CA2423448A1 - Method of diagnosing colorectal adenomas and cancer using proton magnetic resonance spectroscopy - Google Patents

Abstract

One dimensional proton magnetic resonance spectroscopy of human stool can be used in a non-invasive method of detecting the presence of colorectal cancer and/or clinically significant adenomas. The spectrum of a patient's stool is compared in a multivariate analysis with that of stool form non-cancerous subjects, observed differences in spectra being indicative of cancer and/or clinically significant adenomas.

Description

METHOD OF DIAGNOSING COLORECTAL ADENOMAS AND CANCER
USING PROTON MAGNETIC RESONANCE SPECTROSCOPY
This invention relates to a method of detecting colorectal adenomas and cancer, and in particular to a method of detecting such adenomas and cancer using proton magnetic resonance spectroscopy.
Colorectal cancer is one of the most common cancers in the U.S.A. and Canada accounting for approximately 146,000 new cases in 1999. The lifetime risk that an individual in North America will develop colorectal cancer is about 5 -6 %.
Symptoms associated with colorectal cancer, including blood in the stool, anemia, abdominal pain and alteration of bowel habits often become apparent only when the disease has advanced significantly. It is well known that prognosis for a patient depends largely on the stage of the disease at the time of diagnosis. In fact, whereas the five-year survival for a patient whose colorectal cancer is detected at an early stage is 92%, survival decreases to about 60% in patients with regional spread, and to about 6% in those with distant metastasises. Accordingly, it is important to detect the precursor adenomas and cancer as early as possible to increase the chances of successful therapeutic intervention.
Screening for a disease requires that the disease be prevalent in a large segment of the population and that early detection of the disease decreases mortality and improves quality of life. Colorectal cancer meets these requirements (Mandel JS, Church TR, Ederer F, Bond JH, Colorectal cancer mortality:
effectiveness of biennial screening for fecal occult blood. J Natl Cancer Inst 1999;
91:434-437 and Mandel JS, Bond JH, Church TR, Snover DC, Bradley GM, Schuman LH, Ederer F, Reducing mortality from colorectal cancer by screening for fecal occult blood. Minnesota colon cancer control study, N Eng J Med 1993;328;1365-1371 ) and, thus, is an ideal candidate for such a program. The natural history of colorectal cancer, namely the progression from adenoma to adenocarcinoma occurring over a number of years (5 - 15), also makes it a suitable target. The cost benefit analysis for the early detection of colorectal cancer has also been shown to be favourable (Bolin, TD. Cost benefit of early diagnosis of colorectal cancer. Scand J Gastroenterol 1996; 31 Suppl 220:142-146).

The screening technique itself also has to meet a, series of criteria, such as, high sensitivity and speciitcity, law cost, safety and simplicityr: Currently, ~digitai rectal examination (DRE), face! occult blood test (FDBT), barium enema and direct cotc~n visualization (sigmoidoscQpy and rolonoscopy) are used for this purpose.
DRE involves examining the recctum using a finger. This method detects cancers that can beg palpated and are within reach of the finger. A negative DRS
provides little reassurance that a patient is free of cancer, because fewer than 1Q°r6 of colorectal canters can be palpated by the examining finger. .
F~B'f detects hidden blood in the stool by chemical means on the assumption that alt colorectal cancers bleed. Although the feast expensive and the simplest, the F~BT method has low sensitivity, moderate specificity and is usually not good for early detection. According to available data, a major drawback of this technique is that more than half of the cancers discovered by this method fQliovved by x-ray or endoscopy are usuaNy beyond the limit of early staging. Afais~
positive rate of 10-12% is expecked when the patients tested are on an unrestricted diet.
Estimates of the positive predictive value range from 2.2 to 50°~b. The guaiac tests have a very low sensitivity, generally around 6fl°~ (Ra~nsohoff DF, fang CA., Screening for colorectal cancer with the fecal occult blood test; a background paper.
Ann Intem Mad 1997; 12fi:811;822). The use atf FORT is based on the assumption that coloreGtat cancers are associated with bleedin~. However, some colorectal cancers bleed intermittently and others not at all.
A barium enema involves an x-ray of the bowel using a contrast agent. The enema can be a single or double contrast. The main radialogic signs of malignancy include muscosal disruption, abrupt cut-off and shouldering and localized lesions with sharp demarcations from uninvolved areas. The estimated sensltirirty of double contrast barium enema for cancer and large polyps is only about B5 75°fo and even lower for small adent~r'nas. Despite its better diagnostic yield, double contrast barium enema has a false-negative rate of 2-180. Moreover, the method involves exposure to radiation, the repeated use of which may not be safe. Perforation from, barium enema is extremely uncommon, but when it happens it is can be fatal or

