacterial marker combinations for predicting adenoma recurrence and their corresponding calculation methods (baseline versus follow-up comparison)

Table 2 Examples of fecal bacterial marker combinations for predicting adenoma recurrence and their corresponding calculation methods (compared to standard controls)

discuss

This is the first study to demonstrate that fecal bacterial markers can effectively predict the risk of adenoma recurrence after polypectomy. By quantitatively monitoring changes in new bacterial marker levels during colonoscopy compared to levels at the index colonoscopy, the inventors found that these markers were highly accurate in predicting adenoma recurrence, with a sensitivity of 90%. These findings underscore the role of noninvasive fecal markers in improving adenoma surveillance programs.

Microbial markers have been previously identified for the diagnosis of CRC through metagenomic sequencing [7], and qPCR tests were developed for possible clinical applications [5,16]. The qPCR test involved four bacterial markers, including Fn and Ch, which were found to be enriched in the stool of CRC patients, m3, which was enriched in the stool of adenoma and CRC patients, and Bc, which was enriched in the stool of normal subjects. Levels of m3 and the combined 4Bac score showed comparable accuracy in detecting both advanced and non-advanced lesions, suggesting that these bacterial markers are sensitive for detecting small adenomas [5]. Several studies have implicated microbial dysbiosis in the etiology of colorectal adenomas [22]. For example, changes in gut microbial community composition have been reported in fecal samples and adenoma tissue [8,23]. Unlike existing non-invasive CRC screening tests, such as multi-target stool DNA or plasma DNA tests, which target genetic/epigenetic changes in cancer cells and are rarely present in non-advanced adenomas, in The bacterial markers disclosed herein can be used to detect adenomas and are particularly useful in detecting early or small precancerous lesions, which account for more than 30% of adenomas found during surveillance colonoscopies.

Moderate changes in the intestinal microbiota were reported 3 months after adenoma resection, but there were no significant changes in major phyla including Fusobacteria [24]. Although it is expected that the gut microbiota composition of patients with adenomas may not change substantially after lesion resection, the gut microbiota can be readily reshaped by lifestyle, diet, and nutritional intake. Altered gut microbiota can promote or inhibit colorectal carcinogenesis, and changes in microbial communities associated with adenomas may represent early events in the pathway leading to CRC. The results of this study showed that Fn and m3, bacterial markers of CRC or enriched adenomas, were consistently increased in the stools of subjects with adenoma recurrence, whereas Ch was decreased in the stools of subjects without recurrence. Importantly, there were no differences in fecal m3, Fn, and Ch levels in subjects with proximal colon adenoma recurrence compared with subjects with distal colon recurrence, indicating that the sensitivity of these markers is not affected by lesion location. These findings support the possible clinical application of bacterial markers Fn, m3, and Ch in the detection of recurrent adenomas. Furthermore, it may be possible to modulate the gut microbiota to a healthier state to reduce the risk of developing colorectal tumors, although this should be evaluated in prospective studies.

The present inventors developed two strategies to predict adenoma recurrence by including follow-up stools alone or paired baseline and follow-up stools. Their data showed that of the two strategies, m3 outperformed Fn and Ch in predicting adenoma recurrence, while combining Fn and Ch improved the diagnostic performance of m3 in both strategies. The combination of m3, Fn and Ch produced the best AUROC compared to other models. However, adding Bc and FIT did not increase the diagnostic sensitivity for recurrent adenomas. Fn has been shown to induce inflammation and modulate host immune responses to promote tumor development [12,13]. Ch has been shown to promote colon epithelial cell proliferation in mouse models [25]. These bacterial species are believed to trigger host immune responses to further promote the development of recurrent adenomas. Therefore, inhibiting these bacteria can effectively help reduce the risk of adenoma recurrence.

Noninvasive biomarkers are urgently needed to monitor adenoma recurrence. Current guidelines recommend colonoscopies at varying intervals after polypectomy based on the characteristics of the lesions, including size, number, histology, and location [2,19], but colonoscopies are invasive and Compliance is poor and regular check-up rates are low. A recent national survey showed that patients prefer stool-based tests to colonoscopy when it comes to CRC screening [26], emphasizing the importance of considering patient preferences in colorectal screening recommendations.

