Thibodeau, S. N.

4052 Background: 18q LOH may be a useful marker in stage II colon carcinoma to assess who needs adjuvant chemotherapy. The objective of this study was to evaluate the intra- and inter-laboratory reproducibility of an assay for 18q LOH that is similar to the integral assay used in E5202, the current Intergroup phase III stage II colon carcinoma clinical trial. Methods: Specimens from 130 stage II/III colon cancer patients were obtained from the Cooperative Human Tissue Network and evaluated at three different laboratories. Samples were divided into subsamples, coded and distributed. Each laboratory used similar equipment in an established PCR protocol with 5 dinucleotide repeats. Starting materials used both histologic sections and extracted DNA samples of tumor and non-neoplastic tissue obtained only by scalpel microdissection. LOH was defined as tumor/normal peak ratio that was >1.35 or <0.67 without microsatellite instability (MSI). Tumors were categorized as either MSI+, LOH + or -, uninformative/monoallelic, or unevaluable (DNA not amplifiable). Standard tests assessed reproducibility of both the numerical ratios and the tumor 18q LOH status. Results: Sample quality issues prevented analysis of 13 tumors (10%). The frequency of 18q LOH was 64%. The probability of agreement on the same tumor evaluated twice was 92% (Cohen's kappa 0.83). Intra-class correlation coefficients for individual markers were consistently over 0.90. Tissue specimens must have high (>85%) tumor cell content for reproducibility. Differences among patients affected reproducibility much more than effects of laboratory and sample type (DNA vs. histologic section). Conclusions: This study validates reproducibility of the 18q LOH assay for both clinical categorization of status and numerical ratios if samples are high quality and appropriate for scalpel microdissection. [Table: see text]

Abstract Background: Regular use of nonsteroidal anti-inflammatory drugs (NSAID) is associated with lower risk of colorectal cancer. Genome-wide interaction analysis on single variants (G × E) has identified several SNPs that may interact with NSAIDs to confer colorectal cancer risk, but variations in gene expression levels may also modify the effect of NSAID use. Therefore, we tested interactions between NSAID use and predicted gene expression levels in relation to colorectal cancer risk. Methods: Genetically predicted gene expressions were tested for interaction with NSAID use on colorectal cancer risk among 19,258 colorectal cancer cases and 18,597 controls from 21 observational studies. A Mixed Score Test for Interactions (MiSTi) approach was used to jointly assess G × E effects which are modeled via fixed interaction effects of the weighted burden within each gene set (burden) and residual G × E effects (variance). A false discovery rate (FDR) at 0.2 was applied to correct for multiple testing. Results: Among the 4,840 genes tested, genetically predicted expression levels of four genes modified the effect of any NSAID use on colorectal cancer risk, including DPP10 (PG×E = 1.96 × 10−4), KRT16 (PG×E = 2.3 × 10−4), CD14 (PG×E = 9.38 × 10−4), and CYP27A1 (PG×E = 1.44 × 10−3). There was a significant interaction between expression level of RP11-89N17 and regular use of aspirin only on colorectal cancer risk (PG×E = 3.23 × 10−5). No interactions were observed between predicted gene expression and nonaspirin NSAID use at FDR &lt; 0.2. Conclusions: By incorporating functional information, we discovered several novel genes that interacted with NSAID use. Impact: These findings provide preliminary support that could help understand the chemopreventive mechanisms of NSAIDs on colorectal cancer.

