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Background: Colorectal cancers with deficient DNA mismatch repair (dMMR) are presumed to uniformly have dense lymphocytic infiltration that underlies their favorable prognosis and is critical to their responsiveness to immunotherapy, as compared to MMR-proficient (pMMR) tumors. We examined T-cell densities and their potential heterogeneity in a large cohort of dMMR tumors. Experimental Design: CD3+ and CD8+ T-cell densities were quantified at the invasive margin (IM) and tumor core (CT) in 561 stage III colon cancers (dMMR, n=278; pMMR, n=283) from a phase 3 adjuvant trial (N0147). Their association with overall survival (OS) was determined using multivariable Cox analysis. Results: While CD3+ and CD8+ T-cell densities in the tumor microenvironment were higher in dMMR vs pMMR tumors overall, inter-tumoral heterogeneity in densities between tumors was significantly higher by 30–88% among dMMR vs pMMR cancers (P<.0001 for all four T-cell subtypes [CD3+IM, CD3+CT, CD8+IM, CD8+CT]). A substantial proportion of dMMR tumors (26% to 35% depending on the T-cell subtype) exhibited T-cell densities as low as that in the bottom half of pMMR tumors. All four T-cell subtypes were prognostic in dMMR with CD3+IM being the most strongly prognostic. Low (vs high) CD3+IM was independently associated with poorer OS among dMMR (HR 4.76 [95% CI 1.43–15.87]; P=.0019) and pMMR tumors (P=.0103). Conclusions: Tumor-infiltrating T-cell densities exhibited greater inter-tumoral heterogeneity among dMMR than pMMR colon cancers, with CD3+IM providing robust stratification of both dMMR and pMMR tumors for prognosis. Potentially, lower T-cell densities among dMMR tumors may contribute to immunotherapy resistance.

3579 Background: Tumor infiltrating lymphocytes (TIL) indicate a host immune response that may influence survival. Immunoscore was developed using CD3+ and CD8+density and location in primary CC pts with pooled stages, varying treatment and follow-up. We determined if individual immune markers and/or Immunoscore are prognostic in resected stage III CC pts (N=600). Methods: CD3+ and CD8+ T-cell or CD20+ B lymphocyte density in central tumor (CT) and invasive margin (IM) was evaluated by immunostaining and quantified by image analysis. Immunoscore was calculated on a scale of I0 to I4 with high densities of CD3+ and CD8+in both CT and IM scored as I4; low densities scored as I0. Associations with disease-free survival (DFS) were evaluated by multivariable Cox regression adjusting for covariates. Results: Data for CD3+, CD8+ and CD20+ were generated (N=595). Higher density of CD3+ CT, CD3+ IM and CD8+ IM were associated with longer DFS adjusting for covariates (Table). CD3+ IM had the strongest association with DFS, and was stronger in left-sided (HRadj.=0.81, 95% CI, 0.70-0.94, padj.= 0.0049) vs right-sided (HRadj.=0.93, 95% CI, 0.85-1.0 padj.=0.52) tumors (pinteraction=0.039). Higher density of CD3+ IM was associated with older age (p=0.034), T1/2 (p<.0001), N1 (p=0.017), right-sided (p=0.013), high TILs (p=0.0008), and deficient MMR (p=0.0003). Using a prior Immunoscore risk stratification, higher scores were associated with better DFS (HR.=0.62, CI, 0.44-0.87, p=0.006) (Table). Conclusions: Densities of CD3+ and CD8+, especially at IM, are individually prognostic in FOLFOX-treated pts. Association of CD3+IM with prognosis differed by primary CC site. Immunoscore was strongly prognostic, and this result provides validation in a clinical trial cohort. [Table: see text]