This study has a number of strengths. This is the first prospective study of regular stool sample collection for up to 10 years after polypectomy. Each individual sampled in this study underwent a complete colonoscopy, with complete visualization of the colon from rectum to cecum, and colonoscopy is considered the most reliable reference standard for determining the presence or absence of polyps. Polyps removed during colonoscopy were reviewed and classified by an experienced gastrointestinal pathologist. Finally, this study includes predictive algorithms based on bacterial markers known to be enriched/depleted in adenoma and non-adenomatous groups.

In summary, this study demonstrates that fecal bacterial markers, including Fn, m3, and Ch, can be used to diagnose recurrent adenomas after polypectomy. Therefore, this study provides the first fecal microbiome-based colorectal tumor surveillance strategy.

Example II

method

Subject recruitment and stool sample collection for the prospective validation cohort

A further prospective study was conducted, recruiting subjects with a history of adenoma resection within the past five years and scheduled regular surveillance colonoscopy from May to October 2021 at the Prince of Wales Hospital of the Chinese University of Hong Kong, China. By. Consecutive eligible subjects provided follow-up stool samples within 1 week before bowel preparation for surveillance colonoscopy. Stool samples were excluded if they were collected from subjects who had taken antibiotics within 3 months before stool collection. Colonoscopy and histology were performed on the discovery cohort. Subjects collected fecal samples in preservative-filled standard containers (Norgen, Canada), and minimal changes in microbial communities were observed after 7 days at room temperature compared with fresh-frozen samples in a previous study [5]. Samples were delivered to the hospital within 24 hours and then immediately stored at -80°C until further analysis. The study was approved by the NTEC-CUHK Joint Clinical Research Ethics Committee (CREC Ref No: 2021.136). All subjects provided written informed consent.

result

Validation of a recurrence diagnostic model with follow-up stool in a prospective cohort

In the validation cohort, 50 consecutive post-polypectomy subjects were recruited and provided eligible stool samples prior to bowel preparation for surveillance colonoscopy. The qPCR test is performed before a colonoscopy and then compared to the colonoscopy diagnostic results. The same logistic regression model involving m3, Ch and Fn follow-up levels and the same cutoffs from the discovery/training cohort were applied to the validation cohort. Of these 50 subjects, 34 were identified as high risk (score above cutoff) by the m3ChFn model, 23 of whom were confirmed to have adenoma recurrence, resulting in a positive predictive value (PPV) of 67.6 from our model %. Of the other 16 subjects identified as low risk by the m3ChFn model, 11 were confirmed to have no adenoma recurrence, resulting in a negative predictive value (NPV) of 68.8% from our model. Importantly, our model detected 5 patients with recurrent advanced adenomas who were all at high risk (Fig. 5, A1).

The m3ChFn model showed that patients with recurrent adenomas had significantly higher composite scores compared with patients without recurrent adenomas (P=0.03), with an AUROC of 0.679 (95% CI: 0.527-0.831) in the validation cohort (Figure 5 B). The m3ChFn model showed a sensitivity of 82.1% for recurrent adenomas (100% for recurrent advanced adenomas) and a specificity of 50%. FIT, on the other hand, detected only 2 of 28 recurrent adenomas (sensitivity = 7.1%) and 0 of 5 recurrent advanced adenomas (sensitivity = 0%), despite its relatively high specificity (95.5%) (A2 in Figure 5). The overall diagnostic accuracy of our model (68.0%; 34/50) was significantly higher than that of FIT (46.0%; 23/50) (Fisher's exact test P < 0.05).

All patents, patent applications, and other publications cited in this application, including GenBank accession numbers and equivalents, are incorporated by reference in their entirety for all purposes.

