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The prognostic value of immune cell infiltration within the tumor microenvironment (TME) has been extensively investigated via histological and genomic approaches. Based on the positive prognostic value of T cell infiltration, Immunoscore has been developed and validated for predicting risk of recurrence for colorectal cancer (CRC). Also, association between a consensus T helper 1 (Th-1) immune response and favorable clinical outcomes has been observed across multiple cancer types. Here, we reanalyzed public genomic data sets from The Cancer Genome Atlas (TCGA) and NCBI Gene Expression Omnibus (NCBI-GEO) and performed multispectral immunohistochemistry (IHC) on a cohort of colorectal tumors. We identified and characterized a risk group, representing approximately 10% of CRC patients, with high intratumoral CD8(+) T cell infiltration, but poor prognosis. These tumors included both microsatellite instable (MSI) and stable (MSS) phenotypes and had a high density of tumor-associated macrophages (TAMs) that expressed CD274 (programmed death-ligand 1 [PD-L1]), TGF-β activation, and an immune overdrive signature characterized by the overexpression of immune response and checkpoint genes. Our findings illustrate that CRC patients may have poor prognosis despite high CD8(+) T cell infiltration and provide CD274 as a simple biomarker for identifying these patients.

// Jun Gong 1 , May Cho 1 , Marvin Sy 1 , Ravi Salgia 1 and Marwan Fakih 1 1 Department of Medical Oncology, City of Hope National Medical Center, Duarte, CA, USA Correspondence to: Marwan Fakih, email: mfakih@coh.org Keywords: metastatic colorectal cancer, comprehensive genomic profiling, next-generation sequencing, FoundationOne, retrospective Received: September 06, 2016 Accepted: January 16, 2017 Published: February 02, 2017 ABSTRACT Background: Recent molecular characterization of colorectal tumors has identified several molecular alterations of interest that are considered targetable in metastatic colorectal cancer (mCRC). Methods: We conducted a single-institution, retrospective study based on comprehensive genomic profiling of tumors from 138 patients with mCRC using next-generation sequencing (NGS) via FoundationOne. Results: Overall, RAS mutations were present in 51.4% and RAF mutations were seen in 7.2% of mCRC patients. We found a novel KRAS R68S1 mutation associated with an aggressive phenotype. RAS amplifications (1.4% KRAS and 0.7% NRAS ), MET amplifications (2.2%), BRAF L597R alterations (0.7%), ARAF S214F alterations (0.7%), and concurrent RAS + RAF (1.4%), BRAF + RAF1 (0.7%), and rare PTEN-PIK3CA-AKT pathway mutations were identified and predominantly associated with poor prognosis. ERBB2 ( HER2 ) amplified tumors were identified in 5.1% and all arose from the rectosigmoid colon. Three cases (2.2%) were associated with a hypermutated profile that was corroborated with findings of high tumor mutational burden (TMB): 2 cases with MSI-H and 1 case with a POLE mutation. Conclusions: Comprehensive genomic profiling can uncover alterations beyond the well-characterized RAS / RAF mutations associated with anti-EGFR resistance. ERBB2 amplified tumors commonly originate from the rectosigmoid colon, are predominantly RAS / BRAF wild-type, and may predict benefit to HER2-directed therapy. Hypermutant tumors or tumors with high TMB correlate with MSI-H status or POLE mutations and may predict a benefit from anti-PD-1 therapy.

627 Background: Microsatellite instability (MSI) can predict response to pembrolizumab in metastatic colorectal cancer (mCRC). Benefit to PD-1 blockade is limited in microsatellite stable (MSS) mCRC yet MSS tumors represent >95% of all mCRC cases. Evidence suggests that high mutational load remains an important predictor of benefit to checkpoint inhibition across several tumors. We aimed to explore associations between tumor mutational burden (TMB) and various patient factors and mutations of interest to identify features of patients (pts) with MSS mCRC of potential significance to prognosis and PD-1 targeting. Methods: We conducted a retrospective study based on next-generation sequencing (NGS) of MSS metastatic colorectal tumors at our single institution using FoundationOne. TMB was categorized as high ≥ 20 mutations/megabase (Mb), intermediate 6-19 mutations/Mb, and low ≤ 5 mutations/Mb. TMB frequencies were analyzed according to age (≤50, >50, >65, and >70 years old), gender, race, location of primary tumor, and KRAS, BRAF, and PIK3CA mutations. Results: Among 194 MSS tumors with available TMB data, frequencies of tumors with intermediate TMB were significantly different in pts ≤50 years (24.6%, n=69) than in pts >65 (48.9%, n=47, Fisher’s exact (two-tailed) p=0.0096) and >70 (53.1%, n=32, p=0.0067). Frequencies of tumors with low TMB were significantly different in pts ≤50 years (75.4%, n=69) than in pts >65 (46.8%, n=47, p=0.0029) and >70 (43.8%, n=32, p=0.0032). Frequencies of high TMB tumors by age were 0% (≤50 years), 1.6% (>50), 4.3% (>65), and 3.1% (>70) and did not reach significance. Frequencies of high, intermediate, or low TMB tumors were not significantly different based on gender, race, and location of primary tumor. Frequencies of KRAS, BRAF, and PIK3CA mutations were similar across all TMB categories (p=0.7995, 0.8774, and 0.053, respectively). Conclusions: Older individuals with mCRC are less likely to have a low mutation burden by FoundationOne assay. Further validation of these findings in larger cohorts may indicate a potential higher likelihood for elderly colorectal cancer patients to benefit from immune-based strategies.