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614 Background: A consensus interpretation of the IDEA colon cancer study results suggested that risk categories based on T/N stage grouping be used to guide decision-making for duration (3 vs 6 months) of adjuvant FOLFOX or CapeOX chemotherapy. Given the prognostic potential of immune biomarkers, we examined the immunoscore and individual T and B lymphocyte markers in low and high risk T/N subsets of stage III colon carcinoma patients (N=600) treated with adjuvant FOLFOX. Methods: Immunoscore (CD3+, CD8+) and individual T-cell and CD20+ B-cell immunostain densities in central tumor (CT) and invasive margin (IM) of FFPE sections were quantified by image analysis. A predetermined immunoscore categorization was used [high (2-4) vs low (0-1)]. Individual markers were analyzed by backwards selection wherein CD3+ IM was most robust for prognosis and an optimized cutoff was then determined. Associations with disease-free survival (DFS) were analyzed by multivariable Cox regression adjusting for age, T/N stage, sidedness, KRAS/BRAF, and DNA mismatch repair. Results: In low and high risk T/N patient subsets, the immunoscore and CD3+ IM were each significantly discriminant for prognosis. Among low risk (T1-3N1) patients, a high vs low immunoscore was associated with a 91% vs 77% 3-year DFS [HR 0.57, 95% confidence interval (CI) 0.34-0.95, adjusted (adj) P= 0.026]. Among high risk (T4 or N2) patients, a high vs low immunoscore was associated with a 68% vs 54% 3-year DFS (HR 0.64, 95% CI 0.42-0.98, Padj= 0.034]. Similarly, a high vs low intratumoral CD3+ density at the invasive margin (IM) was significantly associated with prognosis in low risk [HR 0.37, 95% CI 0.21- 0.66), Padj< 0.0003] and in high risk [HR 0.47, 95% CI, 0.27- 0.80), Padj< 0.0028] patient subsets. Conclusions: Immunoscore and CD3+ IM were shown to prognostically stratify FOLFOX-treated patients within both low and high risk T/N subsets. These data underscore limitations of the T/N risk classification for adjuvant treatment decisions in stage III patients, and demonstrate the ability of T-cell markers to enhance prognostication to guide clinical decision-making.

3516 Background: Epidemiological data suggest a protective effect of vitamin D on CC risk. However, association of serum vitamin D level with clinical outcome in pts with surgically resected CC remains unknown. We analyzed total circulating 25-hydroxyvitamin D [25(OH)D)] with clinical outcome in pts with resected stage III CC treated with FOLFOX +/- cetuximab in a phase III trial. Methods: Association of pre-treatment level of plasma 25(OH)D with disease-free survival (DFS) and time-to-recurrence (TTR) was analyzed with 25(OH)D as a continuous variable by a relative risk model with splines and as a binary variable (vitamin D deficient [<30 ng/mL]) using multivariable Cox regression. Results: Overall, 49% (291/600) of pts were vitamin D deficient. Prevalence of vitamin D deficiency was significantly higher in women vs men (52% vs. 48%, p=0.030) and in blacks vs whites (74% vs. 45%, p=0.0003). The continuous 25(OH)D level and pt sex were associated by the interaction test (pinteraction=0.044). There were no statistically significant associations with DFS/TTR for either continuous 25(OH)D level (DFS, p=0.22; TTR, p=0.26) nor for vitamin D deficiency (DFS, adjusted hazard ratio [HRadj]=1.01, 95% CI, 0.74-1.38; padj=0.94; TTR, HRadj=1.00; CI, 0.72-1.38; padj=0.99. A comparison of the highest vs lowest tertile showed a significant association between the highest 25(OH)D level and better outcomes in men (DFS, HRadj.=0.18, CI, 0.04-0.76, padj=0.020; TTR, HRadj.=0.12, CI, 0.02-0.64, padj=0.014), but not in women (DFS, pinteraction=0.039; TTR, pinteraction=0.033). Conclusions: Nearly one-half of pts in the clinical trial cohort were vitamin D deficient with lowest 25(OH)D levels found in women and blacks. While vitamin D deficiency was not associated with adverse outcome, high levels of vitamin D in men, but not women, were associated with longer survival. Support: U10CA180821, U10CA180882, U10CA180820, U10CA180863, U10CA180888, U10CA077202, CCSRI 021039. ClinicalTrials.gov Identifier: NCT00079274.

<p>Table S2: Association between immune T-cell subtypes in MMR-deficient and -proficient stage III colon cancers.</p>

<p>Figure S3 Backward selection to identify T-cell subtype(s) independently associated with overall survival in mismatch-repair deficient stage III colon cancers</p>