BIOMARKERS FOR COLORECTAL CANCER

Abstract

The present invention relates to certain volatile organic compounds which can be detected in stool samples of individuals and be used as biomarkers in determining the probability that an individual has colorectal cancer.

Claims

1. A method of determining the probability that an individual has colorectal cancer, the method comprising the steps of: i) analysing a stool sample collected from said individual to detect whether the volatile organic compound isopropanol is present; ii) if isopropanol is present in step i), determining the amount of isopropanol that is present and comparing the amount detected with a control value; wherein the presence of an increased amount of isopropanol in the stool compared to the control value is indicative of an increased probability that the individual has colorectal cancer.

2. A method according to claim 1, wherein step i) further comprises analysing the stool sample collected from said individual to detect for the presence of one or more additional volatile organic compounds and, if one or more additional volatile organic compounds are present, step ii) further comprises determining the amount of the one or more additional volatile organic compounds and comparing the amount detected with one or more control values for each of the one or more additional volatile organic compounds; wherein an increase or decrease in the level of said one or more additional volatile organic compounds relative to the control value is indicative of the probability that the individual has colorectal cancer.

3. A method according to claim 2, wherein the one or more additional volatile organic compounds are organic compounds with a molecular weight of less than or equal to 250 Da.

4. A method according to claim 2, wherein the one or more additional volatile organic compounds are organic compounds with a boiling point of less than or equal to 250° C. at a pressure of 1 atmosphere.

5. A method according to claim 2, wherein the one or more additional volatile organic compounds are selected from: i) 2-hexanone; ii) Butanoic acid, 3-methyl-, ethyl ester; iii) Butanoic acid, 1-methylethyl ester; iv) Pentanoic acid, 1-methylethyl ester; v) p-Xylene; vi) Propanoic acid, 1-methylethyl ester; or vii) Dimenthol.

6. A method according to claim 1, wherein said method comprises the steps of: i) analysing a stool sample collected from said individual to detect whether the volatile organic compounds, isopropanol and butanoic acid, 3-methyl-, ethyl ester, are present; and ii) if isopropanol and butanoic acid, 3-methyl-, ethyl ester are present, determining the amount of isopropanol and butanoic acid, 3-methyl-, ethyl ester present and comparing these amounts with control values for each respective volatile organic compound; wherein an increased amount of isopropanol and butanoic acid, 3-methyl-, ethyl ester compared to their respective control values is indicative of an increased probability that the individual has colorectal cancer.

7. A method according to claim 6, wherein step i) further comprises analysing the stool sample collected from said individual to detect the presence of one or more additional volatile organic compounds and, if one or more additional volatile organic compounds are present, step ii) further comprises determining the amount of the one or more additional volatile organic compounds and comparing the amount with one or more control values for each of the one or more additional volatile organic compounds; wherein an increase or decrease in the level of said one or more additional volatile organic compounds relative to the control value is indicative of the probability that the individual has colorectal cancer.

8. A method according to claim 7, wherein the one or more additional volatile organic compounds are selected from i) 2-hexanone; ii) Butanoic acid, 1-methylethyl ester; iii) Pentanoic acid, 1-methylethyl ester; iv) p-Xylene; v) Propanoic acid, 1-methylethyl ester; or vi) Dimenthol; and wherein an increase in the levels of i) to v) relative to their respective control values and a decrease in the amount of vi) relative to its control value is indicative of an increased probability that the individual has colorectal cancer.

9. A method according to claim 1, wherein said method comprises the steps of: i) analysing the stool sample collected from said individual to detect whether the volatile organic compounds, isopropanol, 2-hexanone and butanoic acid, 3-methyl-, ethyl ester, are present; ii) if isopropanol, 2-hexanone and butanoic acid, 3-methyl-, ethyl ester are present in step i), determining the amount of isopropanol, 2-hexanone and butanoic acid, 3-methyl-, ethyl ester present and comparing the amount with a control values for each respective volatile organic compound; wherein an increased amount of isopropanol, 2-hexanone and butanoic acid, 3-methyl-, ethyl ester compared to their respective control values is indicative of an increased probability that the individual has colorectal cancer.

