Sinicrope, Frank A.

11088 Background: Obesity is associated with an increased risk of colon cancer. However, the influence of body mass index (BMI) upon the prognosis of patients with established colon cancer remains unknown. Methods: We conducted a retrospective study of 1,803 patients with surgically resected stage III colon cancer who were enrolled in five randomized trials of 5-fluorouracil-based adjuvant chemotherapy conducted by the North Central Cancer Treatment Group. Patient height and weight were recorded at study entry and BMI (kg/m2) was calculated and categorized. Cancer recurrence or death were monitored during 5 years of follow-up. The score and likelihood ratio p-values were determined from univariate and multivariate Cox regression models respectively, after stratifying by study. Results: Among stage III colon cancer patients, 19% were obese (BMI 30 kg/m2), 37% were overweight (BMI, 25 to 29.9 kg/m2), 38% were of normal-weight (BMI, 20 to 24.9 kg/m2), and 6% were underweight (BMI < 20 kg/m2). Obese versus normal-weight patients showed higher rates of lymph node (LN) metastasis (>3 LNs; 38% vs. 29%, p <0.01) and tumor site was more likely to be distal versus proximal (52% vs. 45%, p= 0.03). No differences in age, gender, or histologic grade were found. In a univariate analysis, obese patients had significantly worse disease-free survival (DFS) compared with normal-weight patients (hazard ratio 1.25 (95% CI: 1.04 -1.51; p= 0.02). The 5 year DFS rates were 49% in obese patients versus 57% in normal weight subjects. Furthermore, poorer DFS was observed for obese patients after adjusting for age, sex, histologic grade, and tumor site (p= 0.03). Neither overweight nor underweight patients (vs. normal-weight) had significantly different DFS. Analysis of the predictive impact of BMI for 5-FU-based adjuvant therapy is in progress. Conclusions: Obesity (BMI 30 kg/m2) was associated with a greater number of metastatic lymph nodes and poorer disease-free survival in patients with stage III colon cancer, suggesting that obesity influences tumor progression. No significant financial relationships to disclose.

4065 Background: Tumor infiltrating lymphocytes (TILs) and tumor budding (linked to epithelial mesenchymal transition) may influence metastatic potential and patient prognosis. We analyzed these features and their relative contribution to survival among low (T1-3 N1) and high (T4 and/or N2) risk groups, defined by the IDEA study, used to inform the duration of adjuvant chemotherapy in stage III colon cancer. Methods: Among 1,532 patients (low risk n=804; high risk n=728) treated in a phase III adjuvant trial of FOLFOX + cetuximab (x 6 months), intraepithelial TIL densities and tumor budding were quantified at microscopy in routine histologic sections. Optimal cutpoints were determined in association with 5-yr disease-free survival (DFS). Relative contribution of variables to DFS was calculated using χ2 from Harrell’s rms R package based on multivariable Cox regression models. Results: In the overall cohort, the tumor budding/TILs combined variable was more robust for predition of DFS than either alone. Budding/TILs was significantly associated with DFS in both low (HRadj, 1.59; 95% CI, 1.02-2.48; p=.0273) and high (HRadj, 2.82; 95% CI, 1.72-4.63; p<.0001) risk patients. We then determined its relative contribution (%) to DFS (Table). Among low risk, budding/TILs ranked second (24.4%) behind KRAS status (45.5%) and ahead of treatment arm (7.2%) and mismatch repair (MMR) status (6.1%). Among high risk, budding/TILs contributed the most to DFS (45.4%) followed by primary tumor sidedness (13.0%), performance status (12.0%), and MMR (10.4%). Conclusions: Tumor budding/TILs provides robust prognostic stratification by risk group to improve anatomic tumor staging. The relative contribution of budding/TILs to DFS was second only to KRAS status in low risk patients, and was the most important predictor of DFS in high risk patients. Evaluation in patients treated with 3 vs 6 mos of adjuvant chemotherapy is warranted. [Table: see text]

