Michael J. Lawson

Difluoromethylornithine plus sulindac for the prevention of sporadic colorectal adenomas: a randomized placebo-controlled, double-blind trial.

Abstract Preclinical studies of chemoprevention drugs given in combination at low doses show remarkable efficacy in preventing adenomas with little additional toxicities, suggesting a strategy to improve risk to benefit ratios for preventing recurrent adenomas. Three hundred seventy-five patients with history of resected (≥3 mm) adenomas were randomly assigned to receive oral difluoromethylornithine (DFMO) 500 mg and sulindac 150 mg once daily or matched placebos for 36 months, stratified by use of low-dose aspirin (81 mg) at baseline and clinical site. Follow-up colonoscopy was done 3 years after randomization or off-study. Colorectal adenoma recurrence was compared among the groups with log-binomial regression. Comparing the outcome in patients receiving placebos to those receiving active intervention, (a) the recurrence of one or more adenomas was 41.1% and 12.3% (risk ratio, 0.30; 95% confidence interval, 0.18-0.49; P < 0.001); (b) 8.5% had one or more advanced adenomas, compared with 0.7% of patients (risk ratio, 0.085; 95% confidence interval, 0.011-0.65; P < 0.001); and (c) 17 (13.2%) patients had multiple adenomas (>1) at the final colonoscopy, compared with 1 (0.7%; risk ratio, 0.055; 0.0074-0.41; P < 0.001). Serious adverse events (grade ≥3) occurred in 8.2% of patients in the placebo group, compared with 11% in the active intervention group (P = 0.35). There was no significant difference in the proportion of patients reporting hearing changes from baseline. Recurrent adenomatous polyps can be markedly reduced by a combination of low oral doses of DFMO and sulindac and with few side effects.

Enhanced Retrieval of DNA from Human Fecal Samples Results in Improved Performance of Colorectal Cancer Screening Test

Colorectal cancer accounts for more than 10% of all cancer deaths but is curable, if detected early. We reported previously on a stool-based screening test in which DNA from stool samples is subjected to genome analysis; sensitivity of the test has been limited in part by inefficiency of retrieving DNA from stool. Our aim was to test the impact of a new purification method that would increase the yield of human DNA from stool. DNA from 86 cancer and 100 non-cancer subjects (diagnosed by colonoscopy) were purified from stool with a new method for DNA recovery based on sequence-specific capture with acrylamide gel immobilized capture probes as well as with a previously developed magnetic bead-capture procedure. The new purification method gives an average 5.4-fold increase in the quantity of human DNA that can routinely be retrieved from fecal samples. The increased recovery of DNA corresponds with an increase in assay sensitivity from 53% (CI: 42 to 64%) to 70% (CI: 59 to 79%); P = 0.0005 (by McNemars test), with no change in specificity. The newly developed sample preparation method mitigates a major problem in detecting rare cancer-associated genetic changes in heterogeneous clinical samples such as stool.

Randomized phase II trial of sulindac, atorvastatin, and prebiotic dietary fiber for colorectal cancer chemoprevention.

Sulindac, atorvastatin, or prebiotic dietary fiber may reduce colorectal cancer (CRC) risk. However, clinical trial data are currently limited. We conducted a randomized, phase II chemoprevention trial involving subjects 40 years or older, with previously resected colon cancer or multiple/advanced colorectal adenomas. Magnification chromoendoscopy (MCE) was performed to identify and characterize rectal aberrant crypt foci (ACF); eligibility criteria required five or more rectal ACFs at baseline. Intervention assignments were as follows: (a) atorvastatin 20 mg qd; (b) sulindac 150 mg bid; (c) oligofructose-enriched inulin (as ORAFTI®Synergy1) 6 gm bid; or (d) control (maltodextrin) 6 gm bid, for 6 months. Percent change in rectal ACF number (%ΔACF) within arm was the primary endpoint. Secondary endpoints included changes in proliferation (Ki67) and apoptosis (caspase-3), as measured from normal mucosa biopsy samples. Among 85 eligible randomized subjects, 76 (86%) completed the trial per protocol. The median (range) of rectal ACF was 9 (5–34) and 8 (0–37) at baseline and postintervention, respectively. The median (SD) for %ΔACF was 5.6 (−69% to 143%), −18.6 (−83% to 160%), −3.6 (−88% to 83%), and −10.0 (−100% to 117%) in the atorvastatin, sulindac, ORAFTI®Synergy1 and control arms, respectively. Neither within-arm (P = 0.12–0.59) nor between-arm (P = 0.30–0.92) comparisons of %ΔACF were statistically significant. The active and control interventions also seemed to have similar effects on mucosal proliferation and apoptosis (P > 0.05 for each comparison). Data from this multicenter, phase II trial do not provide convincing evidence of CRC risk reduction from 6-month interventions with atorvastatin, sulindac, or ORAFTI®Synergy1, although statistical power was limited by the relatively small sample size. Cancer Prev Res; 4(2); 259–69. ©2011 AACR.