20023 Background: EGFR is a transmembrane receptor tyrosine kinase belonging to the ErbB family of proteins. Anti-EGFR antibodies have shown efficacy as monotherapy and can enhance cytotoxic chemotherapy in patients with advanced colorectal cancers. We studied EGFR expression in primary colon cancers and its association with, microsatellite instability (MSI), clinicopathological variables and patient survival rates. Methods: Archival colon carcinomas were studied from patients with high risk Dukes’ stage B2 (n = 78) and C (n = 230) patients enrolled in a prior adjuvant trial of 5-fluorouracil, leucovorin plus standard dose or high-dose levamisole (NCCTG 91–46–53). EGFR expression (anti-EGFR H11 antibody, DAKO) was analyzed by immunohistochemistry (IHC) and intensity (0–3+), extent and their product (weighted score) were determined. MSI was analyzed using BAT26 and expression of hMLH1, hMSH2 and hMSH6 proteins by IHC. Patients were censored at 5 years after randomization for DFS and censored at 8 years post study randomization for overall survival (OS) data. Results: EGFR protein expression was detected in 216 of 308 (70%) of Dukes’ B2 and C primary colon carcinomas from patients and localized to the plasma membrane. EGFR expression variables (intensity, extent, weighted score) were increased in poor/undifferentiated compared to moderate/well differentiated tumors (p ≤ 0.036). Similar EGFR expression levels were found in MSI-H (n = 27) versus MSS (n = 203) colon cancers and for other clinicopathological variables. EGFR intensity was more likely moderate (2+) or strong (3+) in Dukes’ C versus B2 tumors (p = 0.088). In addition, higher EGFR intensity (2+, 3+) showed a trend toward worse disease-free survival (5 yr DFS %: 62.2 vs. 71.7; p = 0.095), but not overall survival (p = 0.14). Conclusions: EGFR overexpression is associated with increased tumor aggressiveness as indicated by poor tumor cell differentiation and reduced DFS in colon cancer patients receiving adjuvant 5-FU and levamisole. These findings indicate that EGFR may be an important therapeutic target in the adjuvant setting. [Table: see text]

10019 Background: Colon cancers with microsatellite instability (MSI) display resistance to 5-fluorouracil (5-FU), and in vitro resistance can be reversed by restoring DNA mismatch repair proficiency. 5-FU inhibits the thymidylate synthase (TS) enzyme and TS may predict clinical outcome after 5-FU-based chemotherapy. To define molecular predictors of prognosis, we analyzed TS, p53, chromosome 17p allelic imbalance (AI), and patient survival stratified by MSI status. Methods: Primary colon carcinomas from patients enrolled in five 5-FU-based adjuvant therapy trials were analyzed for MSI and 17p AI using 11 microsatellite markers (MSI-H: ≥ 30% of the loci demonstrating instability). For 17p AI, markers included D17S261 and TP53 at or near the p53 locus. Expression of DNA mismatch repair (hMLH1, PharMingen; hMSH2; Oncogene), TS (TS106, Zymed), and p53 (D07, Novacastra) proteins were analyzed by immunohistochemistry. Correlations between markers and associations with overall survival (OS) were determined. Patients were censored at 5 years for DFS and at 8 years post study randomization for overall survival (OS) data. Results: Of 320 Dukes’ stage B2 and C cancers studied, 60 of 320 (19%) were MSI-H. TS expression variables (intensity, extent, weighted score) were similar in MSI-H and MSI stable/low frequency (MSS/MSI-L) cancers; similar results were found using DNA mismatch repair (dMMR) proteins. MSI-H tumors had lower stage (p= 0.0007), fewer metastatic lymph nodes (p= 0.004), and improved OS (vs. MSS/MSI-L tumors; p= 0.01). Loss of dMMR proteins was also associated with better OS (p= 0.006). None of the TS variables were prognostic for OS. Histologic grade (p= 0.0008) and nodal status (p= 0.0002) were associated with OS in contrast to 17p LOH or p53. Only MSI status or dMMR, histologic grade, and tumor stage were independent markers for OS. Conclusions: MSI-H tumors show earlier stage at presentation and better stage-adjusted survival rates. MSI status and TS expression were unrelated and TS was not prognostic, suggesting that TS levels cannot explain therapeutic resistance to 5-FU reported in MSI-H colon cancers. [Table: see text]