Table 3. Nucleotide sequences of primers and probes used in this study

primer Sequence(5'-->3') Bc-F TCCATCCGCAAGCCTTTACT(SEQ ID NO:1) Bc-R GCTTCCGGTGCCATTGACTA(SEQ ID NO:2) m3-F AATGGGAATGGAGCGGATTC(SEQ ID NO:3) m3-R CCTGCACCAGCTTATCGTCAA(SEQ ID NO:4) Ch-F GGGCTGCGGAAGCAACTTA(SEQ ID NO:5) Ch-R GATGACCTCGCCCTGATCAT(SEQ ID NO:6) FN-F TTCAATAAAAGTGGCAGGTCAAG(SEQ ID NO:7) FN-R TAACAACACATGCAGGTCAATGG(SEQ ID NO:8) Ri-F CGGATTTGCAGTGGCAAGTT(SEQ ID NO:9) Ri-R TGATTGCAGACGCCAATGTC(SEQ ID NO:10) C-F CGTCAGCTCGTGYCGTGAG(SEQ ID NO:11) C-R CGTCRTCCCCRCCTTCC(SEQ ID NO:12) probe Sequence(5'-->3') Bc TTCATCATCACAGCCGACAACGCA(SEQ ID NO:13) m3 AAGCCTGCGGAACCACAGTTACCAGC(SEQ ID NO:14) Ch ACCACCACACAGGACGGAAAGATTCTCC(SEQ ID NO:15) FN ACTCGAACCCCCACCCTCGGTTT(SEQ ID NO:16) Ri CGTGAAAATCCGCGCATCTGGC(SEQ ID NO:17) C ttaagtCCCRYAACGAGCGCAACCC(SEQ ID NO:18)

Table 4. Clinical characteristics of subjects recruited in this study

*The size of the largest lesion in each patient was considered.

#Baseline data are missing for 5 patients in Group II.

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25.Xia

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primer Sequence(5-->3) Bc-F TCCATCCGCAAGCCTTTACT(SEQ ID NO:1) Bc-R GCTTCCGGTGCCATTGACTA(SEQ ID NO:2) m3-F AATGGGAATGGAGCGGATTC(SEQ ID NO:3) m3-R CCTGCACCAGCTTATCGTCAA(SEQ ID NO:4) Ch-F GGGCTGCGGAAGCAACTTA(SEQ ID NO:5) Ch-R GATGACCTCGCCCTGATCAT(SEQ ID NO:6) FN-F TTCAATAAAAGTGGCAGGTCAAG(SEQ ID NO:7) FN-R TAACAACACATGCAGGTCAATGG(SEQ ID NO:8) Ri-F CGGATTTGCAGTGGCAAGTT(SEQ ID NO:9) Ri-R TGATTGCAGACGCCAATGTC(SEQ ID NO:10) C-F CGTCAGCTCGTGYCGTGAG(SEQ ID NO:11) C-R CGTCRTCCCCRCCTTCC(SEQ ID NO:12) probe Sequence(5'-->3') Bc TTCATCATCACAGCCGACAACGCA(SEQ ID NO:13) m3 AAGCCTGCGGAACCACAGTTACCAGC(SEQ ID NO:14) Ch ACCACCACACAGGACGGAAAGATTCTCC(SEQ ID NO:15) FN ACTCGAACCCCCACCCTCGGTTT(SEQ ID NO:16) Ri CGTGAAAATCCGCGCATCTGGC(SEQ ID NO:17) C ttaagtCCCRYAACGAGCGCAACCC(SEQ ID NO:18)

SEQ ID NO:19

>A Lachnoclostridium sp.m3(gene ID 482585)