10. A method according to claim 9, wherein step i) further comprises analysing the stool sample collected from said individual to detect the presence of one or more additional volatile organic compounds and, if one or more additional volatile organic compounds are present, step ii) further comprises determining the amount of the one or more additional volatile organic compounds and comparing the amount with one or more control values for each of the one or more additional volatile organic compounds wherein an increase or decrease in the level of said one or more additional volatile organic compounds relative to the control value is indicative of the probability that the individual has colorectal cancer.

11. A method according to claim 10, wherein the one or more additional volatile organic compounds are selected from: i) Butanoic acid, 1-methylethyl ester; ii) Pentanoic acid, 1-methylethyl ester; iii) p-Xylene; iv) Propanoic acid, 1-methylethyl ester; or v) Dimenthol; and wherein an increase in the levels of i) to iv) relative to their respective control values and a decrease in the amount of v) relative to its control value is indicative of an increased probability that the individual has colorectal cancer.

12. A method according to claim 5, wherein the presence and amount of isopropanol and all of the volatile organic compounds listed in claim 5 are determined, and the amounts of each are compared with respective control values.

13. A method according to claim 1, wherein the presence and amount of the volatile organic compound is determined using gas chromatography mass spectrometry (GC-MS).

14. A method according to claim 1, wherein the individual has one or more symptoms associated with colorectal cancer.

15. A method according to claim 14, wherein the symptoms associated with colorectal cancer are selected from rectal bleeding, diarrhea, constipation, abdominal cramps, abdominal pain, fatigue or weight loss.

16. A method according to claim 1, wherein the individual has a predetermined risk factor associated with colorectal cancer.

17. A method according to claim 16, wherein the predetermined risk factor associated with colorectal cancer is determined based on one or more of the following: i) a positive Faecal Occult Blood Test (FOBT); ii) the presence of colorectal polyps; iii) iron deficiency anaemia; iv) a condition requiring radiological imaging of the bowel; v) abnormal bowel habits of the individual (e.g. constipation and/or diarrhoea); vi) a family history of colorectal polyps; or vii) the presence of a polyposis syndrome.

18. A method according to claim 1, wherein said method involves determining the probability that an individual has colorectal adenocarcinoma.

19-22. (canceled)

Description

DESCRIPTION OF DRAWINGS

[0132] Embodiments of the invention will be described, byway of example only, with reference to the accompanying drawings, in which:

[0133] FIG. 1 shows the PLS-DA comparing VOCs emitted from faecal samples from patients with colonic adenocarcinoma and no neoplasia (normal). All identified VOCs were subjected to PLS-DA using Metaboanalyst.

[0134] FIG. 2 shows an ROC curve showing the sensitivity and specificity of the 3-VOC model (isopropyl alcohol; 2-hexanone; butanoic acid 3-methyl, ethyl ester) for the diagnosis of colorectal cancer.

MATERIALS AND METHODS

Participants

[0135] Most participants were recruited from colonoscopy waiting lists at the Royal Liverpool University Hospital.

[0136] Participants were people referred by the Merseyside and Wirral Bowel Cancer Screening Programme with positive FOBt, or patients undergoing colonoscopy for polyp surveillance, planned polypectomy, the investigation of iron deficiency anaemia (IDA), change in bowel habit or abnormal radiological imaging. The FOBt status of the non-BCSP patients was unknown.

[0137] A subset of the faecal samples were provided from a cohort of symptomatic patients undergoing colonoscopy in Sheffield and Plymouth, UK.

[0138] Ethics committee approval for the study was obtained from the NRES Committee South West—Central Bristol (REC reference 14/SW/1162) with R&D approval from University of Liverpool and Broadgreen University Hospital Trust from where patients were recruited over a 12-month period. All patients were supplied with an information sheet and provided written consent.

[0139] Specific permission was also granted by the NHS Bowel Cancer Screening Programme Research Committee Samples collected from Sheffield and Plymouth were acquired in line with existing ethical approval.

Sample Collection and Storage

[0140] Samples were produced within 48 hours of the donor attending their colonoscopy and before commencing the required bowel preparation. Participants were asked to place at least 3 spoonfuls of faeces into a glass vial, before it was sealed and stored in a cool place. The sample was brought to the Endoscopy Department when the patients attended for the colonoscopy, and collected by the researcher. Colonoscopy results, including any histological findings, were recorded. Patients were then categorised as having no neoplasia, adenomatous polyp(s) or cancer. The location, size and number of polyps was recorded. Polyps were assigned to the adenoma group only after histological confirmation. Hyperplastic polyps were classified as no neoplasia.

