WO2016003893A1 - Detection of colorectal cancer with two novel heme-related molecules in human feces - Google Patents

Abstract

The present invention provides a method of assessing the presence or absence of CRC or CRA in a patient. The method involves obtaining a stool sample from the patient and assaying the stool sample to detect the presence or absence of one or both of two peptides referred to herein as X-18565 and X-19549. In another embodiment, the present invention provides an immunogenically-stimulatory composition comprising one or both of X-18565 and X-19549, including a physiologically-acceptable carrier. The invention also provides a method for producing antibodies that can specifically bind to X-18565 and X-19549, respectively, by administering such compositions to animals, which are thereby induced to produce antibodies. In another embodiment, the present invention provides a composition comprising an specific binding partner to either or both of X-18565 and X-19549 and to diagnostic test kits employing such reagents.

Description

DETECTION OF COLORECTAL CANCER WITH TWO NOVEL HEME-RELATED

MOLECULES IN HUMAN FECES

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 62/019,258, filed June 30, 2014, which is incorporated by reference in its entirety herein.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED

ELECTRONICALLY

[0002] Incorporated by reference in its entirety herein is a computer-readable

nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 1,073 Byte ASCII (Text) file named "720850_ST25.TXT," dated May 28, 2015.

BACKGROUND OF THE INVENTION

[0003] Mortality from colorectal cancer (CRC) can be reduced by detecting the cancer or its precursor, colorectal adenoma (CRA), so that it can be removed at an early stage.

Screening stool specimens for blood and especially for hemoglobin, a major component of blood, has been partially effective. For example, the fecal immunochemical test (FIT) for hemoglobin, the best currently available, is positive in stool for about 60% of early-stage and about 85% of advanced CRC cases, with an acceptable false-positive rate (positive without CRC or CRA) of less than 10%. As described in Chubak et al., Preventive Medicine, 57: 671 - 78 (2013), the likelihood that a patient has CRC or high-risk CRA that requires frequent follow-up, evaluations and treatment ranges from 24% to 33% with a positive result on any one of three currently available stool tests. Thus, better stool tests are needed to make screening more accurate.

BRIEF SUMMARY OF THE INVENTION

[0004] The present invention provides a method of assessing the presence or absence of CRC or CRA in a patient. The method involves obtaining a stool sample from the patient and assaying the stool sample to detect the presence or absence of one or both of two peptides referred to herein as X-l 8565 and X-19549. The presence of one, and especially both, of these peptides within the stool sample indicates a high likelihood that CRC or CRA is present within the patient. Conversely, the absence of both X-18565 and X-19549 indicates a high likelihood that CRC or CRA is not present within the patient.

[0005] In another embodiment, the present invention provides an immunogenically- stimulatory composition comprising one or both of X-18565 and X-19549, including a physiologically-acceptable carrier. The invention also provides a method for producing antibodies that can specifically bind to X-18565 and X-19549, respectively, by administering such compositions to animals, which are thereby induced to produce antibodies.

[0006] In another embodiment, the invention provides a composition comprising an specific binding partner to either or both of X-18565 and X-19549 and to diagnostic test kits employing such reagents.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0007] Figures 1A and IB are graphs showing the distributions of intraclass correlation coefficients (ICCs) across 579 metabolites detected in at least 10% of quality-control specimens of human feces with the laboratory methods that were used to discover the peptides X-18565 and X-19549 and the intraclass correlation (ICC) statistical methods described in Sampson et al., Cancer Epidemiol. Biomarkers Prev., 22: 631-40 (2013). (A) Technical ICC is a measure of laboratory variability; 91 % of metabolites have technical ICC >0.7. (B) Within-subject ICC is a measure of stability over 6 months; 44% of metabolites have within-subject ICC >0.5.

[0008] Figure 2 is a quantile-quantile (QQ) plot of observed and expected P-values that test whether each of 1043 metabolites differed significantly, beyond chance, in feces from CRC patients compared to controls. The solid diagonal line indicates mere chance

association, with 95% boundaries around chance association shown in the dotted lines.

Circles outside the dotted lines indicate fecal metabolites that differed significantly between CRC patients and controls.

[0009] Figures 3A and 3B are schematics illustrating the pairwise correlations of 1 1 CRC-associated metabolites, not including the peptides X-18565 and X-19549, in feces of CRC cases (A) and matched controls (B). Double-headed arrows (→) indicate direct (positive) correlations; blocked lines (|— |) indicate inverse (negative) correlations.

Mandelate, / hydroxybenzaldehyde, and palmitoyl-sphingomyelin are directly correlated with CRC; the remainder of the compounds shown in Figures 3A and 3B are inversely correlated with CRC. Line weight indicates strength of correlation coefficient. Cases' metabolites are generally more correlated than controls' metabolites.

[0010] Figure 4 is a graph showing the proportion of metabolites expected to be detected in a case-control study as a function of effect size according to different sample size [n of 500 (dashed line), 1000 (solid line) and 5000 (dotted line)] under Bonferroni-adjusted a-levels (0.05/579). Effect size is defined by the true relative risk (RR, on the x-axis) of disease comparing individuals in the top and bottom deciles of the "usual" metabolite level. A study of n=500 (dashed line), would detect 0.2%, 1.2%, 45%> and 82%> of metabolites that have true relative risks of 1.5, 2.0, 5.0 and 10.0, respectively. In a study of n=5000 (dotted line), the same relative risks would be detected with 1.9%, 8.0%, 99% and 99% of the metabolites.

[0011] Figures 5A and 5B present tandem mass spectrometry (MS ) analyses of metabolites at various mass-to-charge ratios (m/z). (A) Metabolite X- 19549. (B) Theoretical MS² spectrum for a compound having proposed molecular formula C₃₅H₅oOi₂Nio.

[0012] Figure 6 is a fragmentation spectrum of the +2 ion (MS 402) of the intact parent molecule X- 19549 showing the relative abundance of peptide fragments detected at various mass-to-charge ratios (m/z).

[0013] Figure 7 is a fragmentation spectrum of the +1 ion (MS 803) of the intact parent molecule X- 19549 showing the relative abundance of peptide fragments detected at various mass-to-charge ratios (m/z).

[0014] Figures 8A and 8B present tandem mass spectrometry (MS ) analyses of metabolites at various mass-to-charge ratios (m/z). (A) Metabolite X-18565. (B) Theoretical MS² spectrum for a compound having proposed molecular formula C22H41 O9N5.

[0015] Figure 9 is a fragmentation spectrum of the intact parent molecule X-l 8565 (M 518) showing the relative abundance of peptide fragments detected at various mass-to-charge ratios (m/z). (A) C 5H25N4O4_; RDB = 5.5. (B) C16H27N4O5; RDB = 2.5. (C) C,₆H₂₉N₄0₆; RDB = 4.5. (D) C i 8H₃₃N₄0₇; RDB = 3.5. (E) C 18H32N5O7; RDB = 5.5. (F) Neutral loss of C₂H₄0 from precursor. (G) C20H34N5O7; RDB = 6.5. (H) C₂2H38N₅0₈; RDB - 6.5. (I) Neutral loss of C₄H₇N0₂ from precursor. (J) Neutral loss of C₄H₇NO2 from parent. (K) C₂oH₃₆N₅0₈; RDB = 5.5. (L) Neutral loss of C₂H₄0 from parent.

[0016] Figure 10 is a fragmentation spectrum of relative abundance at various mass-to- charge ratios (m/z) of one of the fragment ions (M 3 518—474) generated in the MS 2 spectrum of Figure 9. (A) C₅H₁₀N₃O₃; RDB = 2.5. (B) C_!3H₂₄N₃0₄; RDB = 3.5. (C) C₁₅H₂₅N₄0₄; RDB = 5.5. (D) Ci₆H₂₇N₄0₅; RDB = 2.5. (E) C_I6H₂₉N₄0₆; RDB = 4.5. (F) C_!7H₃oN₅0₆; RDB = 5.5. (G) C₂₀H₃₄N₅O₇; RDB = 6.5. (H) Neutral loss of CH₂0 from precursor. (I) Neutral loss of C₄H₇N0₂ from precursor. (J) Ci₈H₃₂N₅0₇; RDB = 5.5. (K) Neutral loss of C₂H₄0 from precursor.