AATATCCGATATGGCAACGGAGCTCTGGTAGTAGTCCGGGCAAGGGAAAACCTTGTACATGGCGAAGCAGAGCAGATTACCTTCAATACTAAAATATTAGAAAGGTGCGTGAGGCATTTGAGAAATCCGATTGAAGTATTGAAAACTCTACAAGAGAAAGCAGGCAACGAGAACTATCAATTTGAACGCCTGTACCGAAATCTGTACAACGAGGAGTTTTTCCTATTGGCATACGGAAATCTCTCTGCAAAAGAGGGAAATCTG ACCAAGGGAACAGACGGCGCCACAATAGACGGAATGGGAATGGAGCGGATTCGCAAGCTGATTGAAAGCCTGCGGAACCACAGTTACCAGCCGTCCCTGCGAGACGTGCCTATATCCCAAAATCTAATGGAAAACGGCGTCCGTTAGGCATACCCTCTGTTGACGATAAGCTGGTGCAGGAAGTTGTGAGGTTAATTCTCGAAAGTGTGTATGAAAGCAATTTTTCTGAACATTCGCATGGTTTTAGACCGAACAGGAGC TGTCACACGGCACTGACCCAGATTCAAAGAAACTTCACAGGGGTTAAATGGTTCATTGAGGGGGACATCAAAGGTTATTTTGACACCATCGACCACCATATCCTTGTGGATATTTTAAGAAGGCGCATAAAGGACGAATACCTAATCTCGCTGATATGGAAATTTCTGAAAGCCGGATACTTAGAAGACTGGAAATTCAATCCTACCTATTCCGGCACTCCGCAAGGCTCGGTCATCAGTCCAAATACTTGCCAATATCTACCTTAAC GAATTCGATACCTATGTTGAAGAATACATAGAGAAATTCAACCGTGGTAAAAGACGTGAAAGAAACAGTGAGTATCGCTTTTATAGTGATGGCGCATCGAAACTGAGGGTAAAGTACCGCGGGTTATGGGAAATAATGACAGCCGATGAAAAAGAAAAAGCCAAATGTGAAGTAAATGAGCTCATGAAAAAAGCAAAACAGATTCCAGCTATGAATCCGATGGACAGCAATTACCGCCGTCTGCTCTATTGCAGGTATGCGGATG ATTTTATTTGCGGAGTAATCGGAAGCAAGGAAGATGCAGAAACCATCAAGGCTGATTTTAGCGGTACCTGAAAGAAAAGCTGGGACTGGATATGTCGGAAGAAAAGACACTGATTACACACTCAAACGAAAAAGCGGCGTTCCTTGGCTACGAAATCGCTGTTTCCAGAAGCAATGAATACAAAAAGATAAGCAACGGACAGAAGGCAAGAACCTTTAATGGGCGTGTTCATCTATTTATGCCACATAATAAATGGGTTAAGAAG CTGACCAGTTGCGGAGCAATGGAAATCAAACAGCAGGACGGCAAAGAAATATGGAAACCGCAGGCGAGGAAAGACCTCATCAACAAAGAGCCGATTGAAATCCTAAGCATTTACAATGCCGAAATTCGTGGGCTGTACAATTATTGTTTGGCAAGCAACGTATGCAAGCTGCAGAAATATTACTACATCATGGAATACAGCATGTACCAGACGTTTGCAGCGAAGTACCGTGATAATTTGCGGAAAACGATTAACAAGCATACCCG AAACGGCGTGTTTGGTGTCAGCTACACTACAAAAACCGGCAACGAGAAACGGGCGACATTCGTGAAAGGAAGCTTCCAAAAACGGACTGTCAGCTTAGATTACAGTGATGAAATCCCCTCTTATCCTGCCGCAAAATATAGTCGGAAAAACGGCTTAATTGAGCGGTTACAGGGTGGAAAATGTGAACTATGCGGACAGCAGACCGACAATGTAAAAGTTCATCATGTCAGGAAGCTGAAAGAATTAGCCGGTATGAAAGAATGGGAAAGAAAAAT GGTTCAGATGAACAGAAAAACTCTGGTTGTTTGTAATACATGTTATGGAAACATAACAGGCAAGTAA

SEQ ID NO:20

>Clostridium hathewayi (now known as Hungatella hathewayi) (gene ID2736705)