21046 Background: The Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor tyrosine kinase that is important for colon cancer cell growth and represents a therapeutic target. We studied EGFR expression in relation to rates of apoptosis and cell proliferation. Methods: TNM stage II and III colon carcinomas (n= 361) from patients treated in a 5-FU-based adjuvant trial were studied. EGFR expression (EGFR pharmDx™) and extent were analyzed and their relationship to rates of cell turnover, histologic grade, mismatch repair (MMR) status (defective if instability at BAT 26 locus plus loss of hMLH1, hMSH2 or hMSH6 proteins) and clinical outcome were determined. Results: EGFR plasma membrane staining was detected in 214 (59.3 %) colon carcinomas. Increased EGFR intensity (2+, 3+) was associated with a higher number of caspase-3- (p= 0.02) and Ki-67- (p< 0.001)positive tumor cells. Increased EGFR intensity (p= 0.02) and extent (p= 0.03) were associated with poor differentiation, and did not depend upon MMR status (Breslow-Day test p ≥ 0.46). Diffuse (vs. focal) EGFR extent was increased in tumors with defective MMR [22 of 32 (69%)] compared to those with intact MMR [136 of 271 (50%)] (p= 0.047). EGFR intensity was unrelated to MMR status. Neither EGFR intensity nor extent was significantly associated with patient survival. Conclusions: Stage II and III colon cancers with EGFR overexpression show adverse prognostic variables, i.e., increased rates of cell turnover and poor histologic grade, suggesting that EGFR may be an important target for adjuvant therapy. *Chi-square test for categorical data, Wilcoxin rank-sum test for continuous data [Table: see text] No significant financial relationships to disclose.

Abstract Background Regular use of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) is associated with lower risk of colorectal cancer (CRC). Genome-wide interaction analysis (GxE) has identified a few variants that may modify the effects of NSAIDs on CRC risk. However, limited statistical power remains a concern. Restricting analyses by using functional genomic information to aggregate variants into biologically relevant sets can reduce the number of tests, thereby increasing statistical power. We tested the interactions between variant models of gene expression and NSAIDs use on CRC risk. Methods Functional weights of each variant were estimated using PrediXcan based on jointly measured transcriptomes and genomes data of transverse colon tissues from the Genotype-Tissue Expression (GTEx) Project for all genes with sufficient heritability (≥1%). A mixed-effects model was used to assess the GxE effects in a gene among 9,917 incident CRC cases and 10,533 controls from 17 (nested) case-control studies from the Colon Cancer Family Registry (CCFR) and Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO). GxE analysis was done by modeling the interaction between predicted gene expression and NSAID use (fixed effects), and residual variant-specific GxE effects that are not accounted for (random effects). Regular user of aspirin and/or non-aspirin NSAIDs was compared to non-regular users. False discovery rate (FDR) was used to account for multiple comparison and FDR≤0.2 was considered genome-wide significant. Secondary analysis was performed on significant genes and duration of use among users. Results Regular use of aspirin was higher among controls (30.3%) compared to cases (24.3%). Among the 4,842 genes tested, SORD significantly modified the effect of regular use of aspirin on CRC risk (p-interaction = 1.45×10-5; FDR=0.07). The SORD gene encodes sorbitol dehydrogenase (SORD), which oxidizes sorbitol to fructose in the polyol pathway. Decreased SORD concentrations were previously observed to dramatically increase in colorectal adenoma cells when compared to normal mucosa cells, suggesting the involvement of dysregulated polyol metabolisms in colorectal tumorigenesis. However, the duration of aspirin use was not statistically significantly associated with SORD gene expression on CRC risk (p=0.149) among 5,560 aspirin users. No significant interactions were observed between genetically determined colon gene expression levels and any NSAID use or non-aspirin NSAID use at FDR&lt;0.2. Conclusions Incorporating functional information, we discovered a novel gene that may interact with aspirin use to confer CRC risk. These findings provide preliminary support for new biological insights that could help understand the chemopreventive mechanisms of aspirin on CRC. We aim to replicate these findings in additional studies. Citation Format: Xiaoliang Wang, Yu-Ru Su, Andrew T. Chan, Stephanie Bien, Sonja I. Bernt, Hermann Brenner, Graham Casey, Jenny Chang-Claude, Steven J. Gallinger, Robert W. Haile, Tabitha A. Harrison, Michael Hoffmeister, Mark A. Jenkins, Amit Joshi, Yi Lin, Noralane M. Lindor, Loic Le Marchand, Hongmei Nan, Polly A. Newcomb, John D. Potter, Martha L. Slattery, Steve N. Thibodeau, Emily White, Li Hsu, Ulrike Peters. Functionally informed genome-wide interaction analysis of nonsteroidal anti-inflammatory drugs on colorectal cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2965.