The evolution to stool DNA testing for colorectal cancer

Despite a variety of screening strategies and recent trends showing death rate stabilization, colorectal cancer still remains the second leading cause of overall cancer death. Current screening tools suffer from performance limitations, low patient acceptability, and marginal reliable access within the health care system. Noninvasive strategies present the lowest risk with the highest potential for patient satisfaction. However, serious implementation barriers exist requiring consistent programmatic screening, strict patient adherence, and poor sensitivity for adenomas. Colonoscopy remains an invasive screening test with the best sensitivity and specificity, but faces large financial costs, manpower requirements, patient access and adherence. Development of advanced molecular techniques identifying altered DNA markers in exfoliated colonocytes signify early or precancerous growth. Stool‐based DNA testing provides an entirely noninvasive population‐based screening strategy which patients can perform easier than faecal occult blood testing (FOBT). Large‐scale prospective randomized control trials currently pending should help characterize accurate test performance, screening intervals, cost‐effectiveness, direct comparison to FOBT and analysis of patient adherence. As tumour development pathways and potential target genes are further elucidated, refinements in multi‐assay stool‐based DNA testing portend enhanced test characteristics to detect and treat this genetically heterogeneous disease.

Correlation of Ki-67, p53, and Adnab-9 immunohistochemical staining and ploidy with clinical and histopathologic features of severely dysplastic colorectal adenomas.

Variations of Ki-67, p53, and Adnab-9 monoclonal antibody reactions in colonic adenomas may be associated with colonic cancer risk. We studied the predictive value of these markers for adverse behavior in severely dysplastic colorectal adenomas, such as an associated carcinoma, multiplicity of adenomas, and subsequent development of adenomas. For this purpose we compared the clinical, gross, and histologic characteristics of highly dysplastic index polyps in 42 patients with Ki 67, p53, and Adnab-9 immunostaining and other molecular markers. Polyps were removed endoscopically, and severely dysplastic polyps were stained immunohistochemically with Ki-67, Adnab-9, and p53 protein by the avidin biotin conjugate (ABC) technique. Quantitative DNA (QDNA) was analyzed by computer-assisted image analysis. Ki-67 immunohistochemistry showed reversal of normal distribution of nuclear staining from the normal basal position to the upper third of the colonic crypts. This abnormality of immunostaining in dysplastic adenomas was the earliest detected by the panel we used. A statistically significant correlation was seen between invasiveness of carcinoma in the index polyp and polyp size (P = 0.003), sessile morphology (P = 0.037), and villous or tubulovillous histology (P = 0.019). In the index adenoma, p53 positivity was correlated with multiplicity at initial examination (P = 0.053), villous histology (P = 0.053), invasiveness of carcinoma (P < 0.003), and recurrence of colorectal adenomas (P = 0.025). Although p53 positivity and aneuploidy were correlated with invasiveness of carcinoma in the index polyp (P = 0.025), Adnab-9 positivity was not. However, Adnab-9 positivity in the index polyp was associated with multiplicity of adenomas (P = 0.04) as well as recurrence of adenomas (P < 0.024). In conclusion, in addition to the morphologic and histologic markers already known, Ki-67, Adnab-9 antibody, and p53 protein may be prognostic indicators useful in follow-up of patients with severely dysplastic colorectal adenomas. Adnab-9 antibody may identify a field defect in above-average-risk adenoma-bearing patients.

Colorectal cancer and noncancer patients have similar labeling indices by microscopy and computed image analysis

The labeling index (LI), a microscopic measurement of proliferative activity in colonic crypts, is proposed as an indicator of colonic cancer risk. Computed image analysis of proliferative regions is less labor intensive and more objective than is direct microscopy but has not been validated for labeling indices by direct comparison. The authors compared colonic crypt proliferation in 26 cancer and 13 noncancer patients by using Ki-67 monoclonal antibody (McAb) labeling of flat mucosa obtained from surgically removed, frozen specimens. In cancer patients, the mucosa specimen was excised 10 cm away from the tumor, and the LI was determined microscopically for the whole crypt, the upper two thirds, and the upper one third of 15 crypts. Nuclear antigen levels of 15 whole crypts were determined by using the CAS-200 computed image analyzer (Cell Analysis Systems, Elmhurst, IL). Cancer and noncancer specimens were compared as were microscopically determined LI and stained nuclei specimens by using image analysis. No statistically significant difference in proliferative activity of whole crypts, or the upper two thirds of crypts, was observed between cancer specimens and noncancer specimens from using either technique. However, a significant correlation existed between microscopic analysis and computed image analysis of labeled nuclei. Computed image analysis using Ki-67 McAb labeling can be used instead of microscopy to determine crypt LI, but neither method can be used to distinguish cancer specimens from noncancer specimens.

