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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 < .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 < .02). Increasing numbers of +LNs and lower continuous Immunoscore were associated with poorer DFS that achieved significance (both Ps< .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.

4026 Background: Patients (pts) with locally advanced esophageal or gastroesophageal junction (GEJ) adenocarcinoma commonly receive neoadjuvant chemoradiotherapy (chemo-RT). Despite this approach the rate of recurrence remains high. Given the difficulties of postoperative therapy, the efficacy of extended neoadjuvant therapy was assessed. Methods: Eligibility criteria included T3-4,N0 – Tany,N(+) disease amenable to radiation and surgery. Pts were randomized to either arm A (docetaxel 60 mg/m2 day 1 , oxaliplatin [Oxal] 85 mg/m2 day 1, and capecitabine 1250 mg/m2/day days 1-14 x 2 cycles [DOC] followed by 5-FU 180 mg/m2/day continuous IV through radiation + Oxal 85 mg/m2 days 1,15,29 + 50.4 Gy radiation (chemo-RT)) or arm B (chemo-RT alone). Randomization was stratified by ECOG PS (0/1 vs 2) and stage (II vs III/IVA). Primary endpoint was pathologic complete response (PCR) rate, defined as no gross or microscopic tumor identified in the surgical specimen. Interim analysis assessed efficacy and futility of the experimental intervention. Wilcoxon rank sum and Fisher’s exact tests were used to compare clinical/pathologic factors between arms. Results: Baseline and stratification factors were well balanced between arms. Of 42 pts included in the interim analysis (86% male; age [median 63, range 38-88], 100% PS 0/1; 71% stage III; 55% esophagus, 40% GEJ; 36% measurable disease), 4 and 1 pts in arms A and B, respectively, did not have surgery due to death (A, 2), progressive disease (A, 1), alternative treatment (A, 1) or adverse event (B, 1). Among 21 arm A pts, 21, 20, and 19 pts started 1st cycle of DOC, 2nd cycle of DOC and chemo-RT, respectively. All arm B pts received chemo-RT. 33% (7/21) of arm A and 48% (10/21) of arm B pts achieved PCR (p=0.53). Among pts undergoing surgery, 94% (16/17) and 100% (20/20) of arm A and B pts had complete resection (p=0.46). 38% and 24% of arm A and B pts experienced at least one grade 4+ adverse event at least possibly related to treatment (p=0.51). Conclusions: Extended neoadjuvant therapy in pts with locally advanced esophageal or GEJ adenocarcinoma failed to improve the PCR rate. Follow-up in regard to survival and rate of recurrence is ongoing. Clinical trial information: NCT00938470.

3618 Background: Current prognostic tools and staging systems in CC use relatively few patient (pt) characteristics; including additional covariates may improve prediction. Using the large ACCENT database, we constructed clinically based NGs for OS and TTR in stage III CC that better separate pts into risk groups compared to AJCC v7 staging. Methods: 15,936 stage III pts accrued to phase III clinical trials since 1989 were used to construct Cox models for TTR and OS. Variables included age, sex, race, BMI, performance status, tumor grade, tumor stage, ratio of positive lymph nodes to nodes examined, number/location of primary tumors (any multiple versus single left, right, or transverse), and treatment (5FU variations vs. 5FU with oxaliplatin or irinotecan). Missing data (<18%) were imputed, continuous variables modeled with splines, and clinically relevant pairwise interactions included if p < 0.001. Final models were internally validated via bootstrapping for corrected calibration and C-indices for survival data. NG-defined risk tertiles were compared to AJCC v7 stage III for observed 3-year (yr) TTR and 5-yr OS for a subset of 7400 pts with complete data. Results: All variables were statistically and clinically significant for OS; age and race did not predict TTR. No meaningful interactions existed. NGs for OS and TTR were well calibrated and associated with C-indices of 0.66 and 0.65, respectively, vs. 0.58 and 0.59 for AJCC. NG risk tertiles were better separated than AJCC groups, (3-yr TTR, 5-yr OS below), with fewer mid-risk NG pts. Removing treatment from NGs did not affect performance (C=0.66 for OS and 0.65 for TTR). Conclusions: The proposed ACCENT NGs are internally valid and have the potential to aid prognostication, decision-making, and patient/physician communication in pts with stage III CC. [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.