[0017] Figure 11 is a fragmentation spectrum of relative abundance at various mass-to- charge ratios (m/z) of one of the fragment ions (M³ 518— >430) generated in the MS² spectrum of Figure 9. (A) C₄H₈N₃0₂; RDB = 2.5. (B) C₅H₁₀N₃O₃; RDB = 2.5. (C) C₈H₁₅N₂0₃; RDB = 2.5. (D) C_i3H₂₄N₃0₄; RDB = 3.5. (E) Ci₅H₂₅N₄0₄; RDB = 5.5. (F) Ci₆H₂₇N₄0₅; RDB = 2.5. (G) Ci₆H₃₀N₅O₄; RDB = 4.5. (H) Ci₇H₂8N₅0₅; RDB = 6.5. (I) Neutral loss of C₂H₅N0₂ from precursor. (J) Neutral loss of C0₂ from precursor. (K) Neutral loss of H₂0 from precursor. (L) Ci₇H₃₀N₅O₆; RDB = 5.5. (M) Neutral loss of CH₂0 from precursor. (N) Ci₈H₃₀N₅O₆; RDB = 6.5 (loss of H₂0 from precursor).

[0018] Figure 12 is a fragmentation spectrum of a positive ion equivalent candidate to X- 18565 (M² 520) showing the relative abundance of peptide fragments detected at various mass-to-charge ratios (m/z).

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention provides a method of assessing the presence or absence of CRC or CRA in a patient. The method involves obtaining a stool sample from the patient and assaying the stool sample to detect the presence or absence of one or both of X-18565 and X- 19549.

[0020] The stool sample can be either fresh or frozen prior to assaying. For example, one or more stool samples can be collected from a patient in a hospital or at a clinic during a visit with a health care provider, or the patient can collect his or her own stool (e.g., at home). When collected remotely from the time of assay, it is preferred for the sample to be frozen (e.g., on dry ice). For assessment in accordance with the inventive method, a single stool sample can be employed, or samples from a patient can be collected over a period of time (e.g., two days, or three or more throughout a week, or even longer if desired) and pooled for assay. Lyophilization of the stool sample is preferred, as such can help assure equal loading of dry weight for the assay. Lyophilized samples can be stored for extended times, for example, at -40° C or -80° C. Preferably, the stool sample includes approximately one gram lyophilized feces. Collection and storage of such samples has been reported in the literature (see, e.g., Schiffman et al., Cancer Res, 49, 1322-6 (1989) and Schiffman et al., Cancer Res, 49, 3420-4 (1989)).

[0021] Typically, the inventive method includes a processing step prior to assaying the stool sample. For example, an extract comprising peptides and other low molecular-weight material can be obtained from the stool sample, which extract is then assayed. To obtain such an extract comprising peptides and other low molecular-weight material, the stool sample can be reconstituted (e.g., if lyophilized) and treated with an organic solvent such as methanol. A suitable extraction protocol involves non-targeted single methanol extraction. Following the extraction, the processing desirably includes precipitation of peptides and other low molecular-weight materials.

[0022] After processing, the extract comprises proteinaceous material (e.g., proteins and peptides) and also can include other molecules including, but not limited to, amino acids, carbohydrates, fatty acids, steroids, and xenobiotics. Volatile molecules, such as short chain fatty acids, can be lost during lyophilization (if the sample is lyophilized) and/or extraction. However, such loss is generally equivalent across specimens and would not affect detection of peptides including X- 18565 and X- 19549.

[0023] In accordance with the inventive method, the extract is assayed to detect the presence or absence of either or both of X-l 8565 and X-l 9549. Chromatographic methods can be employed, and a suitable assay for use in the inventive method involves gas chromatography coupled with tandem mass spectrometry (MS ). An embodiment in which the assay involves ultra high-performance liquid phase chromatography and gas

chromatography coupled with tandem mass spectrometry (HPLC-GC/ MS²) is preferred. Where such technique is employed, X-l 9549 can be identified as having the isotope +2 spectrum pattern depicted in Figure 5 A. Also, X-l 9549 can be identified via +2 ion (MS² 402) fragmentation spectrum as having the ions indicated as detected in Figure 6 and via +1 ion (MS² 803) fragmentation spectrum as having the ions indicated as detected in Figure 7. Likewise, X-18565 can be identified via positive ion (MS 520) fragmentation spectrum as having the ions indicated as detected in Figure 12.

[0024] X-18565 and X-19549 alternatively can be identified by ascertaining the sequence of peptides within the extract. X-19549 is a peptide consisting of the following amino acid sequence: Val-Gly-Ala-His-Ala-Gly-Glu-Tyr (SEQ ID NO: l). X-18565 is a peptide consisting of the following sequence: Ser-Thr-Val-X-Thr, wherein X at position 4 is He or Leu (SEQ ID NO:2). Alternatively or additionally, X-18565 and X-19549 can be identified as a peptide having the molecular formula of C₂₂H₄iN₅0₉ and C3₅H₅oNioOi2, respectively.

[0025] Another type of assay for use in the inventive method involves the use of specific binding partners preferentially binding to either X-18565 or X-19549, respectively. Thus, the assay can involve the use of a first specific binding partner that can preferentially, e.g., specifically, bind to X-19549, a second specific binding partner that can

preferentially/specifically bind to X-18565, or both of said first and said second specific binding partners. A "specific binding partner" is a molecule that can bind with measurably higher affinity to either X-18565 or X-19549, respectively, than to other molecules. The specific binding partner can be, e.g., an immunoglobulin (also referred to herein as an "antibody") or an antigen-binding portion of the immunoglobulin. The immunoglobulin for use in the inventive method can be of any type. For instance, the immunoglobulin can be of any isotype, e.g., IgA, IgD, IgE, IgG, IgM, etc. The antibody can be monoclonal or polyclonal. The antibody can be a naturally-occurring antibody, e.g., an antibody isolated and/or purified from a mammal, e.g., mouse, rabbit, goat, horse, chicken, hamster, human, etc. Alternatively, the antibody can be a genetically-engineered antibody, e.g., a humanized antibody or a chimeric antibody. The antibody can be in monomeric or polymeric form.

[0026] In an embodiment, the specific binding partner is an antigen binding portion of any of the immunoglobulins described herein. The antigen binding portion can be any portion of the immunoglobulin that has at least one antigen binding site. In an embodiment, the antigen binding portion is a Fab fragment (Fab), F(ab')2 fragment, diabody, triabody, tetrabody, single-chain variable region fragment (scFv), or disulfide-stabilized variable region fragment (dsFv). A single-chain variable region fragment (scFv), which is a truncated Fab fragment including the variable (V) domain of an antibody heavy chain linked to a V domain of a light antibody chain via a synthetic peptide, can be generated using routine recombinant DNA technology techniques (see, e.g., Murphy et al. (eds.), Murphy 's

Immunobiology, 7^th Ed., Garland Science, New York, NY (2008)). Similarly, disulfide- stabilized variable region fragments (dsFv) can be prepared by recombinant DNA technology (see, e.g., Reiter et al., Protein Engineering, 7, 697-704 (1994)). The immunoglobulins of the invention, however, are not limited to these exemplary types of antibody fragments.

[0027] Using such specific binding partners that can specifically/preferentially bind to either X-18565 or X-19549, respectively (or both types of specific binding partners), the assay can use immunochemical methods to detect the presence of either or both of X-18565 or X-19549 in the extract. Such types assays are known to persons of ordinary skill in the art and include methods such as immunoprecipitation, immunonephelometry, radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescent immunoassay (FIA), and the like. A commonly-performed assay of this type, which can be employed in the inventive method using one or more specific binding partners that can specifically bind to either X-18565 or X- 19549, respectively (or both types of specific binding partners) is an enzyme-linked immunosorbent assay (ELISA) or enzyme immunoassay (EIA). Thus, detection of either or both of X-18565 and X-19549 can be undertaken similarly to fecal immunochemical tests that have been employed (see, e.g., Allison et al., J. Natl . Cancer Inst, 99, 1462-70 (2007). It is believed that the use of such immunochemical tests can provide higher sensitivity in preinvasive cases than chromatographic methods . Also, the inventive method can be used in conjunction with other methods as well, such as detection of methylated or mutated CRC- associated oncogenes in feces (Imperiale et al, N. Engl. J. Med. 3/370(14): 1287-97 (2014)).