Colorectal Cancer Risk: The Impact of Evidence of a Field Effect of Carcinogenesis on Blinded Diagnosis Using an Anti-Adenoma Antibody Test Performed on Colonoscopic Effluent

To better define high-risk populations for colorectal cancer screening, we used anti-adenoma antibody Adnab-9, comparing colonic effluent and tissue field effects from 45 high-risk and 11 control patients. We included Adnab-9 binding at the tissue level to elucidate the impact of a field effect of carcinogenesis contributing to the outcome of the effluent binding test. In high-risk patients, 64% of the left-sided effluent samples were positive (P < 0.002); 67% showed a field effect (P < 0.006); 82% of combined tests were positive (P < 0.001), as compared to 9%, 18%, and 27% respectively, of controls. Adnab-9 binding correlates with increased colorectal cancer risk associated with a field effect of carcinogenesis.

Small Early Tubular Adenomas and Mixed Colonic Polyps Found on Screening Flexible Sigmoidoscopy Do Not Predict Proximal Neoplasia in Males

BACKGROUND & AIMS Distal colonic adenomas found on flexible sigmoidoscopy are associated with proximal neoplasias, thus requiring a complete colonoscopic examination, but data regarding the association of distal mixed polyps with proximal neoplasia are lacking. We conducted this study to elucidate the significance of distal mixed colonic polyps in predicting proximal neoplasia. METHODS We retrospectively analyzed data from patients who underwent a flexible sigmoidoscopic examination for colorectal cancer screening and a follow-up colonoscopic examination because of distal colonic polyps. Distal index polyps were classified by a pathologist as early tubular adenoma (ETA), serrated, or true mixed categories. Index polyps also were immunostained with a monoclonal antibody, Adnab-9, a marker for the colorectal adenoma carcinoma sequence. RESULTS In 636 patients with distal index polyps, 6% were malignant, 55% were adenomas, 13% were ETAs, 6% were serrated, 4% were true mixed, and 17% were hyperplastic. Compared with distal hyperplastic index polyps, distal malignant polyps (P = 0.0006) and adenomas (P = 0.001) were associated with a significantly increased number of synchronous proximal neoplasia, but the small distal mixed, serrated, or ETA did not predict the increased incidence of proximal neoplasia. Large distal polyps of each category were significantly associated with an increased number of synchronous proximal neoplasias. In support of these findings, immunostaining of distal polyps with Adnab-9 showed predictability for proximal neoplasia only in the adenoma category (P < 0.05). CONCLUSIONS Small ETAs, serrated, or mixed polyps found on flexible sigmoidoscopic examination do not increase the probability of synchronous proximal neoplasia.

Measuring cell proliferation in the rectal mucosa: comparing bromodeoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA) assays

Cell proliferation in the human colorectum can be measured using bromodeoxyuridine (BrdU) or proliferating cell nuclear antigen (PCNA) assays. Using data from the National Cancer Institutes Polyp Prevention Trial, these two assays are compared using correlation coefficients and variance components analysis. Adjusting for fixed as well as for the random effects of between-biopsy and scoring variation, the estimated correlation is 0.46 for the log labeling index and 0.45 for log proliferative height. This is an estimate of the highest correlation that can be achieved by taking multiple biopsies scored by multiple scorers. For single biopsies, the estimated correlation is 0.16 and 0.10, respectively. There are significant differences between the variance components for the two assays. For example, for log labeling index, PCNA has a lower variation between biopsies than BrdU, but higher variation between scorings. When used in a clinical or epidemiological setting, it is important to take multiple biopsies at multiple time points.

Colonic retinoblastoma protein and proliferation in cancer and non-cancer patients#

Both suppressor oncogene and proliferative activity are believed to indicate colon cancer risk. The retinoblastoma (Rb) gene is a suppressor oncogene affecting cell differentiation. Retinoblastoma gene inactivation is associated with tumour development. However, the relation of the Rb protein to cell proliferation and colon tumour formation is unknown. Retinoblastoma protein quantity was correlated with proliferative activity in flat, unaffected mucosa specimens from 36 cancer patients, 21 non‐cancer control subjects and in 29 tumour tissue samples from cancer patients. Nuclear Rb protein was measured by using automated CAS‐200 image analysis of monoclonal antibody labelled frozen sections from fresh, surgically removed tissue. All colon cells within 15 whole crypts were imaged. Proliferative activity was also measured by using image analysis with Ki‐67 monoclonal antibody. Retinoblastoma protein content correlated directly with proliferative activity in flat mucosa of non‐cancer control subjects (r= 0.63; P < 0.001; n= 21). A significant correlation was also found in flat mucosa specimens of non‐metastatic (Dukes stages A and B) cancer patients (r=0.52; P < 0.01; n= 22). However, Rb protein did not correlate with proliferation in flat mucosa from metastatic (Dukes stages C and D) cancer patients (r=0.03; NS; n=14) or in cancer tissue (r=0.068; NS; n=29). Mucosal Rb protein in the colon normally increases as proliferation increases. Dissociation between Rb protein and colon proliferation may occur in flat mucosa in patients with a higher risk of metastatic tumour growth. Future studies comparing Rb protein quantity and proliferative activity may help identify high‐risk colon cancer patients.