[0141] Demographic details, smoking status and antibiotic use was also recorded

Laboratory Analysis

[0142] Samples were taken from the Endoscopy Department directly to the laboratory and 450 mg of unadulterated faeces aliquoted into a new 10 ml headspace vials and sealed with magnetic caps (Supelco, UK)..sup.[19] Both the sample intended for analysis, and the residual faeces, were then stored at −20° C. until GCMS analysis was performed.

[0143] Headspace VOCs analysis was performed using a Combipal (CTC, Switzerland) and carboxen/polydimethylsiloxane solid phase microextraction fibre (Sigma Aldrich, Dorset, UK). The fibre was exposed to the headspace above the faeces for 20 minutes.

[0144] VOCs were analysed by GCMS (Perkin Elmer Clarus 500 quadrupole, Beaconsfield, UK): VOCs were thermally desorbed from the fibre at 220° C. in the injection port of the GCMS for 5 minutes. Injection was made in splitless mode and a split of 50 ml/min was turned on two minutes into the run. Helium carrier gas of 99.996% purity (BOC, Guildford, UK) was passed through a helium purification system, Excelasorb™ (Supelco, Poole, UK) at 1 ml min.sup.−1. The GC column was a 60 metre long Zebron ZB-624 capillary column with an inner diameter of 0.25 mm. The column was lined with a 1.4 μm think film, specifically designed for the separation of VOCs (Phenomenex, Macclesfield, UK) consisting of 94% dimethyl polysiloxane and 6% cyanopropylphenyl. The GCMS temperature program of the run was as follows: initial oven temperature was held at 40° C. for 2 minutes then the temperature was ramped up at a rate of 5° C./min to 220° C., with a 4 minute hold at this temperature to give a total run time of 42 minutes. The mass spectrometer was run in electron impact (EI) ionization mode, scanning the mass ion range 10-300 at 0.05 scan/sec. A 4 minute solvent delay was used at the start of the run..sup.[19-21]

Data Processing

[0145] The GCMS data were processed using a pipeline involving the Automated Mass Spectral Deconvolution and Identification System software (AMDIS, Version 2.71, 2012), the NIST mass spectral library (version 2.0, 2011) and the R (R core team, 2013) package Metab..sup.[21]

[0146] AMDIS and NIST software were used to build a VOC library containing 162 metabolites present in the stool samples analysed in this study. A forward and reverse match of 800/1000 and above was used for assigning tentative compound identifications. Using this VOC library, AMDIS was then applied to deconvolute chromatograms and identifying metabolites.

[0147] The report generated by AMDIS was further processed by Metab in order to confirm the identity of metabolites and recalculate their relative abundances based on the intensity of a specific ion mass fragment per metabolite. In order to develop robust parsimonious statistical models, those compounds found to be present in fewer than 20% of the patients in both groups were removed..sup.[20,21]

Statistical Analysis

[0148] Data analysis was performed in R, Stata and Metaboanalyst,.sup.[3] utilising Student's t test, Fisher's exact test, ANOVA, false discovery rate correction, Partial Least Squared Discriminant Analysis (PLS-DA), factor analysis and Receiver Operator Characteristic (ROC) analysis.

[0149] Logistic regression modelling, along with 10-fold cross-validation was used to test potential biomarkers.

[0150] When Metaboanalyst was used the data was normalised by median and log transformed.

Results

Participant Demographic

[0151] There were a total of 137 patients included in this study: the average age was 64.3 years; 56% were male. The mean age was least in those with no neoplasia and greatest those the cancer, p=0.02.

[0152] None of the participants reported being smokers or vegetarians.

[0153] Self-reported ethnicity was noted: all but one patient reported themselves to be White British. BCSP participants were the largest group of donors (31.5%).