[0028] The presence of one, and especially both, of these peptides within the stool sample indicates a high likelihood that CRC or CRA is present within the patient. Conversely, the absence of both X-18565 and X-19549 indicates a high likelihood that CRC or CRA is not present within the patient. For example, in connection with the experimental work discussed below, using HPLC-GC/ MS², X-18565 was detected in 67% of CRC cases. This 67% sensitivity of X-18565 for CRC overall ranged from 50%o for non-invasive CRC cases to 75%o for invasive, non-metastatic CRC cases. Specificity of X-18565 was 99%, as it was detected in only 1 %_> of control patients who did not have CRC (e.g., false positives). X-19549 was detected in 48%> of CRC cases, and this 48%) overall sensitivity ranged from 40% for non- invasive and to 55% for invasive, non-metastatic CRC cases. Specificity of X-19549 was 97%, as it was detected in only 3% of controls patients who did not have CRC (e.g., false positives). Only partial overlap in detection of heme, X-18565, and X-19549 was observed. Thus, upon performing the inventive method, the absence of both X-18565 and X-19549 from the stool sample (or extract) indicates a greater than 95% likelihood that CRC or CRA is not present within the patient from which the stool sample is obtained. Conversely, if either or both of X-18565 and X-19549 are detected within the stool sample (or extract) in accordance with the inventive method, siich result indicates a likelihood that CRC or CRA is present within the patient of at least about 25%, which is ten-fold higher than the 2.5% prevalence of CRC and high-risk CRA in the general population. In some embodiments, such as those that are based on assaying one or both of X-18565 and/or X-19549 such as using specific-binding partners to such peptides as described herein (e.g., the test kits discussed herein), even greater sensitivity and specificity can be achieved (such as a two-fold improvement in both sensitivity and specificity). Thus, in such embodiments it is possible that a positive detection of either or both of X-18565 and X-19549 are detected within the stool sample (or extract) in accordance with the inventive method can indicate a likelihood of at least about 30%, such as at least about 35%, at least about 40%, at least about 45%, or at least about 50% (e.g., from about 25% to about 50%). The likelihood that CRC or CRA is present within the patient in light of such positive detection of either or both of X-18565 and/or X-19549 can be calculated using methods known to persons of ordinary skill (see, e.g., Chubak et al., Preventive Medicine, 57: 671 -78 (2013) and Allison et al. J Natl Cancer Inst, 99: 1462-70 (2007).

[0029] It will be understood that if either or both of X-18565 and X-19549 are detected within a stool sample (or extract) in accordance with the inventive method, the patient from which the stool sample has been obtained should be provided with follow up evaluations and treatment, once a positive diagnosis is confirmed.

[0030] In another aspect, the invention provides reagents for generating antibodies that can specifically (e.g., preferentially) bind to either X-l 8565 or X-19549. In this respect, the invention provides a composition comprising immunologically-stimulatory concentration of X-18565, X-19549, or both, and a physiologically-acceptable carrier. The composition can be administered to an animal (typically a mouse, goat, rabbit, or other animal commonly employed for generating antibodies). The immunologically-stimulatory concentration of the composition is sufficiently concentrated to challenge the immune system of the animal with either or both of the X-18565 or X-19549 present in the composition. The physiological carrier can be a buffered saline or other media typically employed to administer antigenic substances for the production of antibodies. The composition also can comprise one or more adjuvants to assist the animal in mounting an immune reaction to the X-18565 or X-19549 (or both) present in the composition to increase the likelihood that immunoglobulins capable of specifically binding to such molecules will be generated.

[0031] Many assays for determining an immunologically-stimulatory concentration are known in the art. For purposes of the invention, an assay, which comprises comparing the extent to which antibodies are secreted by B cells in the mammal upon administration of a given concentration of X-l 8565 or X-19549 to a mammal among a set of mammals in which each is given a different concentration of X-18565 or X-19549, could be used to determine a starting concentration to be administered to a mammal. The extent to which antibodies are secreted by B cells upon administration of a certain concentration can be assayed by methods known in the art.

[0032] Thus, using such immunological compositions, the invention provides a method of producing an antibody (including multiple antibodies) that can specifically (e.g.,

preferentially) bind to X-18565 by administering (e.g., by intravenous injection or other suitable route) the composition comprising the immunologically-stimulatory concentration of X-18565 and a physiologically-acceptable carrier to an animal under conditions sufficient for the animal to develop an immune response to the X-18565. Similarly, the invention provides a method of producing an antibody (including multiple antibodies) that can specifically (e.g., preferentially) bind to X-l 9549 by administering the composition comprising the

immunologically-stimulatory concentration of X-19549 and a physiologically-acceptable carrier to an animal under conditions sufficient for the animal to develop an immune response to the X-19549. In this context, the animal's immune response includes the generation of one or more immunoglobulins that can specifically (e.g., preferentially) bind to X-l 8565 or X- 19549, respectively.

[0033] Standard immunological techniques can be employed in producing

immunoglobulins that can specifically (e.g., preferentially) bind to X-18565 or X-19549, respectively, in accordance with the present invention. Methods of producing antibodies in non-human animals are described in, e.g., U.S. Patents 5,545,806, 5,569,825, and 5,714,352, and U.S. Patent Application Publication No. 2002/0197266 Al . For example, once the animal is inoculated and has developed the immune response (producing one or more immunoglobulins that can specifically bind X-l 8565 or X- 19549, respectively), serum can be harvested from the animal which comprises the immunoglobulin(s) of interest. The serum can be further processed, if desired, to concentrate or stabilize the immunoglobulin(s). The resulting composition is typically a polyclonal antibody composition.

[0034] Alternatively, once the animal is inoculated and has developed the immune response (producing one or more immunoglobulins that can specifically bind X-l 8565 or X- 19549, respectively), one or more splenocytes can be harvested from the animal, which can then be fused with one or more immortal cell(s) (e.g., myeloma cells) to form one or more hybridomas. The hybridoma is then cultured (and typically proliferated into a population), such that it then secretes the immunoglobulins that can specifically (e.g., preferentially) bind to X-l 8565 or X-l 9549 into the culture media in which the hybridoma is grown. The culture medium can then be harvested and further processed, if desired, to concentrate or stabilize the immunoglobulin(s). Also, the hybridomas can be cultured initially, or subcultured, at a sufficiently dilute density to establish clonal populations, which can facilitate the production of monoclonal antibodies. Standard hybridoma methods are described in, e.g., ohler and Milstein, Eur. J. Immunol, 5, 51 1-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, 2nd Ed., CSH Press (2013), and Murphy et al. (eds.), Murphy 's

Immunobiology, 7^th Ed., Garland Science, New York, NY (2008)). Epstein-Barr virus (EBV)-hybridoma methods are described in Haskard and Archer, J. Immunol. Methods, 74(2), 361 -67 (1984), and Roder et al., Methods Enzymol., 121 , 140-67 (1986).

[0035] The affinity of binding of the resulting immunoglobulins can be assessed, for example, by exposing a substrate coated or impregnated with one or both of X-l 8565 or X- 19549 to the putative specific binding partner (e.g., immunoglobulin) and exposing a negative control (e.g., a blank substrate and/or one coated with or impregnated with another molecule (e.g., albumin)) to the putative specific binding partner under like conditions. It will be understood that the substrate(s) can be the same, such as a Western blot having spots or bands of several molecules. After exposure to the putative specific binding partner, the substrate(s) can be washed to remove non-specific binding and then the presence of the specific binding partner bound to the of X-18565 or X-19549 can be verified, e.g., by using a labeled secondary antibody or other suitable technique.

[0036] The immunoglobulins produced in accordance with the present invention can be derivitized to produce other specific binding partners for specifically binding X-18565 or X- 19549 (e.g., an antigen binding portion of the immunoglobulin as described herein with respect to other aspects of the invention). Also, if desired, the immunoglobulins and other specific binding partners that can specifically bind X-18565 or X-19549 can be bound to or conjugated with any one or more of a detectable label such as, for example, radioactive or luminescent labels, a substrate for enzymatic detection, and other moieties typically used in immunological assays. Further examples of detectable labels may include, but are not limited to, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and element particles (e.g., gold particles).