363 Background: Irinotecan (CPT-11) has demonstrated antitumor activity against metastatic colorectal cancer used alone or with 5-fluorouracil (5FU)/leucovorin (LV). Two arms with CPT-11, 5FU, and LV (FOLFIRI) +/- cetuximab (Cmab) were originally included in N0147. However, after CALGB 89803 (J Clin Oncol. 25:3456, 2007), PETACC-3 (J Clin Oncol. 27:3117, 2009), and Accord02 (Ann Oncol. 20:674, 2009) showed no benefit to the three-drug combination in adjuvant therapy, the CPT-11 arms of N0147 were discontinued. We report the outcomes for patients given FOLFIRI +/- Cmab. Methods: Following a signed informed consent patients with resected stage III colon cancer were randomized to one of 6 arms including 12 biweekly cycles of CPT-11 180 mg/m2 d1 with LV 400 mg/m2, 5FU 400 mg/m2 bolus IV, then 46-hr IV 5FU 2,400 mg/m2 on d1-2 without (Arm B, FOLFIRI) or with Cmab (Arm E) 400 mg/m2 d1 cycle 1 then Cmab at 250 mg/m2 d1 and 8. Primary endpoint was 3-year disease-free survival (DFS). Secondary endpoints included overall survival (OS) and toxicity. Results: 156 patients (Arm B-111, Arm E-45) were enrolled; median follow-up on 81 patients in Arm B was 60.3 months and 58.2 months in Arm E for 41 patients. wtKRAS (vs mt) status was associated with improved DFS (HR=0.6 [95% CI 0.4-1.1], p = 0.09) and OS (HR 0.7 [95% CI 0.4-1.5], p = 0.38). The addition of Cmab improved DFS and OS in the overall group and within wtKRAS pts. Grade greater than III non-hematologic adverse effects were significantly increased in the Cmab arm (46% vs. 64%, p = 0.05). Conclusions: In this randomized phase III trial adjuvant FOLFIRI resulted in a 3-year DFS lower than that expected for FOLFOX. Trends for improved DFS and OS with the addition of Cmab were observed in patients with resected stage III colon cancer patients, regardless of KRAS status. Supported by NIH Grant CA25224, Bristol-Myers Squibb, ImClone, Sanofi-Aventis, and Pfizer. [Table: see text] [Table: see text]