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t'x ~° ~ fr~ "' t:l~ ~
yf"4~. X27 C1~3,~U4' leads to serious long term probiems.as a result of barium spiilage.irtto the abdominal cavity.
A variety of instruments {collectively called endc~scopes) are used for examining the bowel. Endascapes can be rigid or flexible wifh varying Lengths.
Flexible. sigmoidoscopes are 60 cm long. A colonascope is a 130 - 960 cm flexible viewing instrument for examining the entire colon. Biopsies are taken from suspicious looking areas while viewing the colon through the endoscope. The flexible sigmoidoscapy examination is limited to the left side of the colon and rectum.
At least 1J3 of neoplastio tumors occur in areas proximal to the splenic flexure that 1D are inaccessible by sigmoidoscopy. Colonoscopy has a high sensitivity, and remains the gold standard for visualization of the colon and the detection of neoplastic abnormalities. However, it is invasive, quite expensive, and exposes the subject to risks of bowel perFaration.
Magnetic resonance spectroscopy (MRS} is a technique that has the potential to detect small and early biochemical changes associated with disease processes, arid has been proven to be useful in the study of tissue biopsies from cancer patients (Smith LC.P, Bezabeh T, Tissue NMR Ex Vivo.ln: Young fR, ad Methods In Biomedical magnetic resonance imaging and spectroscopy, Chicester, UK; Wiiey, 200a_89~-7). It is particularly useful to detect small, mobile chemical species in a 2D given bic~logi~cal sample that are of diagnostic interest, Obtaining tis$ue biopsies for such an examination, however, usually involves an invasive proce~tufe.
There are a number of currently available methods for detecting cancer in its stages. Biophysics! methods such as conventional X-rays, nuclear medicine, rectilinear scanners, ultrasound, CAT and MRl all play an important race in early detection and treatment of cancer. Clinical laboratory testing for tumor markers can also be used as an aid in early cancer detection. Tumor marker tests measure either tum~ar-associated antigens or other substances present in cancer patients which aid in diagna$i$, staging, disease progression, monitoring response to therapy and detection of recurrent dis~ase. Unfortunately, most tumor marker tests do riot possess sufficient specificity to be used as screening tools in a cost-effective manner. Even highly specific tests suffer from poor predictive value, because the

3 AIUiinf~iL?~fl ~~E~E~' prevalence ofi a partiauiar cancer is retatlvely low in the general population. The majority of available tumor marker tests are not useful in diagnosing cancer in symptomatic patients because elevated levels of markers. are also seen in a variety of benign diseases. The main clinical value of tumor markers Is in tumor staging, monitoring therapeutic responses, predicting patient outcomes and detecting recurrence of cancer.
United States Patents Nos. 4,812,050 and 4,91$,021, issued to E.T. Fassel on March 27, 1990 and April 17, 1990, respectively dtsclase a teehntque for detecting cancer by proton nuclear magnetic resonance (NMR) of blood, blood serum or biovd plasma United States Patent No. 5,261,405, issued to ti~e same inventor on November 16, 1993 describes an apparatus and method for aertomating the pracess_ United States Patent No. 5,318,D31, issued to Mour~tford et al on ,tune 7, 1897 describes a method far~d~xtennining chemical states of living animal or human tissue using NMR and 2D-COSY (two-dimensional cvrrefatlon) NMR spectroscopy, and comparing measured values to reference measurements of normal, abnormal and transitions state tissue.
C. L, Lean et of (Magn. Reson Mad 20:306-311, 1991; Biochemistry 3;11095-11105, 1992 and Magn Reson Mad 30:525-533, 1992) describe the use of magnetic 2t) resonance spectroscopy toy examine colon cells and tissue specimer~.
However, a need still exists far an inexpensive, non-invasive method of detecting cx~iorectal cancer and colorectal adenomas. Tyre object of the present invention i$ to provide a relatively simple, non-invasive method of detecting cofo~tal adenomas and cancer which masts flee above defined criteria of high sensitivity and specificity, low cost and safety.
. Accordingly, the invention refutes to a. method of detecting the presence of caforec~at adenomas and colorectal cancer in a patient comprising the steps of subjecting a stoat sample from the patient to magnetic resonance spectroscopy;
and comparing the resulting spectrum with the magnetic fesonance sof stool from non-cancerous subjects, with observed differences in spectra being indicative of cancer or clinically significant adenomas.