ATGCCTATACTTCAGCAGCTTCTCACATTAGTAGAGCAGCACTTCGGTAACAAATGCGAAATCGTGCTTCATGATCTGACAAAGGATTACAACCATACCATTGTCGATATCCGAAACGGAGACATTACCCATCGTTCCATCGGGGGCTGCGGAAGCAACTTAGGGCTGGAAGTCCTGCGCGGAACCGTGCTGGATGGGGATCGTTTTAACTATGTTACCACCACACAGGACGGAAAGATTCTCCGTTCCTCATCGATCTATCTAAA AAATGATCAGGGCGAGGTCATCGGATCGATCTGCGTGAACCTGGATATCACAGAGACACTTCAGTTTGAAGGGTATTTACGCCAGTTTAACCAGTTTGACAGCTTTACTTCCAACGACGAGGAGATTTTCGCTCCCGACGTGAATAATCTTCTCAGCCATCTGATTCAGATGGGACAGGAACAGATCGGAAAGCCTGCGCTGGAGATGAACAAGAACGAGAAGATTGAGTTTATCCGTTTCCTTGACCAGAAAGGAGCAT TCCTCATCACGAAGTCCGGGGAACAGATCTGTGAACTTCTGGGAATCAGCAAATTTACCTTTTATAATTACCTTGAAAGCAGCCGCAGCCAGTCGGATTCG

SEQ ID NO:21

>Fusobacterium nucleatum(Fn)(gene ID 1704941)

TCTGCAAAAGAAAAAAGTTGCTGCATTAGTTGCTGCATTAAAAGCAGATGGATATGATTTTACTGTTGGTATCCCTCTTGATACACCAATAGGAAAATCTGAAAGAGTTGTAAGTGCTGGTAAAGGGATTGGAGATAAAAAGAATATGAAGCTAATTGAAAACTTAGCAAAACAAGCTGGAGCTTCTATTGGTTCTTCTCGTCCAGTGGCAGAAACATTGCAATATGTACCTCTTGACCGTTATGTAGGAATGTCAGGACAAAAAAA ATTTGTTGGAAACCTTTATAGCTTGTGGAATTTCAGGAGCTTTACAACATTTAAAAGGAATTAAAGATGCAACAACAATAGTTGCTATAAATACAAACTCAAATGCTCCAATATTTAAGAATGCAGACTATGGAATAGTTGGAGATTTAGCAGAAATTTTACCTTTTATTAACTAAGGAATTAGATAATGGAGAAGCTAAAAAAGATGCACCACCTATGAAGAAAATGAAGAGAGTTATACCTAGAGTAGTGTATAGTCCTCATGT ATATGTAGTAGTGGTTGTGGACATGAATACAATCCTGATTTAGGAGATGAAGATTCTGACATAAAACCAGGAACTAGATTTAAAGATTTACCAGAAGATTGGACTTGTCCTGATTGTGGAGATCCAAAATCTGGATATATAGATGCAAAAAAATAA

SEQ ID NO:22

>Bacteroides clarus(Bc)(gene ID 370640)

ATGAAAATCAAACAATTAGCGAAAAGCGCATCATTCTTGCTGGTGGCAGGTTTTATCAGTTTTACTATTCCGTCGTGTAGCAGTGAAGAAGAAATCATCATCCTTCAGGATGTAAAAGTAAACAGTGAAAGCTTCAATCTGGCCGAAGACGGCAGTACGACCATAGAAGTCAAGGTAGTACCCGAAAATACTCCAATAGCCAAAGCCGTACTCAGCACATCATTATTTAATGAAAGCGGTGTTTTTCGAAGTAACCCGACTCACTCCC AAAGGTAACGGTGTATGGCAGATAGCAGCAAAAGTAAAGGACTTCTCACGCATTCAAAACGGTCAGGACGTAATACTTTCCGTCTATCAGGAAGATAATATGTATATCCAAACCACATTGAAATAAACGACCCATATAGCATCGAGGGTAAATATACACCGGTCCATCCGCAAGCCTTTACTTTCTACAGTGCCGAAGACGGCAAACTGATGGAGATTCCGTTCATCACAGCCGACAACGCAGCCGACCTTGCCGCCATCAGC TACGACAATATAAAGGTAGTCAATGGCACCGGAAGCTCTACACCCAGCATAAGTATCACACATTTCGCAATAGCTCCGATGACAGGTAAAACAGGCTTCTATCTGCAAGTGGATAACGCCCAACTCGAAACGGTAAAAAAAGCCATCACAACCATCGCTTTTTGGACTGCCGGGTTATGATAACCGGCCCTAACGGCCGTGTTGCCTATACTCCTGTGCGCCTCATTGTTTCTTCTCCGAAGTGCATCATCAAGGACGACCAACT CAGCCTGCTGCATACAGAATTGTCCGCCCCGGAGTTTAATAGACAAATCACCATAGATATGACCCACGATTTTTATCGTTTGGGCAAACAGAATGATAAAACAACCTTTGAGGCGTTTGAAAACCGAGGCTTGTATAACTCACAAGGAGAAATGGCAGATGCAGACCCTCAGTTCATTTCGTTGGGTTATACCACTCAGGGCAAAAATACAACATGTAACGTAACTTTAAAACATGATGCCACAATTCCTGCAATCGGCACTTACCACATGGTAG AACGCCTAAAAGGATATTGGGAATATGACGGAAAGAAATATCCGACCGTTTGTACAGACCTGCAATTCCAAATCACGATTAAATAA