CRA3507 Background: FOLFOX is standard adjuvant therapy for stage III CC. Adding Cmab to FOLFOX benefits pts with metastatic CC WT KRAS tumors. N0147 assessed the potential benefit of Cmab added to FOLFOX. Methods: 21-56 days following resection and informed consent, KRAS status was centrally determined. Pts with wtKRAS CC were randomized to 12 biweekly cycles of oxaliplatin 85 mg/m2 d1, with leucovorin 400 mg/m2, 5FU 400 mg/m2 bolus IV, then 46-hr IV 5FU 2,400 mg/m2 on d1-2 (mFOLFOX6), without (arm A) or with Cmab (arm D) 250 mg/m2 d1&8, with Cmab at 400 mg/m2, cycle 1, d1. Primary endpoint was 3-yr disease free survival (DFS). Secondary endpoints included overall survival (OS) and toxicity. Planned accrual of 2,070 wtKRAS pts provided 90% power to detect hazard ratio (HR) of 1.33 with 2-sided α=0.05; with interim analyses after 25%, 50%, and 75% of planned events. Results: 1,760 wtKRAS pts (Arm A-858, Arm D-902) were enrolled at the time of closure; median follow-up on 1,624 pts is 15.9 months. Trial closed to accrual when preplanned interim analysis after 50% of planned events demonstrated no benefit to addition of Cmab. 3-yr DFS favored FOLFOX alone (HR 1.18, 95% CI 0.92-1.52; p=0.33). No benefit of Cmab was observed in any subgroups assessed. Any grade ≥ 3 AE, diarrhea, and failure to complete 12 cycles was significantly increased in arm D. Increased toxicity and greater differences in all outcomes were observed in pts aged ≥ 70 ( Table ). Conclusions: In this randomized phase III trial the addition of Cmab to mFOLFOX6 was of no benefit for pts with resected stage III wtKRAS CC. Supported by NIH Grant CA25224, Bristol-Myers Squibb, ImClone, Sanofi-Aventis, and Pfizer. [Table: see text] [Table: see text]

Abstract Background: Family studies have served as a cornerstone of genetic research on colorectal cancer. Materials and Methods: The Colorectal Cancer Family Registry (Colon CFR) is an international consortium of six centers in North America and Australia formed as a resource to support studies on the etiology, prevention, and clinical management of colorectal cancer. Differences in design and sampling schemes ensures a resource that covers the continuum of disease risk. Two separate recruitment strategies identified colorectal cancer cases: population-based (incident case probands identified by cancer registries; all six centers) and clinic-based (families with multiple cases of colorectal cancer presenting at cancer family clinics; three centers). At this time, the Colon CFR is in year 10 with the second phase of enrollment nearly complete. In phase I recruitment (1998-2002), population-based sampling ranged from all incident cases of colorectal cancer to a subsample based on age at diagnosis and/or family cancer history. During phase II (2002-2007), population-based recruitment targeted cases diagnosed before the age of 50 years are more likely attributable to genetic factors. Standardized protocols were used to collect information regarding family cancer history and colorectal cancer risk factors, and biospecimens were obtained to assess microsatellite instability (MSI) status, expression of mismatch repair proteins, and other molecular and genetic processes. Results: Of the 8,369 case probands enrolled to date, 2,602 reported having one or more colorectal cancer–affected relatives and 799 met the Amsterdam I criteria for Lynch syndrome. A large number of affected (1,324) and unaffected (19,816) relatives were enrolled, as were population-based (4,108) and spouse (983) controls. To date, 91% of case probands provided blood (or, for a few, buccal cell) samples and 75% provided tumor tissue. For a selected sample of high-risk subjects, lymphocytes have been immortalized. Nearly 600 case probands had more than two affected colorectal cancer relatives, and 800 meeting the Amsterdam I criteria and 128, the Amsterdam II criteria. MSI testing for 10 markers was attempted on all obtained tumors. Of the 4,011 tumors collected in phase I that were successfully tested, 16% were MSI-high, 12% were MSI-low, and 72% were microsatellite stable. Tumor tissues from clinic-based cases were twice as likely as population-based cases to be MSI-high (34% versus 17%). Seventeen percent of phase I proband tumors and 24% of phase II proband tumors had some loss of mismatch repair protein, with the prevalence depending on sampling. Active follow-up to update personal and family histories, new neoplasms, and deaths in probands and relatives is nearly complete. Conclusions: The Colon CFR supports an evolving research program that is broad and interdisciplinary. The greater scientific community has access to this large and well-characterized resource for studies of colorectal cancer. (Cancer Epidemiol Biomarkers Prev 2007;16(11):2331–43)