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The pertorming of spectral analysis Qn humor! stop! offers a significant advantage over other methods, because the collecfic~n afthe specimen is non-invasive and presents no risk to the patient. Moreover, no speciai~prQCessing afthe sample is required prior to analysis.
While the inventors reported earlier that the use of 2D-Gc~SY spectroscopy is preferred, further 'testing has revealed that one-dirnensianal proton magnetic resonance spectroscopy is preferred. In '1D MRS, the early onset of cotoreckal cancer is determined by the deteatian of spectral prafileslfeatures characteristics of colonic neaplasia by performing multivariate analysis on.one-dimensiana!
proton '10 magnetic resonance spectra of human stool.
METHt,?D
tine hundred and twenty-two subjects, who were scheduled for calanoscopy or surgery were recruited to donate a single sample of stool. Table 1 proviaes a breakdown of the cases.
Table 1 (Breakdown of subjects recruited}
Cases Colorectal cancer 3.4 Narmai 50 Adenomatous Polyps 3s The group referred tcs as "hlormal" includes same subjects with colonic conditionslabnormalittes that are ran-neaplastic. F~camples include divertiaulosis, hyperplastic polyps and internal hemorrhoids. Specimens from subjects with inflarnmatQry bowel disease ace not included in the analysis.
5taol samples were collected at the University of Texas M.D. Anderson Cancer Genter. The samples were kept frozen in the patients' refrigerators for an average of 24-48 hours prior to their delivery to the hospital in small ice chests (mailers). They were then stared in a -7g °C freezer until being shipped to the hfational Research Caunoil Institute for Biodiagnostics, Winnipeg, Canada. All samples were shipped blindly in dry ice and kept froaen at -70°C until the time of the S
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exp~riment. There was no significant difference in the lengths of time~for which.the samples were kept frozen.
SAMPLE PREPARATION
For MRS experiments, samples were thawed and homogenized, -and .an aliquot portion was taken and suspended in PBSIDztJ bufFer. The suspension was then put inside a capillary tube (fitted to approximately one-third of its volume) with one end closed with ~ Teflon (trademark for polytetrafluoroethylene) plug.
This was then put in a standard 5 mm MR tube contain#ng~p-amino benzoic acid (PABA) that served as a chemical shift reference.
'10 MRS E~1'ERiMEN'T~
All experiments were performed at 3fi0 MHz (Broker Instruments) at 25°C
with presaturation of the water signal. The 1 D acquisition parameters include 80°
pulse at g Ns; number of scans, NS=256; recycle delay, RD = 2.41 s; time domain data paints, TD = 4K; spectral width, SW = 51700 Hz.
DATA PI~G1CESSiNG
The' 0.5-2_5 ppm region (30Q data points) of each spectrum is used for the ana~iysis, to minimize the influence of spectwal artifacts created by suppression of the water peak at 4.6 ppm and the resonance at 3.7 ppm due to polyethylene glycol contained in the colon lavage solution used to flush the colon as part of the preparation for calonoscopy and for surgery in some patients. Each magnitude spectrum is normalized by dividing every data point by the total spectral area or by rank ordering the spectral intensities, and aligned an a r~offerenae peak.
The classification strategy used has been developed speci~cafiy to deal with the discr#mirtat#on of spectra of bic~m~licai origin. The strategy vomprises three stages. The first stage is a preprocessing step, found to be essential for reliable classification. ~tt consists of selecting firom the spectra a few maximally discriminatory subregions, using an optimal rs~gfon selection (ORu) algorithm, based on a genetic algorithm (GA)-driven apt4mization method (Bezabeh, T, et al, The use af'H Magnetic Resonance Spectroscopy in (nflamn latc~ry Bt~wel Disease:
Distinguishing Ulcerative Co#otis From Crohn's Disease, Am. J. Gastroenterol 2001;
fi ~, ~a~~~~'a~?:.' w~e~~..~
jyi~i 96: 442-443 and Somorjai, R.L. et al, Distinguishing Normal from Rejecting Renal Allographs: Application of a Three-Stage Classification Strategy to MR and IR
Spectra of Urine, in press). For reliability of classification, the number of these subregions should be an order of magnitude smaller than the number of samples to be classified. To avoid the overly optimistic classification results that a straight resubstitution approach would give, the inventors have developed a cross-validation method, using a bootstrap methodology.
The bootstrap method repeatedly partitions (with replacement) the data into approximately equal sized random training and test subsets. For each of the random training subsets an optimal classifier is found, and its accuracy validated on the random test subset. The process is repeated a number of times (250 times at the less critical ORS preprocessing stage, 1000 times for the final classifier). Once the optimal subregions are identified, the second stage finds the ultimate classifier as the weighted average of the classifier coefficients of the 1000 individual component classifiers. This approach effectively uses all n samples. A
standard multivariate statistical method, Linear Discriminant Analysis (LDA) is the choice for all classifiers, at all stages, because of its speed and robustness. The concept of crispness of a classifier is also used because the inventors' classifiers produce class probabilities. The inventors call a 2-class classification of a sample crisp if the class assignment probability for that sample is >75%.
For difficult classification problems, a third stage consists of combining the outcomes of several classifiers via aggregation methods (computerized consensus diagnosis, CCD) into an overall classifier that is more reliable and accurate than the individual classifiers. The particular classifier aggregation used by the inventors is Wolpert's Stacked Generalizer (WSG). WSG uses the output class probabilities obtained by the individual classifiers as input features to the ultimate classifier. For 2-class problems the number of features is 1 per classifier (with K
independent classifiers this gives K probabilities as input features). The overall classification quality is generally higher. The crispness of the classifier is invariably greater. This is important in a clinical environment because fewer patients will have to be re-examined.