3526 Background: Prior observational studies have suggested that smoking is associated with poorer overall survival after colon cancer (CC) diagnosis. Using data from N0147, a phase III randomized adjuvant trial of patients with stage III CC, we assessed the relationship between smoking status and cancer outcomes (disease-free survival (DFS) and time to recurrence (TTR)), accounting for possible heterogeneity by patient and tumor characteristics. Methods: Patients completed a risk factor questionnaire at baseline prior to randomization to FOLFOX or FOLFOX+cetuximab (N=1968). Information was collected on smoking and other lifestyle factors, including alcohol consumption, body mass index (BMI), and physical activity. Multivariate Cox models assessed the association between smoking history and the primary trial outcome of DFS, as well TTR, adjusting for other lifestyle and clinical factors. The median follow-up was 3.5 years. Results: Overall, 52% of patients were former or current smokers. Compared to never smokers, ever smokers experienced significantly shorter DFS [3-year DFS: 70% vs 74%, hazard ratio (HR)=1.21, 95% confidence interval (CI): 1.02-1.42]. This association persisted after adjusting for age, sex, tumor subsite, number of nodes involved, T-stage, mismatch repair deficiency, BRAF mutation status, performance score, physical activity, BMI, and alcohol consumption (HR=1.23, 95% CI: 1.02-1.49). There was significant interaction in this association by BRAF mutation status (p=0.02): smoking was associated with shorter DFS in BRAF wildtype CC patients (HR=1.25, 95% CI: 1.04-1.51) but not in BRAF mutant CC patients (HR=0.72, 95% CI: 0.47-1.10). Patterns of association with smoking, overall and by BRAF status, were identical in analyses of TTR. Conclusions: Overall, smoking was significantly associated with shorter DFS and TTR in CC patients. These adverse relationships were most evident in patients with BRAF wildtype CC.

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.

e13052 Background: The combination of GEM and CDDP has shown activity in a variety of cancers, including biliary tract/gallbladder. Preclinical testing shows a potential benefit to the addition of EV. Methods: Using a standard 3+3 design, the MTDs of GEM + EV (Cohort I) and GEM + CDDP + EV (Cohort II) were determined as shown in the table. The MTD was defined as the dose level below the lowest dose that induces a DLT in at least one-third of pts. At the MTD of Cohort II, 10 patients were enrolled with biliary tract/gallbladder cancer (Cohort III). A weekly CBC was obtained. Assessments occurred every 3 weeks and imaging for response at every other cycle. Results: In Cohort I (N=12), no DLT occurred at dose level 0, and in dose level 1, grade 3 thrombocytopenia was found in 2 of 6 pts. The MTD for Cohort I was determined to be dose level 0. Responses were seen in 3 pts: 2 CRs (primary peritoneal, pancreatic) and 1 PR (breast). In Cohort II (N=15) DLTs at dose level 0 were neutropenia and thrombocytopenia in 2 of 3 pts and at dose level -1, thrombocytopenia in 2 of 6 pts. At dose level -2, 1 of 6 pts experienced a DLT (grade 3 thrombocytopenia), establishing this dose level as the MTD. Responses were seen in 2 pts, both PRs (ampullary, pheochromocytoma). All 10 pts have been enrolled in Cohort III with 2 DLTs (neutropenia and thrombocytopenia). Stable disease seen in 5 of 7 evaluable pts. Conclusions: Gem + EV was well tolerated at dose level 0, though dose escalation was limited by thrombocytopenia. GEM + CDDP + EV had DLTs of neutropenia and thrombocytopenia leading to the MTD of dose level of -2. Cohort III is fully accrued and the 2 DLTs are hematologic. [Table: see text]

3640 Background: Prior economic analysis of a 12-gene assay (Oncotype DX), compared with patterns of care reported in the NCCN database of patients with stage II, T3, DNA mismatch repair proficient (MMR-P) colon cancer, predicted that the assay would save medical costs and improve patient well-being (Hornberger et al. Value Health 2012). This study assessed the validity of those findings with actual adjuvant chemotherapy (aCT) recommendations. Methods: Outcomes and costs were estimated for patients with stage II, T3, MMR-P colon cancer using a Markov model. A study of 141 patients from 17 sites in the Mayo Clinic Cancer Research Consortium collected data on aCT recommended before and after knowledge of the 12-gene assay results (Srivastava et al. abstract). Quality-adjusted life years (QALY) and medical resource use after recurrence were computed using guideline-validated state-transition probability estimation methods. Risk of progression and incidence of adverse events with different aCT regimens were based on published literature. Drug and administration costs for aCT were obtained from 2012 Medicare fee schedules. One-way sensitivity analyses were conducted to evaluate parameter influence on economic impact. Results: After receiving the 12-gene assay results, physician recommendations in favor of aCT decreased 22% (95% CI 11%-32%; McNemar test p<0.001) from 73 (52%) to 42 (30%) patients. Oxaliplatin aCT and 5-FU monotherapy recommendations each declined 11%. Average aCT costs decreased $3,228 for drugs, $750 for administration, and $3,168 for adverse events management. Overall, average total direct medical costs decreased $1,683. The net effect on average patient well-being was a gain of 0.102 QALYs. Total change in medical costs is most influenced by the cost of death due to colon cancer, time-preference discount rate, and the change in aCT recommendations. Savings are expected to persist even if the cost of oxaliplatin dropped by >75% due to generic substitution. Conclusions: The 12-gene assayhas been shown to alter aCT recommendations for patients with stage II, T3, MMR-P colon cancer. This study provides real-world confirmation that these aCT changes reduce direct medical costs and improve patient well-being.

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.