[0037] Phage display furthermore can be used to generate an antibody. In this regard, phage libraries encoding antigen-binding variable (V) domains of antibodies can be generated using standard molecular biology and recombinant DNA techniques. See, for instance, Green et al. (eds.), Molecular Cloning, A Laboratory Manual, 4^th Edition, Cold Spring Harbor Laboratory Press, New York (2012) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY (2007). Phage encoding a variable region with the desired specificity are selected for specific binding to the desired antigen, and a complete or partial antibody is reconstituted comprising the selected variable domain. Nucleic acid sequences encoding the reconstituted antibody are introduced into a suitable cell line, such as a myeloma cell used for hybridoma production, such that antibodies having the characteristics of monoclonal antibodies are secreted by the cell (see, e.g., Murphy et al., supra, Huse et al., Science, 246, 1275-81 (1989), and U.S. Patent 6,265,150).

[0038] The inventive immunoglobulin(s) and other specific binding partner(s) for specifically binding X-18565 or X- 19549 can be employed as reagents in standard immunochemical assays for the detection of X-l 8565 or X-19549, such as from a stool sample as described herein (e.g., FITs), or from other biological sources. Thus, the present invention provides a composition comprising one or more immunoglobulins or other specific binding partner(s) that can specifically (e.g., preferentially) bind to X-18565 or X-19549 in isolated form or including a carrier. The carrier can include suitable lyoprotectant substances, if the composition is to be lyophilized. Alternatively, the carrier can be aqueous and include buffers, preservatives, chelating agents, if desired, to enhance stability and to facilitate their use in immunochemical assays.

[0039] The invention also provides a test kit comprising the inventive immunoglobulin(s) and other specific binding partner(s) that can specifically (e.g., preferentially) bind to X- 18565 or X-19549, or both. The test kit can include, for example, one or more of a substrate onto which the specific binding partner is bound or affixed, a reagent for facilitating binding of X-18565 and/or X-19549 within a sample to the specific binding partner(s), a reagent for detecting X-18565 and/or X-19549 specifically bound to the specific binding partner(s) (e.g., one or more secondary antibody(ies) that can specifically bind to the specific binding partner(s), which can be conjugated to an enzymatic substrate moiety, fluorescent moiety, or radioactive moiety, as well as suitable enzymes and apparatus for detecting the fluorescence or radioactivity, respectively), a positive control sample (e.g., a composition comprising X- 18565 and/or X-19549), a negative control sample (e.g., a composition comprising a peptide other than X-l 8565 or X-19549) and other materials and reagents commonly employed in immunochemical diagnostic test kits and apparatus.

[0040] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLES

[0041] The following materials and methods were employed for Examples 1 -4. Study participants and specimens

[0042] The study employed data and stored frozen specimens from a colorectal cancer (CRC) case-control study of fecal mutagens that was reviewed and approved by an

Institutional Review Board at the National Cancer Institute (Schiffman et al., Cancer Res., 49: 1322-6 (1989); Schiffman et al., Cancer Res., 49: 3420-4 (1989)). Briefly, for a period of four years at three hospitals, following signed informed consent, patients suspected to have CRC were recruited before surgery or initiation of treatment. Only newly diagnosed, histologically confirmed cases of adenocarcinoma of the colon or rectum were retained.

Likewise, contemporaneous patients awaiting elective surgery for non-oncologic, non- gastrointestinal conditions at these hospitals were recruited as controls. A median of 6 days (IQR 3-13 days) prior to hospitalization and surgery, participants completed diet and demographic questionnaires and provided two-day fecal samples that were frozen at home on dry ice and subsequently lyophilized. The two-day lyophilates were pooled, mixed and stored at -40 °C.

[0043] Of 69 cases and 1 14 controls in the original study (Schiffman et al., Cancer Res., 49: 1322-6 (1989); Schiffman et al., Cancer Res., 49: 3420-4 (1989)), the case-control analysis included 48 cases and 102 controls for whom at least 100 mg of lyophilized feces was available. Controls were frequency matched to cases by gender and body mass index (BMI). Tumors in the cases were classified by stage and site in the large intestine.

[0044] To quantify technical variability of the metabolomics assay, 48 identical, masked aliquots of lyophilized feces were prepared from the same patients, including 11 from each of 2 controls, and 26 from 7 other controls. To quantify within-subject variability over time, half of the 48 replicate control specimens were collected six months after the baseline specimens using identical collection and handling methods.

Laboratory methods

[0045] A range of small molecules (most <1000 Daltons) was detected in the lyophilized fecal specimens by ultra high-performance liquid phase chromatography and gas

chromatography coupled with tandem mass spectrometry (HPLC-GC/ MS ) (Metabolon, Inc., North Carolina, USA) as described previously (Evans et al., Anal. Chem., 81 : 6656-67 (2009); Sha et al., FASEB J., 24: 2962-75 (2010)). Briefly, non-targeted single methanol extraction was performed, followed by protein precipitation. Individual molecules and their relative levels were identified from the mass spectral peaks compared to a chemical reference library generated from 2,500 standards, based on mass spectral peaks, retention times, and mass-to-charge ratios. The molecules include, but are not limited to, amino acids, carbohydrates, fatty acids, lipids, and xenobiotics. Volatile molecules, such as short chain fatty acids but not peptides, may be lost during lyophilization or extraction. However, such loss is generally equivalent across specimens, and lyophilization is optimal for fecal specimens to assure equal loading of dry weight.

Assay performance statistical methods

[0046] As overall reliability and validity of this platform for serum and urine were previously evaluated (Sampson et al., Cancer Epidemiol. Biomarkers Prev., 22: 631-40 (2013)), herein the technical, within-subject, and between- subject variability of the fecal metabolite data were assessed by calculating intraclass correlation coefficients (ICC) for the fecal quality control samples, as follows. The total variance of each metabolite, σ , was decomposed into three different components: the between-subject variance, σ|, which represents the variance of the "usual" level for subjects in a population; the within- subject variance, σ^, which represents the variability over time around the "usual" level within an individual; and the technical variance, σ|, which is the variance introduced by measurement error in the laboratory procedures.

[0047] From these three variance components, the following additional quantities were defined:

1) Technical ICC: the proportion of the total variance that is attributed to biological variance, as opposed to random laboratory variation. High technical ICC indicates high laboratory reproducibility.

2) Within-subject ICC: the ratio of between-subject variance / total variance. Higher Uj indicates higher stability over time in vivo and thus higher power to act as a marker for long-term risk analysis.

Case-control statistical methods

[0048] Demographic data for the cases and controls was compared by Fisher's exact test for categorical variables. For this pilot study of fecal metabolites, the primary analysis modeled the association between each metabolite and CRC by unconditional logistic regression, adjusting for BMI, age, gender, race and hospital. Metabolite values below the level of detection were assigned the minimum observed value for that metabolite. For metabolites present in less than 80% of the individuals, the metabolite was characterized as present or absent (i.e., dichotomous) and the associated odds ratio (OR) and its P-value from a likelihood ratio test were reported. For metabolites present in at least 80% of individuals, the odd ratios (ORs) comparing the 90^th to the 10^th percentile of the metabolite values, the corresponding confidence intervals (CIs), and the P-value from the likelihood ratio test comparing models with and without the metabolite were reported. Letting X90, X₁₀, and β denote the 90th percentile, 10th percentile, and the log(OR) from the logistic regression, the OR of interest was defined by _e ^P(X™^~Xw) . The q-values, which reflect the False Discovery Rate (FDR), were calculated separately for metabolites present in less than 80% and metabolites in at least 80% of individuals.

[0049] The proportion of metabolites associated with cancer was examined by a quantile- quantile (QQ) plot. On a loglO-scale, the expected P-values (n/(n+l ), (n-l)/(n+l),...,l/n) were plotted against the observed P-values, ordered from largest to smallest

(P ' P P ) ' where n is the number of metabolites. A point-wise 95% confidence interval was also plotted showing the range of that can occur by chance. Specifically, 1000 permuted datasets were created by randomly assigning case/control status, calculated ( Ο ' * ₎ »-» ) ^{for each} Permutation b e {1,...,1000} , and then extracted the 2.5^th and 97.5^th quantiles of each p₍ ^b _i) .