Abstract Background The American Joint Committee on Cancer staging and other prognostic tools fail to account for stage-independent variability in outcome. We developed a prognostic classifier adding Immunoscore to clinicopathological and molecular features in patients with stage III colon cancer. Methods Patient (n = 559) data from the FOLFOX arm of adjuvant trial NCCTG N0147 were used to construct Cox models for predicting disease-free survival (DFS). Variables included age, sex, T stage, positive lymph nodes (+LNs), N stage, performance status, histologic grade, sidedness, KRAS/BRAF, mismatch repair, and Immunoscore (CD3+, CD8+ T-cell densities). After determining optimal functional form (continuous or categorical) and within Cox models, backward selection was performed to analyze all variables as candidate predictors. All statistical tests were two-sided. Results Poorer DFS was found for tumors that were T4 vs T3 (hazard ratio [HR] = 1.76, 95% confidence interval [CI] = 1.19 to 2.60; P = .004), right- vs left-sided (HR = 1.52, 95% CI = 1.14 to 2.04; P = .005), BRAF V600E (HR = 1.74, 95% CI = 1.26 to 2.40; P &lt; .001), mutant KRAS (HR = 1.66, 95% CI = 1.08 to 2.55; P = .02), and low vs high Immunoscore (HR = 1.69, 95% CI = 1.22 to 2.33; P = .001) (all P &lt; .02). Increasing numbers of +LNs and lower continuous Immunoscore were associated with poorer DFS that achieved significance (both Ps&lt; .0001). After number of +LNs, T stage, and BRAF/KRAS, Immunoscore was the most informative predictor of DFS shown multivariately. Among T1–3 N1 tumors, Immunoscore was the only variable associated with DFS that achieved statistical significance. A nomogram was generated to determine the likelihood of being recurrence-free at 3 years. Conclusions The Immunoscore can enhance the accuracy of survival prediction among patients with stage III colon cancer.

4071 Background: Selection of patients for HER2-targeted therapy is based on HER2 analysis in primary EACs. Since EACs metastasize via regional lymph nodes, concordance of HER2 gene amplification results between primary tumors and their metastatic lymph nodes (MLN) is a clinically important issue. Methods: Resected EACs (N = 103) having at least three distinct regional MLNs (total 508 MLNs; median 4 [range 3-11]) were sectioned using routine procedures and tested for HER2 amplification by fluorescence in situ hybridization (FISH). Primary tumors were also evaluated for HER2 protein expression by immunohistochemistry (IHC) and by FISH. Amplification was defined as HER2/CEP17 ≥ 2. IHC was scored as 0, 1+, 2+, or 3+. Primaries whose HER2 status was positive by both FISH and IHC (ie, amplified and IHC3+), or negative by both (ie, nonamplified and IHC0-1+), were selected. HER2 status was compared between primaries and their MLNs (kappa). Results: Concordant HER2 results between primaries and their MLNs were found in 92% (95/103) of EACs (Table; κ = .76 [95% CI .60 - .92]). However, among patients with HER2-positive primaries, 19% (4/21) had HER2-nonamplified MLNs; among these cases, either all MLNs were HER2-nonamplified (n = 2), or both HER2-nonamplified and –amplified MLNs were present (n = 2). Among HER2-negative primary EACs, 5% (4/82) of cases had MLNs that were all HER2-amplified (n = 3) or both HER2-nonamplified and -amplified (n = 1). Conclusions: A patient subset whose primary EACs were HER2-positive (by both FISH and IHC) were unexpectedly found to lack HER2 amplification in corresponding MLNs. Conversely, a subgroup with HER2-negative primaries had HER2-amplified MLNs, and would have been deemed ineligible for HER2-targeted therapy. These preliminary data suggest that evaluating MLNs in resected EACs has the potential to better identify patients who benefit from HER2-targeted therapy. [Table: see text]

3617 Background: In metastatic colorectal cancer (CRC), previous studies suggested potential relationships between certain drug metabolizing (DM) genes (DPYD, ERCC2, GSTP1 and TYMS) and QOL prior to receipt of chemotherapy (Sloan, Plenary Session ASCO 2004). The current study is the first to examine relationships between similar DM genes and QOL in stage III CC pts. Methods: 1,583 CC pts on a randomized adjuvant stage III, phase III trial received FOLFOX, FOLFIRI, or either +/- Cetuximab. Genomic DNA and baseline QOL data via linear analogue self assessment (LASA) scales with 4 QOL variables were obtained. 53 DM SNPs were selected for known functional effect within potential regulatory sites. Two-sample t-tests assessed differences in QOL between pts with variants and wild-type status. Differences of at least 10 points on a 0-100 point QOL score were considered clinically significant. Results: 14 relationships between DM genes and QOL were detected at the nominal p < 0.05 level; six of these were related to DPYD, ERCC2, GSTP1 and TYMS. Relationships were also observed at the 0.05 level with polymorphisms of genes DPYS, ERCC1, MGMT, MTHFR, OPRT and UGT1A1 based on unadjusted and adjusted association tests. GSTP1 I105V and OPRT 5’UTR 28 A>G markers differed on fatigue scores (p=0.04, 0.005, respectively). Variants on MTHFR R594Q were associated with higher mental acuity scores (p=0.02). These relationships remained after adjusting for age, gender, race, T stage, PS, disease site, lymph nodes involved, and grade. The robustness of the observed associations varied with the multiple comparison adjustment method. Conclusions: Similar to the earlier study of stage IV CRC, we observed a possible relationship in pts with Stage III CC with regard to DM genes and QOL, although these relationships were more modest than observed in Stage IV pts. These DM genes may be in linkage disequilibrium with other genetic variants that more directly affect behavior and QOL. Further genome wide association studies are needed to clarify these relationships.