Claims (35)

1. A method for assessing an individual's risk of colorectal adenoma recurrence after colorectal cancer or adenoma resection, comprising the following steps: (a) Lachnoclostridium species carrying genetic marker m3 (m3), Fusobacterium nucleatum (Fn), Clostridium hathawayii (Ch ) and baseline levels of one or more of the four bacterial species Bacteroides clarusii (Bc); (b) obtaining follow-up levels of one or more of the four bacterial species from a second stool sample collected from the individual after resection of the colorectal cancer or adenoma; (c) calculating a composite score based on the baseline level and the follow-up level of any one or more of the four bacterial species Fn, m3, Bc and Ch; and (d) Detecting that the value is higher than a standard control value determines that the individual is at increased risk of colorectal adenoma recurrence. 2. The method of claim 1, wherein the composite score for the baseline and follow-up levels for any one, two, three or four of the four bacterial species is represented by in Table 1 listed methods to calculate. 3. The method of claim 1, wherein the genome of m3 comprises the nucleotide sequence of SEQ ID NO: 19, or wherein the genome of Ch comprises the nucleotide sequence of SEQ ID NO: 20, or wherein the genome of Fn The nucleotide sequence comprising SEQ ID NO:21, or wherein the genome of Bc comprises the nucleotide sequence of SEQ ID NO:22. 4. The method of claim 1, wherein the individual has a colorectal adenoma removed by polypectomy. 5. The method of claim 1, wherein steps (a) and (b) each comprise obtaining all DNA, RNA or proteins unique to at least one of the bacterial species Fn, m3, Bc and Ch. level of description. 6. The method of claim 1, wherein steps (a) and (b) each comprise a polymerase chain reaction (PCR) for determining the level of the bacterial species. 7. The method of claim 6, wherein the PCR is quantitative polymerase chain reaction (qPCR) or reverse transcription-polymerase chain reaction (RT-PCR). 8. The method of claim 1, wherein the second fecal sample is collected from the individual about one year to about five years after the resection of the colorectal cancer or adenoma. 9. The method of claim 8, wherein the second stool sample is collected from the individual approximately one year after the resection of the colorectal cancer or adenoma. 10. A method for assessing an individual's risk of colorectal adenoma recurrence after colorectal cancer or adenoma resection, comprising the steps of: (a) Obtain the following values from a stool sample collected from said individual after resection of a colorectal cancer or adenoma: or (2) A composite score of the levels of m3 and Ch of the two bacterial species, calculated by I2+β5*m3+β6*Ch; or (3) Comprehensive score of the levels of Fn, m3 and Ch of the three bacterial species, which is calculated by I3+β7*Fn+β8*m3+β9*Ch; or (4) Comprehensive score of the levels of Fn, m3, Bc and Ch of the four bacterial species, which is calculated by the following I1+β1*Fn+β2*m3+β3*Bc+β4*Ch; and (b) Detecting that the value is higher than a standard control value determines that the individual is at increased risk of colorectal adenoma recurrence. 11. The method of claim 10, wherein the genome of m3 comprises the nucleotide sequence of SEQ ID NO: 19, or wherein the genome of Ch comprises the nucleotide sequence of SEQ ID NO: 20, or wherein the genome of Fn The nucleotide sequence comprising SEQ ID NO:21, or wherein the genome of Bc comprises the nucleotide sequence of SEQ ID NO:22. 12. The method of claim 10, wherein the individual has a colorectal adenoma removed by polypectomy. 13. The method of claim 10, wherein step (a) includes obtaining said level of DNA, RNA or protein unique to at least one of said bacterial species Fn, m3, Bc and Ch. 14. The method of claim 10, wherein step (a) includes polymerase chain reaction (PCR) for determining the level of the bacterial species. 15. The method of claim 14, wherein the PCR is quantitative polymerase chain reaction (qPCR) or reverse transcription-polymerase chain reaction (RT-PCR). 16. The method of claim 10, wherein the fecal sample is collected from the individual about one year to about five years after resection of a colorectal cancer or adenoma. 