[0050] A standard pathway analysis was performed. Specifically, whether the metabolites within predefined pathways were associated with the outcome of CRC by analysis of variance (globalAncova (Hummel et al., Bioinformatics, 24: 78-85 (2008)) was evaluated. P-values were calculated by permutation and are therefore valid. Pearson pairwise correlations were also calculated, overall and by case-control status, of all FDR- significant metabolites, using natural log-transformed values for metabolites detected in at least 80%o of specimens, else presence vs absence for lower prevalence metabolites.

Statistical power calculations

[0051] Using the observed estimates of technical, within-subject, and between-subject variability, the expected power was estimated for a case-control study nested within a cohort focused on a single outcome. Specifically, it was assumed that a nested study will have n participants, with an equal number of cases and controls. It was further assumed that the study will use a t test to compare the metabolite levels between cases and controls to detect associations between metabolites and disease, using a Bonferroni-corrected significance threshold. The effect size was defined as the relative risk (RR) of disease comparing individuals in the top to the bottom quartiles of the "usual" metabolite level. The mean probability of detecting a statistically significant association was calculated at a given effect size, across metabolites, and accounting for the 3 sources of variability. This average probability, or the average power, indicates the proportion of true metabolite-disease associations that were expected to be discovered in a given prospective study. Statistical power then was applied to selected metabolites that were observed to be associated with CRC in the case- control study.

[0052] All analyses were performed with SAS software version 9.1.3 (SAS Institute, Cary, NC) and the R statistical language version 3.0.1.

EXAMPLE 1

[0053] This example demonstrates the detection of small molecules in fecal specimens.

[0054] In the fecal specimens, there were 1043 small molecules detected. These included 773 characterized and 270 uncharacterized ("X") molecules. Of the 579 molecules detected in at least 10% of the fecal specimens, overall laboratory reproducibility in masked replicate specimens was high, with the technical ICCs exceeding 0.7 for 527 ( 1 %) of the metabolites (Figure 1A).

[0055| In addition to technical reproducibility, within-subject and between-subject variance contribute to total variance. Considering the between-subject/total variance ratio, which is equivalent to within-subject ICC, the within-subject ICC was relatively low (Figure IB). Only 44% of the metabolites had a within-subject ICC >0.5. Only 5% of the metabolites had a within-subject ICC >0.7.

EXAMPLE 2

[0056] This example demonstrates the characteristics of colorectal cancer patients and controls with fecal metabolite data. [0057] The 48 CRC cases tended to be older than the 102 frequency matched controls (mean 62.9 vs 58.3 years, P=0.06), but they did not differ by sex (60% male), BMI, race, smoking history, attained education, or hospital of recruitment (Table 1). The primary CRC tumors arose in approximately equal proportions in the proximal colon, distal colon, and rectum (29%, 33%, and 27%, respectively). The cases included 35% with metastases at diagnosis (Dukes' stage C/D), 42% with local invasion but no known metastases (Dukes' stage B), and 21% with only localized disease (Dukes' stage A).

TABLE 1

NA = Not applicable; SD = standard deviation.

EXAMPLE 3

[0058] This example demonstrates the association of fecal metabolites with CRC.

[0059] Global assessment indicated that many of the 1043 fecal metabolites differed significantly between cases and controls (Figure 2). The prevalence of all 1043 fecal metabolites in cases and controls was determined. Of these, 41 (3.9%) were significantly associated with CRC at FDR=0.10. The prevalence of two of these metabolites, X-19549 and X-18565, as well as heme, is presented in Table 2. As summarized in Table 3, CRC was over-represented with cofactors and vitamins (P=0.032, especially tocopherol-related), xenobiotics (P=0.006, especially drugs and food components / plants), and marginally with lipids (P=0.064) and metabolites that were not previously characterized, including X-19549 and X-18565 (P=0.074).

TABLE 2

Prevalence (%)

Metabolites Overall Cases Controls

X-18565 22 67 1

X-19549 17 48 3

Heme 1 1 29 2

TABLE 3

^*By false discovery rate (FDR) 0.10. P values calculated by global Ancova.

[0060] Heme was detected in 29% of cases and 2% of controls (OR 16.55, Table 4). Heme met the Bonferroni threshold for statistical significance, as did two heme-related metabolites (Table 4 footnote) - X-18565 (67% vs 1 %, OR 345) and X-19549 (48% vs 3%, OR 30). The likelihood of CRC was very high with detection of X-19549 (OR 29.81 , Table 4) and especially with detection of X-18565 (OR 345.3, Table 4). The heme-related molecules were highly correlated (X-19549 with heme, R=0.36; X-18565 with heme, R=0.49; and X-19549 with X-18565, R=0.73). In combination analyses, CRC risk was increased 24-fold (58% vs 5%) with detection of heme or X-19549, 97-fold (69% vs 3%) with detection of heme or X-18565, and 49-fold (71 % vs 5%) with detection of any of these heme-related metabolites.

TABLE 4

Prevalence (%)*

Metabolite P-value† Odds ratio (Cl)†

Cases Controls

Heme-related

Heme 29 2 1.5E-05 16.55 (3.46-79.09)

X-18565t 67 1 <1.0E-10 345.3 (35.43-3364.92)

X-19549:}: 48 3 1.8E-10 29.81 (7.99-1 1 1 .14)

Cof actors and vitamins

a-Tocopherol 96 100 6.0E-03 0.25 (0.08-0.74) γ-Tocopherol 98 100 1 .8E-03 0.26 (0.1 -0.64)

Pterin 90 99 4.0E-03 0.33 (0.15-0.73)

Xenobiotics

4-Acetamidophenol 92 98 9.1 E-06 0.04 (0.01 -0.27)

2-Hydroxyacetaminophen sulfate 0 10 1 .4E-03 0 (0-lnfinity)

3-Cystein-S-YL-acetaminophen 2 23 7.6E-04 0.07 (0.01 -0.58) p-Acetamidophenylglucuronide 0 8 1.9E-03 0 (0-lnfinity)

p-Aminobenzoate (PABA) 98 100 8.8E-04 0.22 (0.08-0.57)

N-2-Furoyl-glycine 98 100 3.8E-03 0.26 (0.1 -0.69)

Sitostanol 90 99 3.0E-04 0.20 (0.08-0.5)

p-Hydroxybenzaldehyde 100 99 1 .6E-03 3.96 (1 .56-10.05)

Mandelate 96 94 5.1 E-03 3.32 (1 .36-8.08)

Lipids

Palmitoyl-sphingomyelin 98 95 2.1 E-06 13.6 (3.9-47.41 )

Conjugated linoleate-18-2N7 96 100 2.3E-04 0.16 (0.06-0.47)

3-Dehydrocarnitine 92 98 1 .6E-03 0.35 (0.18-0.7)

Amino acids

Histidine 100 99 2.7E-05 7.46 (2.56-21 .8)

C/s-Urocanate 100 99 6.6E-04 5.19 (1 .88-14.37)

Peptides Prevalence (%)*

Metabolite P-value† Odds ratio (Cl)†

Cases Controls

Tryptophyl-glycine 100 97 1 .7E-06 14.34 (4.09-50.35)

Leucyl-tryptophan 100 94 5.6E-05 7.96 (2.58-24.56)

Alanyl-histidine 60 28 4.1 E-05 5.28 (2.29-12.16)

Histidyl-glycine 67 32 3.7E-05 4.94 (2.23-10.94)

Tyrosyl-glutamine 96 91 5.7E-04 4.97 (1 .84-13.45)

Histidyl-alanine 92 82 3.3E-04 6.87 (2.22-21 .28)

Valyl-aspartate 100 99 3.6E-04 7.10 (2.18-23.12)

Pyro-glutamyl-glycine 19 5 1.8E-03 7.12 (1 .98-25.68)

Alanyl-ieucine 100 99 2.9E-03 4.94 (1 .63-14.95)

Alanyl-tryptophan 100 94 3.4E-03 4.09 (1 .51 -1 1 .08)

Histidyl-phenylalanine 96 92 1.7E-03 5.33 (1 .76-16.19)

Leucyl-glutamate 100 99 2.5E-03 5.12 (1 .67-15.65)

Leucyl-serine 100 99 2.1 E-03 5.34 (1 .7-16.79)

a-Glutamyl-valine 100 99 4.6E-03 3.39 (1 .39-8.3)

Prolyl-alanine 100 99 2.3E-03 3.21 (1 .45-7.08)

Valyl-histidine 90 79 3.0E-03 4.43 (1 .61 -12.22)

Uncharacterized molecules

X_19558 92 84 9.3E-05 9.11 (2.76-30.1 1 )

X_16343 96 98 4.6E-03 3.77 (1 .44-9.86)

X_17749 98 100 7.0E-04 0.22 (0.09-0.57)

X_19232 96 100 4.9E-04 0.22 (0.09-0.55)

X_19136 77 86 1 .8E-03 0.19 (0.06-0.55)

^* Fraction above the limit of detection.