3514 Background: Activating mutations in the BRAF oncogene (mutBRAF) are associated with deficient MMR (dMMR) in sporadic colon cancers and are mutually exclusive with KRAS mutations (mutKRAS). We evaluated BRAF and KRAS in relation to MMR and disease-free survival (DFS) in stage III pts treated with adjuvant FOLFOX +/-cetuximab; study was amended mid-way to require prospective KRAS testing (Alberts SR, ASCO 2010). Methods: Extracted DNA from archival tumors was analyzed for BRAF exon 15 (V600E) and KRAS exon 2 mutations by allele specific RT-PCR. Tumors with loss of any MMR protein (MLH1, MSH2, MSH6) were categorized as dMMR vs proficient (pMMR). DFS was censored at 4 yrs; median follow-up was 3.1 yrs. Results: Among 2,686 pts, 12% (314/2580) of tumors were dMMR; 28% (716/2579) had mutKRAS, and 14% (346/2515) had mutBRAF of which 43% (150/346) were dMMR. mutBRAF was associated with high grade, proximal site, T3-4, >4 LNs, and dMMR (all p<0.002); mutKRAS with low grade, proximal, <3 LNs, and pMMR (all p<0.02). Among dMMR tumors, mutKRAS was more frequent in distal vs proximal cancers (28% vs. 8%, p=0.0004). Both mutKRAS (HR=1.42, p=0.0001) and mutBRAF (HR=1.34, p=0.02) were associated with worse DFS and remained significant after adjustment for treatment, MMR, grade, site, T stage and LNs. mutBRAF (p=0.003) and mutKRAS (p=0.003) were each prognostic for DFS in pMMR, but not dMMR tumors (KRAS, p=0.79; BRAF, p=0.60). While MMR was not significant univariately, proximal dMMR tumors had favorable DFS [HR 0.8, 95% CI (0.6-1)] and distal dMMR tumors had poor DFS [HR 2 (1.1-3.4)] after adjusting for KRAS, BRAF, grade, T stage and LNs (pinteraction=0.02). pMMR/mutBRAF (p=0.027) and pMMR/mutKRAS (p<0.001) had worse DFS vs pMMR/bothWT. Conclusions: mutBRAF and mutKRAS were significantly associated with worse DFS independent of treatment, covariates, and each other, with the DFS impact restricted to pMMR tumors. Prognostic impact of MMR was dependent upon tumor site after adjusting for covariates. Support: NIH Grant CA 25224, NCI K05CA 142885, Bristol-Myers Squibb, ImClone, Sanofi-Aventis, and Pfizer.

3576 Background: BRAF encodes a serine-threonine kinase that is a downstream effector of activated RAS. A point mutation (V600E) in BRAF occurs in a subset of colorectal cancers (CRCs) and is associated with adverse outcome and may predict non response to anti-EGFR antibodies. Detection of a BRAFV600E mutation in a CRC with microsatellite instability indicates a sporadic origin and excludes Lynch Syndrome. While BRAFV600E mutation status is determined using a DNA-based assay, antibodies against the BRAFV600E protein have recently been developed. We examined mutant BRAFV600E protein expression and its concordance with mutation status. Methods: Primary stage III colon carcinomas (50 BRAFV600E mutation carriers and 25 wild-type cases) were studied from a completed phase III adjuvant trial comparing FOLFOX +/- cetuximab (NCCTG N0147). In archival resection specimens, immunohistochemistry (IHC) was performed using a pan-BRAF antibody and a V600E mutation-specific antibody raised against an immunogenic synthetic peptide derived from the internal region of the BRAFV600E protein. BRAFV600E mutations in codon 15 were analyzed in extracted DNA using a multiplex, allele specific PCR–based assay. BRAF staining was scored independently by two pathologists blinded to mutation status. Results: In primary colon carcinomas stained with a pan-BRAF antibody, diffuse cytoplasmic staining for BRAF proteins was detected in 74 of 75 carcinomas with one case deemed non-evaluable. Using the mutation-specific BRAFV600E antibody, diffuse cytoplasmic staining was detected in 49 of 74 tumors without appreciable heterogeneity of expression. Among these 49 tumors expressing mutant BRAFV600E proteins, all (100%) were found to carry a BRAFV600E mutation according to a DNA-based assay. In contrast, absent BRAFV600E staining was observed in all 25 tumors that were found to have wild-type copies of BRAFV600E detected using a PCR-based assay. Conclusions: For the detection of mutant BRAFV600E, complete concordance was found between IHC and a DNA-based method in colon carcinomas. This finding supports the use of IHC as a simplified strategy to screen CRCs for mutant BRAFV600E proteins in routine clinical practice to inform clinical decision-making.