TABLE-US-00001 TABLE 1 Demographic and clinical features of participants recruited in Liverpool, Sheffield and Plymouth. Total No-neoplasia Adenoma Cancer Number 137 60 56 21 Mean age, years (range) 64.3 61.9 65.6 72.7 (22-85) (22-85) (41-84) (64-78) Gender Male 69 25 36 7 Female 57 34 20 3 Smoker (Yes) 0 0 0 0 Indication for colonoscopy BCSP 38 13 22 3 IDA 27 16 6 5 Change in bowel 16 11 4 1 habit-diarrhoea Surveillance previous 35 10 24 1 neoplasia/FH IBD assessment/ 9 9 0 0 surveillance GI bleeding 1 1 0 0 Unknown 11 0 0 11

Volatile Organic Compound (Biomarker) Identification

[0154] A total of 162 VOCs were identified in the whole sample set. The mean number of VOCs identified in the entire cohort was 56.7, with no significant difference in those with or without neoplasia, p=0.2.

[0155] Biomarker identification focused on higher risk neoplastic disease, namely established colorectal cancer, a single adenoma >1 cm in size and >4 individual polyps of any size.

[0156] Analysis was performed using quantitative and qualitative data.

VOCs as a Biomarker for Colonic Adenocarcinoma—Quantitative Analysis

[0157] PLS-DA comparing those with no neoplasia and those with colonic adenocarcinoma showed a separation that suggested potential diagnostic utility (FIG. 1). Potential candidates for biomarker analysis can be seen in Table 2.

TABLE-US-00002 TABLE 2 VOCs with p values <0.05, identified from Student's t test comparing no neoplasia and cancer. The q value was found after correcting for multiple comparisons. Abundance direction in VOC p value q value cancer: Isopropanol <0.0001 0.004 Increased 2-hexanone 0.01 0.77 Increased Butanoic acid, 3-methyl-, 0.03 0.77 Increased ethyl ester Butanoic acid, 1-methylethyl 0.03 0.77 Increased ester Pentanoic acid, 1-methylethyl 0.03 0.77 Increased ester p-Xylene 0.03 0.77 Increased Propanoic acid, 1-methylethyl 0.04 0.77 Increased ester dl-Menthol 0.05 0.77 Decreased

[0158] Isopropanol showed the most promise as a biomarker for colorectal cancer. In isolation, it achieves an Area under Receiver Operating Characteristic (AUROC) of 0.76.

[0159] Calculating ratios of all possible metabolite pairs and then choosing top ranked ratios, based on p values, allowed for further biomarker assessment. This technique identified the combination of butanoic acid, 3-methyl- and isopropanol as having the greatest potential: data from 21 patients with cancer and 60 with no neoplasia were modelled using logistic regression and 10-fold cross-validation, based upon the abundance of butanoic acid, 3-methyl- and isopropanol (FIG. 2): AUROC is 0.86, sensitivity 87.9% (95% CI 0.87-0.99) and specificity 84.6% (95% Cl 0.65-1.0).

VOCs as a Biomarker for Colonic Adenocarcinoma—Qualitative Analysis

[0160] Principle components analysis and a non-orthogonal rotation feature analysis was applied to qualitative (presence/absence) data for VOCs. The solution could not be extracted due to convergence issues until the number of extracted factors had been reduced to 17. This process highlighted the combination of isopropanol, 2-hexanone and 3-methyl-butanoic acid ethyl ester as a key predictor. Using all 3 VOCs as a biomarker panel predicts cancer versus all others with a p value of 0.001 and an area under the curve of 0.73, and predicts cancer versus normal with a p value of 0.006 and an area under the ROC curve of 0.70.

TABLE-US-00003 TABLE 3 Prevalence of isopropanol, 2-hexanone and 3-methyl- butanoic acid ethyl ester in stool from patients with colonic adenocarcinoma, adenomatous colonic polyps and no neoplasia. Adenoma Cancer Normal Mean number of 1.16 2.0 1.33 these three VOC's Proportion with none 23.2% (13/56)    0% (0/21) 20.0% (12/60) of these three VOCs With just 1 42.8% (24/56) 23.8% (5/21) 36/7% (22/60) With just 2 28.6% (16/56) 52.4% (11/21) 33.3% (20/60) With all three  5.4% (3/56) 23.8% (5/21) 10.0% (6/60)

[0161] Pure reference solutions of isopropanol, 2-hexanone and butanoic acid, 3-methyl-, ethyl ester confirmed the identification within the stool samples was correct.

[0162] While specific embodiments of the invention have been described herein for the purpose of reference and illustration, various modifications will be apparent to a person skilled in the art without departing from the scope of the invention as defined by the appended claims.

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