†Odds ratios, corresponding confidence intervals (CI) and P-values were calculated by logistic regression, adjusted for continuous age, body mass index, sex, race and hospital. Odds ratios compared 10^th vs 90^th percentiles for common metabolites (prevalence >80%), else presence vs absence for less common metabolites.

X X-18656 was found to have a neutral accurate mass of 519.29139±0.0026; a proposed molecular formula of C22H4-1O9 5, RDB=5; and a proposed identification as the peptide Ser-Thr-Val-X-Thr, wherein X at position 4 is lie or Leu (SEQ ID NO:2) from human hemoglobin. X-19549 was found to have neutral accurate mass of 802.36235 ±0.004; a proposed molecular formula of C35H50O-12N-10, RDB=16; and a proposed identification as the peptide Val-Gly-Ala-His-Ala-Gly-Glu-Tyr (SEQ ID NO:1 ) from human hemoglobin.

[0061] As shown in Table 4, the CRC risk was reduced with four acetaminophen metabolites. Pairwise correlations of the four acetaminophen metabolites ranged from R=0.29 to R=0.82 (median R=0.40). CRC risk also was low with 2-hydroxyhippurate (salicylulate), which did not meet FDR (OR=0.27, P-0.004), but not with salicylate, salicyluric-glucuronide, ibuoprofen, or hydroxy-ibuprofen (P=0.023 - 0.081).

[0062] Eighteen previously characterized fecal peptides/amino acids were associated with increased CRC risk, and none was associated with decreased risk. These 18 peptides/amino acids were highly correlated with each other (median R=0.51, interquartile range R=0.33 to R=0.67). Tryptophylglycine had very strong correlations (R=0.73 to R=0.88) with eight other dipeptides. One uncharacterized molecule (X_16343) had very strong correlations (R=0.70 to R=0.85) with tryptophylglycine and five other dipeptides. The strongest inverse correlation observed was between tryptophylglycine and sitostanol (R=-0.45).

[0063] The 1 1 other CRC-associated molecules, which may functionally contribute to the disease, included pterin, 2 tocopherols, 5 xenobiotics, and 3 lipids. Of these 1 1 , eight were associated with lower CRC risk and three with higher risk (Table 4). Table 5 presents the mean levels of the eight reduced-risk and three increased-risk metabolites. Figures 3A and 3B present the pairwise correlations of these metabolites. Nearly all correlations were positive (arrows). In both cases and controls, the metabolite network was centered around p- aminobenzoate (PABA). Cases had lower levels (Table 5) but many more and stronger correlations of reduced-risk metabolites compared to controls (Figures 3A and 3B).

TABLE 5

Mean of normalized levels

Cases Controls

Reduced risk PABA 0.7075237 1 .1867376

Sitostanol 0.5775664 0.9263738

Pterin 0.7567347 0.9910276 γ-Tocopherol 0.5877252 1 .0049531 a-Tocopherol 0.8024419 1 .7186395

Dehydrocarnitine 0.64101 13 0.9472227

N-2-Furoyl-glycine 0.7958184 1 .1051999

Linoleate-18-2N7 0.676362 1 .3850176

Increased risk p-Hydroxybenzaldehyde 1 .3493867 0.9959933

Mandelate 1 .1 190748 0.849791 1

Palmitoyl-sphingomyelin 1 .8041617 0.7672291 [0064] Among potential candidate molecules (Sears et al., Cell Host Microbe, 15: 317- 328 (2014)), ursodeoxycholate was marginally associated with lower CRC risk (OR=0.32, P=0.03), but six other fecal bile acids were unrelated (P=O.30 - 0.98). CRC was not associated with 3-aminobutyrate, 3-aminoisobutyrate, or 3-methyl~2-oxobutyrate (P=0.03 - 0.74). The 5 uncharacterized CRC-associated molecules included a pair (X_19232 and X_17749) associated with very low CRC risk that had the strongest direct correlation that was observed (R=0.93).

[0065] None of the previously characterized metabolites that were strongly associated with CRC differed significantly by tumor site (Table 6A). Two of them differed by stage of the tumor (Table 6B). Prevalence of heme was directly related to stage: 10% without invasion (Dukes' stage A), 20% with local invasion (Dukes' stage B), 47% with metastases (Dukes' stage C/D,

Palmitoyl-sphingomyelin level above median had a similar trend: 60% without invasion, 80% with local invasion, 94% with metastases (Ptrend-0.03).

TABLE 6A

^* P-values by Cochran-Armitage test with controls as reference. TABLE 6B

^* P-values by Cochran-Armitage test with controls as reference.

EXAMPLE 4

[0066] This example demonstrates the statistical power of fecal metabolomics.

[0067] With the observed technical, within-subject and between-subject variances, the study power to detect relative risks (RRs) of 1.5, 2.0, 5.0 and 10.0 in prospective, nested case-control studies using a Bonferonni adjusted a-level of 0.05/579 was estimated. As shown in Figure 4, a study with 250 cases and 250 controls (500 participants) is expected to detect 0.2%, 1.2%, 45% and 82% of the metabolites with true RRs of 1.5, 2.0, 5.0 and 10.0, respectively. In a study of 1000 participants, the proportions of metabolites detected with these RRs increases to 0.6%o, 6.5%, 84% and 96%, respectively. With a sample size of 5000, approximately 100% of metabolites with RRs of 5.0 and 10.0 are expected to be detected (Figure 4). With the very high associations with CRC observed for X-19549 (OR 29.81 , Table 4) and X-18565 (OR 345.3, Table 4), the statistical power to detect CRC with them approaches 100%.

[0068] Table 7 illustrates the impact of instability over time (within-subject ICC) on study power, using as examples the 1 1 potentially functional, previously characterized metabolites associated with CRC in this case-control study. In a study of 500 participants, power to detect a true RR=5.0 (or the converse, RR=0.20) would be good (>0.89) for six metabolites - 3-dehydrocarnitine, PABA, a-tocopherol, γ-tocopherol, pterin and N-2-furoyl- glycine. However, even for these metabolites, power would be low for a true RR=2.5 (or RR=0.40). In a larger study of 1000 participants, power for a true RR=2.5 would be good only for 3-dehydrocarnitine and PABA. At RR=5.0, power would be excellent for nine metabolites and good for palmitoyl-sphingomyelin (0.84) among 1000 participants. None of these metabolites could reliably detect a true RR=1.5 even among 1000 participants.

TABLE 7

EXAMPLE 5

[0069] This example demonstrates the characterization of the fecal metabolite X-19549 identified in Example 3. [0070] The metabolite X- 19549 identified in Example 3 was analyzed by tandem mass spectrometry (MS²). X-19549 was determined to have a neutral accurate mass of 802.36235 ± 0.004, a proposed molecular formula of C3₅H₅oOi₂Nio, and a number of rings or double bonds (RDB) of 16. The experimental neutral accurate mass matched the proposed neutral accurate mass of the proposed molecular formula within 1.8 parts per million (ppm; a standard measure of error, calculated by dividing the difference between the theoretical results and experimental results by the theoretical mass and then multiplying the resultant number by 1 x 10⁶).

[0071] The spectrum for the experimental +2 isotope pattern measured and the spectrum for the theoretical isotope +2 pattern of the proposed molecular formula are shown in Figures 5A and 5B, respectively. As shown in Figures 5A and 5B, the experimental isotope pattern observed for X-19549 (Figure 5 A) matched the theoretical isotope pattern (Figure 5B) for the proposed molecular formula.