17. The method of claim 10, wherein the fecal sample is collected from the individual approximately one year after resection of a colorectal cancer or adenoma. 18. The method of claim 1 or 10, further comprising, after determining that the individual is at increased risk for colorectal adenoma recurrence, performing periodic colonoscopies to monitor the individual or administering an inhibitor to the individual The step of reagent, said inhibitor inhibits or eliminates one or more of said bacterial species Fn, m3 and Ch in said individual. 19. The method of claim 1 or 10, wherein the inhibitor is a small inhibitory RNA that specifically targets at least one gene of one or more of the bacterial species Fn, m3 and Ch. Or an antisense oligonucleotide, or an expression cassette directing the expression of the inhibitory RNA, or a viral vector including the expression cassette. 20. The method of claim 19, wherein the expression cassette is included within a viral particle. 21. A kit for assessing an individual's risk of colorectal adenoma recurrence after colorectal cancer or adenoma resection, comprising: (1) A first container containing a reagent for determining the level of bacterial species Fn; and (2) A second container containing reagents for determining the level of bacterial species m3. 22. The kit of claim 21, further comprising a third container containing one or more reagents for determining the level of bacterial species Bc. 23. The kit of claim 21, further comprising a third container containing one or more reagents for determining the level of bacterial species Ch. 24. The kit of claim 21, wherein the reagents in each of the containers are reagents for polymerase chain reaction (PCR). 25. The kit of claim 24, wherein the PCR is qPCR or RT-PCR. 26. The kit of claim 21, wherein the reagents in each of the containers are reagents for detecting proteins specific to the bacterial species. 27. A method for reducing the risk of colorectal adenoma recurrence in an individual following resection of colorectal cancer or adenoma, comprising administering to said individual an effective amount of an inhibitor that inhibits or eliminates the risk of colorectal adenoma recurrence in said individual. One or more of the bacterial species Fn, m3 and Ch. 28. The method of claim 27, wherein the inhibitor is a small inhibitory RNA or a transgene that specifically targets at least one gene of one or more of the bacterial species Fn, m3 and Ch. sense oligonucleotide, or an expression cassette directing the expression of the inhibitory RNA, or a viral vector including the expression cassette. 29. The method of claim 28, wherein the expression cassette is included within a viral particle. 30. The method of claim 27, further comprising the step of determining the level of one or more of the bacterial species Fn, m3 and Ch in the feces of the individual after the step of administering. 31. The method of claim 27, wherein the step of administering is performed within about one year after resection of the colorectal cancer or adenoma. 32. The method of claim 27, wherein the step of administering is repeated from about one year to about five years after resection of the colorectal cancer or adenoma. 33. A kit for reducing the risk of recurrence of colorectal adenomas, comprising: (1) A first container containing one or more reagents for determining levels of one or more of bacterial species Fn, m3, and Ch; and (2) A second container containing a composition including an effective amount of an inhibitor that inhibits or eliminates one or more of the bacterial species Fn, m3, and Ch. 34. The kit of claim 33, wherein the first container includes PCR reagents for determining the levels of DNA or RNA of one or more of the bacterial species Fn, m3 and Ch. 35. The kit according to claim 33, wherein the inhibitor is a small inhibitory RNA or a transgene that specifically targets at least one gene of one or more of the bacterial species Fn, m3 and Ch. sense oligonucleotide, or an expression cassette directing the expression of the inhibitory RNA, or a viral vector including the expression cassette.