RESULTS
There were notable differences between the COSY spectra of stool specimens from normal subjects and those with colorectal carcinoma. Of particular interest is the appearance of a crosspeak at 1.3-4.3 (attributed to the methyl-methine couplings of bound fucose) that was suggested to serve as a marker for the presence of colorectal cancer.
Based on the presence or absence of this crosspeak, specimens were identified as being positive or negative for malignancy. The sensitivity, specificity and positive predictive value (PPV) for the analysis are indicated in Table 2.
The reported sensitivity of FOBT extends from 24-78°!° (Young, G.P.
et al, Clinical Methods for Early Detection: Basis, Use and Evaluation; Chapter 13, pp 242-270 in Prevention and Early Detection of Colorectal Cancer, ed Young, G.P. et al, London, 1996). These values are lower for adenomatous polyps. The 2D COSY MR results yield a much higher accuracy than those of the FOBT technique. The 2D results are from earlier work by the inventors, and are presented for comparison purposes only.
The 2D COSY technique, which has reasonable accuracy and casts light on the nature of the diagnostic compound, has the disadvantage of long acquisition time (one to several hours). Furthermore, the absence of a peak can have sources other than the absence of the compound. The inventors, therefore turned to multivariate analysis of the corresponding one-dimensional spectra, a method which has been extremely successful for cancer biopsies (Somorjai, R. et al, J. Mag.
Reson. Imaging 6, 437 (1996)). These spectra require only minutes for acquisition, provide a wide variety of data points, and can be analyzed automatically once the diagnostic algorithm has been authenticated.
The results of the two-dimensional and the one-dimensional approach are shown in Table 2. The ID results demonstrate a higher sensitivity and specificity than given by the two-dimensional approach. The values for the 2D COSY results drop significantly when polyps are included with the cancers, as discussed above.

Table 2 Sens. Spec. PPV Sens. Spec. PPV % Crispness Cancer vs. Normal 83 85 91 Cancer + Adenomatous Polyps vs. Normal 77 85 94 98.7 96.4 97.4 99.2 a. 2D COSY results for 69 patients b. 1 D MRS results for 122 patients CONCLUSION
The results show that 1 D MRS can be used as a screening tool for colorectal carcinoma and adenoma.

Claims

WE CLAIM:

1. A method of detecting the presence of colorectal adenomas and colorectal cancer in a patient comprising the steps of subjecting a stool sample from the patient to one-dimensional proton magnetic resonance spectroscopy; and subjecting the resulting spectrum to multivariate analysis in which the spectrum is compared with the magnetic resonance spectra of stool from non-cancerous subjects, observed differences in spectra being indicative of cancer or clinically significant adenomas.

2. A method according to claim 1, wherein the stool sample is subjected to 360 MHz magnetic resonance spectroscopy.

3. A method of detecting the presence of colorectal adenomas and colorectal cancer in a patient comprising the steps of collecting a sample of the patient's stool;
subjecting the sample to one-dimensional proton magnetic resonance spectroscopy;
and subjecting the resulting spectrum to multivariate analysis in which the spectrum is compared with the magnetic resonance spectra of stool from non-cancerous subjects, observed differences in spectra being indicative of cancer or clinically significant adenomas.

4. A method according to claim 3, wherein the stool sample is subjected to 360 MHz magnetic resonance spectroscopy.

5. A method according to claim 2, including the steps of selecting from the spectra resulting from the one-dimensional magnetic resonance spectroscopy maximally discriminatory subregions; repeatedly partitioning the data into approximately equal sized random training and test subsets, finding an optimal classifier for each random training subset and validating the accuracy of the optimal classifier an the random test subset; and determining the ultimate classifier as the weighted average of the classifier coefficients of a large number of individual component classifiers.

6. A method according to claim 1, wherein a liquid suspension of the stool sample is subjected to spectroscopy.

9. A method according to claim 3, including the step of forming a liquid suspension of the stool sample before subjecting the sample to spectroscopy.