[0072] Fragmentation spectra were also obtained for X-19549. The fragmentation spectra of the intact parent (intact unknown molecule X-19549) ion (MS²) are shown in Figures 6 and 7. Table 8 shows the theoretically possible fragment ions (all accurate mass) detected for X-19549 for the +2 ion (MS 402), with the ions that were actually detected in the experimental spectrum underlined, and with the abbreviations VGAHAGEY (SEQ ID NO: 1 ), corresponding to the peptide with amino acid sequence Val-Gly-Ala-His-Ala-Gly- Glu-Tyr (SEQ ID NO: 1). Table 9 shows the theoretically possible fragment ions (all accurate mass) detected for X-19549 for the +1 ion (MS² 803), with the ions that were actually detected in the experimental spectrum underlined, and with the abbreviations VGAHAGEY (SEQ ID NO: 1), corresponding to the peptide with amino acid sequence Val- Gly-Ala-His-Ala-Gly-Glu-Tyr (SEQ ID NO: 1). Excellent coverage of the peptide backbone was obtained with accurate mass fragmentation.

TABLE 8

N- terminal

a - 129.1022 200.1394 337.1983 408.2354 465.2568 594.2994 - a^+¾ - - - 169.1028 204.6213 233.1321 297.6534 - b-H₂0 - - - - - - 604.2838 - b-H₂0^+/ - - - - - - 302.6455 - b - 157.0972 228.1343 365.1932 436.2303 493.2518 622.2944 - b⁺ - - - 183.1002 218.6188 247.1295 31 1.6508 - b+H₂0 - - - - - 511.2623 640.3049 - b+H₂0⁺ⁱⁱ 256.1348 320.6561 -

V G A H A G E Y

C- terminal

Y - 704.2998 647.2784 576.2413 439.1823 368.1452 31 1 .1238 182.0812

Y⁺ - 352.6536 324.1428 288.6243 - - - -

Y-H₂0 - 686.2893 629.2678 558.2307 421 .1718 350.1347 293.1132 - γ-Η₂0^+¾ - 343.6483 315.1375 279.6190 - - - -

TABLE 9

[0073] The mass of X- 19549, the elemental composition, and the fragmentation spectra all indicate that X- 19549 is a peptide. Because the backbone of the peptide fragmented effectively, the peptide was successfully sequenced by hand as VGAHAGEY (SEQ ID NO: l). A search in the National Center for Biotechnology Information (NCBI) BLAST non- redundant (nr) GenBank protein database for this peptide sequence identified a peptide within hemoglobin as a primary hit.

[0074] These data support the identification of X- 19549 as the peptide VGAHAGEY (SEQ ID NO: l ) from human hemoglobin.

EXAMPLE 6

[0075] This example demonstrates the characterization of the fecal metabolite X-l 8565 identified in Example 3. [0076] The metabolite X-l 8565 identified in Example 3 was analyzed by tandem mass spectrometry (MS ). X-18565 was determined to have a neutral accurate mass of 519.29139 ± 0.0026 and a proposed molecular formula of C22H41 O9N5, and a RDB of 5. The experimental neutral accurate mass matched the proposed neutral accurate mass of the proposed molecular formula within 1.9 ppm.

[0077] The spectrum for the experimental +2 isotope pattern measured and the spectrum for the theoretical isotope +2 pattern of the proposed molecular formula are shown in Figures 8A and 8B, respectively. As shown in Figures 8A and 8B, the experimental isotope pattern observed for X-18565 (Figure 8 A) matched the theoretical isotope pattern (Figure 8B) for the proposed molecular formula.

[0078] The fragmentation spectra of the intact parent (intact unknown molecule) ion (MS/MS (MS²)) is shown in Figure 9. The MS² 518 spectrum (Figure 9) showed very limited fragmentation detail. The fragmentation spectrum of one of the fragment ions

2 3

generated in the MS spectrum (M 518— »474) showed that X-l 8565 could readily lose two separate C₂H₄0 moieties and also showed the ability to lose C4H7NO2 (Figure 10). Neutral loss is a mechanism of molecule fragmentation that often involves fragmenting off a section of the molecule in neural form. The "parent molecule" refers to the intact unknown molecule, while the "precursor" is the ion from which other ions were generated in multiple rounds of fragmentation (MS", where "n" is the number of rounds of fragmentation). Within the many fragmentation pathways of the molecule, an acid loss and a loss of C₂H₃NO were also observed (Figures 10 and 1 1).

[0079] The following transitions were also monitored: MS³ 518 => 373; MS³ 518 => 417; MS⁴ 518 => 430 => 325; MS⁴ 518 => 430 => 382; MS⁴ 518 => 430 => 142; MS⁴ 518 => 430 => 412; MS⁴ 518 => 430 => 160; MS⁴ 518 => 430 => 130; MS⁴ 518 -> 430 => 187; MS⁴ 518 => 430 => 400; and MS⁴ 518 => 474 => 355. These MS¹¹ data were complex.

[0080] It was suspected that X-l 8565 was a peptide. Therefore, the positive channel was used to look for a corresponding accurate mass positive ion signature because peptides tend to fragment in a more "sequenceable" fashion in positive ion mode as compared to negative ion mode. Two positive ions were identified as having an accurate mass and retention time that is similar to that of X-l 8565. [0081] One of these positive ions was a peptide with a sequence of STVXT, wherein X at position 4 is He or Leu (SEQ ID NO:2), which is from human hemoglobin (Figure 12). Table 10 shows the theoretically possible fragment ions (all accurate mass) detected for X-18565 (MS² 520), with the ions that were actually detected in the experimental spectrum underlined, and with the abbreviations STVLT (SEQ ID NO: 2, wherein X at position 4 is Leu), corresponding to the peptide with amino acid sequence Ser-Thr-Val-X-Thr, wherein X at position 4 is He or Leu (SEQ ID NO: 2). MS is not able to distinguish He from Leu.

TABLE 10

[0082] The second positive ion identified on the positive channel was less readily sequenceable and appeared to be a mix of peptides. As a result, this second positive ion candidate has several positive sequences, including VTLTS (SEQ ID NO:3), LTVTS (SEQ ID NO:4), and TLVTS (SEQ ID NO:5). Two of these match various immunoglobulin proteins in the National Center for Biotechnology Information (NCBI) BLAST non- redundant (nr) GenBank protein database.

[0083] Given the sequence from hemoglobin, STVXT, wherein X at position 4 is He or Leu (SEQ ID NO:2), the fragmentation of the negative ion unknown was able to be traced. It was determined that it was a very good match to STVXT, wherein X at position 4 is He or Leu (SEQ ID NO:2). The consecutive losses of C₂H₄0 were from the two threonine side chains, and the loss of C4H7NO2 was the loss of the N-terminal threonine residue. Further MS" fragmentation data showed a single loss of CH?0 arising from the serine side chain and the loss of C₂H3NO arose from the loss of the glycine-like residue after the Thr side chain had been cleaved off in a previous round of fragmentation.

[0084] These data support the identification of X-l 8565 as the peptide STVXT, wherein X at position 4 is He or Leu (SEQ ID NO:2).

EXAMPLE 7

[0085] This example demonstrates that the commercially available colorectal cancer detection test kit OC-Light (Polymedco, Cortland Manor, NY) does not detect Val-Gly-Ala- His-Ala-Gly-Glu-Tyr (SEQ ID NO: l) or Ser-Thr-Val-X-Thr, wherein X at position 4 is lie or Leu (SEQ ID NO:2).

[0086] It was investigated whether Val-Gly-Ala-His-Ala-Gly-Glu-Tyr (SEQ ID NO: 1 ) or Ser-Thr-Val-X-Thr, wherein X at position 4 is He or Leu (SEQ ID NO:2) could be detected with a commercially available colorectal cancer detection test kit. To do so, the following three peptides were synthesized and purified by high performance liquid chromatography by New England Peptide (Gardner MA):

• H₂N-VGAHAGEY-OH (SEQ ID NO: 1) (lot# 2818-38), 94% purity, mass 802 (calculated mass 803);

• H₂N-STVLT-OH (SEQ ID NO: 2 wherein X at position 4 is Leu) (lot # 2818- 47Q), 88% purity, mass 519 (calculated mass 520); and

• H₂N-STVIT-OH (SEQ ID NO: 2, wherein X at position 4 is He) (lot# 2818-48Q), 95% purity, mass 519 (calculated mass 520).