3523 Background: The association of KRAS or BRAFV600E mutations with prognosis in colon cancers has been inconsistent. Since primary tumor site may influence outcome, we analyzed KRAS and BRAFV600E stratified by tumor site in resected stage III colon cancers from a randomized adjuvant trial of mFOLFOX6 + cetuximab chemotherapy (N= 2,686) where no survival difference by treatment was found. Methods: 2,580 tumors were analyzed for mutations in BRAFV600E (exon 15) or KRAS (codons 12, 13), and for deficient DNA mismatch repair (dMMR). Cox models were used, adjusting for mutation status, age, sex, treatment, T-stage, histologic grade, nodal status, tumor site, and MMR. After study initiation, eligibility was restricted to patients (pts) with KRAS wild-type (WT) tumors. At a median follow-up 4.1 yrs, 83% of pts are alive. Results: KRAS and BRAFV600E mutations were detected in 716 (28%) and 346 (14%) tumors, respectively; dMMR was found in 314 (12%). Proximal (to splenic flexure) tumors (50%) were associated with older age and were more likely to be high grade (33 vs 18%), T-stage3,4 (88 vs 82%), mutated for KRAS (33 vs 23%) or BRAFV600E (23 vs 4%), and dMMR (21 vs 3%)[all p <0.02]. Pts with distal vs proximal tumors showed improved disease-free survival (DFS)[ HR 0.7, 95% CI (0.6-0.9); p<0.01 unadjusted for KRAS or BRAF;]. Mutant KRAS [HR=1.5 (1.2-1.7); p<.0001] or mutant BRAF [HR=1.4 (1.1-1.7); p=0.02] were each independently associated with worse DFS. The KRAS and tumor site interaction was significant (unadjusted pinteraction=0.02; adjusted pinteraction=0.07), with poorer DFS among distal tumors having mutant KRAS [HR 1.7 (1.3-2.2); p<.0001]. No interaction was observed for BRAFV600E and tumor site, despite worse DFS for mutant BRAFV600E among proximal tumors [HR 1.3 (1-1.8); p=0.04]. Conclusions: The adverse prognostic impact of KRAS, but not BRAFV600E, mutations was confined to the distal colon. This finding underscores the importance of tumor site in the interpretation of the prognostic impact of KRAS status, and motivates similar analyses in pts with advanced disease.

3510 Background: Prediction of 5-FU-related adverse events (5FU-AEs) continues to be problematic. Pharmacogenetic studies on the rate-limiting enzyme in 5-FU metabolism, dihydropyrimidine dehydrogenase (DPD), suggest a link between three variants and both decreased enzyme activity and increased toxicity: c.1905+1 G>A (DPYD*2A; rs3918290), c.1679 T>G (I560S; DPYD*13; rs55886062), and c.2846A>T (D949V; rs67376798). Since the adverse impact of DPYD variants on 5-FU toxicity remains controversial, we determined associations between the three known DPYD variants and 5FU-AEs in stage III CC patients receiving FOLFOX or FOLFIRI (+ cetuximab) after curative resection. Methods: 2886 patients were genotyped by multiplexed single-base extension assays using the IPLEX Gold Kit and analyzed on the Sequenom MassARRAY system. Grade 3+ AEs were recorded per CTCAE v3. Fisher’s exact test, unequal variance two-sample t-test, and Wilcoxon rank sum test were used to compare categorical variables, continuous variables, and counts between patients with wild-type and mutant status. Logistic regressions were used to assess univariate and multivariate associations. Results: Patients displayed the following characteristics: male gender 53.2%, median age 58 [19-86], proficient DNA mismatch repair status 88.6%, PS-0 76.6%, + irinotecan 8.1%, and + cetuximab 45.9%. A total of 27 (0.9%), 4 (0.1%), and 32 (1.1%) patients carried the DPYD*2A, I560S, and D949V variants, respectively. Analysis identified significant associations between DPYD*2A and D949V variants and toxicity, with grade 3+ 5FU-AEs identified in 22 DPYD*2A carriers (OR=11.9, 95% CI 4.0-32.7, p<0.0001) and 22 D949V carriers (OR=5.5, 95% CI 2.5-12.1, p< 0.0001). No interaction effect was found between DPYD*2Aand D949V on grade 3+ 5FU-AEs (p = 0.98), nor on overall grade 3+ AEs (p = 0.97). No significant association was identified between I560S and grade 3+ 5FU-AEs. Conclusions: In the largest study to date, statistically significant associations were found between DPYD*2A and D949V variants and increased incidence of grade 3+ 5FU-AEs, suggesting utility in 5-FU toxicity prediction.