[0087] As per the manufacturer's recommendations, 1.0 mg of each peptide was solubilized in 1.0 mL ultrapure water. From these stock solutions, 1 : 10 and 1 : 100 dilutions were made with ultrapure water.

[0088] The stock solutions and the dilutions (in duplicate) were tested with fecal immunochemical test (FIT) strips (OC-Light) that were obtained from the manufacturer (Polymedco, Cortland Manor, NY). Following the manufacturer's instructions, after 5 minutes incubation, the presence/absence of the control band and the peptide-related band was read. All 15 FIT strips (three at 1 : 1 plus duplicates at 1 : 10 and 1 : 100 for each of the three peptides) demonstrated clear control bands but no peptide-related bands. [0089] It was concluded that these commercially available FIT strips do not detect Val- Gly-Ala-His-Ala-Gly-Glu-Tyr (SEQ ID NO: l) or Ser-Thr-Val-X-Thr, wherein X at position 4 is He or Leu (SEQ ID NO:2).

[0090] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0091] The use of the terms "a" and "an" and "the" and "at least one" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (for example, "at least one of A and B") is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly

contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

100921 Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):

1. A method for assessing the absence of colorectal cancer (CRC) or colorectal adenoma (CRA) in a patient, the method comprising:

(a) obtaining a stool sample from a patient,

(b) processing the stool sample to produce an extract comprising peptides, and

(c) assaying the extract to determine the presence or absence of X-l 8565, X-19549, or both X-18565 and X-19549 within the extract;

wherein the absence of both X-18565 and X-19549 within the extract indicates a greater than 95 % likelihood that CRC or CRA is not present within the patient.

2. A method for assessing the presence of colorectal cancer (CRC) or colorectal adenoma (CRA) in a patient, the method comprising:

(a) obtaining a stool sample from a patient,

(b) processing the stool sample to produce an extract comprising peptides, and

(c) assaying the extract to determine the presence or absence of X-18565, X-19549, or both X-18565 and X-19549 within the extract;

wherein the presence of X-18565, X-19549, or both X-18565 and X-19549 within the extract indicates a greater than 25 % likelihood that CRC or CRA is present within the patient.

3. The method of claim 1 or 2, wherein the stool sample is fresh.

4. The method of claim 1 or 2, wherein the stool sample is frozen prior to processing.

5. The method of claim 1 or 2, wherein the stool sample is lyophilized prior to processing.

6. The method of any of claims 1 -5, wherein the processing comprises methanol extraction.

7. The method of claim 6, wherein the extraction comprises non-targeted single methanol extraction.

8. The method of claim 6 or 7, wherein the processing comprises protein precipitation following the extraction.

9. The method of any of claims 1-8, wherein the assay comprises gas

chromatography coupled with tandem mass spectrometry.

10. The method of claim 9, wherein the assay comprises ultra high-performance liquid phase chromatography and gas chromatography coupled with tandem mass

spectrometry (HPLC-GC/ MS²).

1 1. The method of claim 10, wherein X-l 9549 is identified as having the isotope +2 spectrum pattern depicted in Figure 5A.

12. The method of claim 10 or 1 1 , wherein X-l 9549 is identified via +2 ion (MS² 402) fragmentation spectrum as having the ions indicated as detected in Figure 6.

13. The method of any of claims 10-12, wherein X-l 9549 is identified via +1 ion

2

(MS 803) fragmentation spectrum as having the ions indicated as detected in Figure 7.

14. The method of claim 10, wherein X-l 8565 is identified as having the isotope +2 spectrum pattern depicted in Figure 8A.

15. The method of claim 10 or 14, wherein X-18565 is identified via positive ion (MS 520) fragmentation spectrum as having the ions indicated as detected in Figure 12.

16. The method of any of claims 1 -15, wherein X-l 9549 is identified as a peptide having the molecular formula of C35H50N 10O12 and consisting of the following sequence: Val- Gly-Ala-His-Ala-Gly-Glu-Tyr (SEQ ID NO: l ).

17. The method of any of claims 1 -16, wherein X-l 8565 is identified as a peptide having the molecular formula of C22H4| N₅0₉ and consisting of the following sequence: Ser- Thr-Val-X-Thr, wherein X at position 4 is He or Leu (SEQ ID NO:2).

18. The method of any of claims 1 -8, wherein the assay comprises the use of a first specific binding partner that can specifically bind to X- 19549, a second specific binding partner that can specifically bind to X-l 8565, or both of said first and said second specific binding partners.

19. The method of claim 18, wherein one or both of said first or second specific binding partners is an immunoglobulin or an antigen-binding portion thereof.

20. The method of claim 19, wherein the assay comprises immunoprecipitation, immunonephelometry, radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescent immunoassay (FIA).

21. The method of claim 20, wherein the assay is an enzyme-linked

immunosorbent assay (ELISA).

22. A composition comprising an immunologically-stimulatory concentration of X-l 8565 and a physiologically-acceptable carrier.

23. A composition comprising an immunologically-stimulatory concentration of X-l 9549 and a physiologically- acceptable carrier.

24. A composition comprising an immunologically-stimulatory concentration of both X-l 8565 and X-19549 and a physiologically-acceptable carrier.

25. The composition of any of claims 22-24 comprising an immunostimulatory adjuvant.

26. A method for producing an immunoglobulin that can specifically bind to X-

18565, comprising administering the composition of claim 22 to an animal under conditions sufficient for the animal to develop an immune response to the X-l 8565.

27. A method for producing an immunoglobulin that can specifically bind to to X- 19549, comprising administering the composition of claim 23 to an animal under conditions sufficient for the animal to develop an immune response to the X-19549.

28. The method of claim 26 or 27, comprising harvesting serum from the animal after the animal has developed the immune response, wherein the serum comprises the immunoglobulin.

29. The method of claim 26 or 27, comprising harvesting a splenocyte from the animal after the animal has developed the immune response, fusing the splenocyte with an immortalized cell to form a hybridoma which secretes the immunoglobulin into culture medium, culturing the hybridoma, and harvesting the culture medium containing the immunoglobulin.

30. The method of claim 29, wherein the hybridoma is cultured at an initially dilute density sufficient to establish a clonal population.

31. A composition comprising an immunoglobulin or antigen-binding fragment thereof, which can specifically bind X-18565 or X-19549.

32. The composition of claim 31 , which is in lyophilized form.

33. The composition of claim 31 or 32, wherein the immunoglobulin or antigen- binding portion thereof is monoclonal.

34. A test kit comprising:

(a) a first specific binding partner that can specifically bind X-18565,

(b) a second specific binding partner that can specifically bind X-19549, or both (a) and (b), and one or more of:

(c) a substrate onto which (a) and/or (b) is bound or affixed,

(d) a reagent for facilitating binding of X-18565 and/or X-19549 within a sample to (a) and/or (b),

(e) a reagent for detecting X-l 8565 and/or X-19549 specifically bound to (a) and/or

(b), (f) a positive control sample, and

(g) a negative control sample.

35. The method of claim 2, wherein the assay comprises the use of a first specific binding partner that can specifically bind to X-19549, a second specific binding partner that can specifically bind to X-18565, or both of said first and said second specific binding partners.

36. The method of claim 35, wherein one or both of said first or second specific binding partners is an immunoglobulin or an antigen-binding portion thereof.

37. The method of claim 36, wherein the assay comprises immunoprecipitation, immunonephelometry, radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescent immunoassay (FLA).

38. The method of claim 37, wherein the assay is an enzyme-linked

immunosorbent assay (ELISA).

39. The method of any of claims 2, or 35-38, wherein the presence of X-18565, X- 19549, or both X-18565 and X-19549 within the extract indicates a greater than 50 % likelihood that CRC or CRA is present within the patient.

40. The method of any of claims 2, or 35-38, wherein the presence of X-18565, X- 19549, or both X-18565 and X-19549 within the extract indicates an about 25% to about 50%> likelihood that CRC or CRA is present within the patient.