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484 Background: The histopathological growth patterns (HGPs) of liver metastasis reflect the complicated and varied interactions between tumor cells and the host microenvironment. We studied HGPs of gastric cancer liver metastasis (GCLM) and sought to predict them using radiomic and clinical variables. Methods: Hepatectomy was performed in 62 patients with GCLM at our university hospital between 2011 and 2021. HGPs were evaluated according to international consensus guidelines, and were classified as either desmoplastic HGP (dHGP) or non-dHGP. CD4, CD8, VEGF and CD31 were analyzed by immunohistochemistry. Eight hundred and fifty-one radiomics features were extracted from CT portal venous phase images and the optimal radiomics signature was determined by univariate analysis and LASSO regression. Multivariable regression analyses and ROC curves were used to assess the predictive performance of HGPs with a radiomic and clinical combined model. Results: Among 62 patients, 14 were categorized as dHGP, and 48 as non-dHGP. Non-dHGP vs. d-HGP was associated with higher tumor N stage and CEA level, and with decreased CD8 + T cells, and lower VEGF and CD31 expression. HGP status was independently associated with patient cancer-specific survival (CSS) (P＜0.05) multivariately.Analysis of percent relative contribution revealed that HGP ranked first for CSS (35.9%), ahead of T (28.9%) and N (28.9%) stage. A model with combined radiomic and clinical features demonstrated robust performance with area under the curve (AUC) values of 0.993 and 0.933 in the training and validation sets, respectively. Conclusions: Non-invasive determination of radiomic and clinical features was shown to predict HGPs types. Compared to dHGP, GCLM with non-dHGP exhibited significantly lower immune cell infiltration and decreased angiogenesis. Among features examined in GCLM, HGP was the most robust for prognostication. Univariate and multivariate analysis of prognostic factors for cancer-specific survival. Characteristics Univariate analysis Multivariate rergression analysis HR (95%CI) P HR (95%CI) P Gender ( Women vs Men ) 1.13(0.61-2.09) 0.71 Age ( >60years vs ≤60years) 1.08(0.60-1.70) 0.98 HGP ( non-desmoplastic HGP vs desmoplastic HGP ) 3.63(1.78-7.41) 0.00 3.48(1.37-8.88) 0.01 T stage ( T3-T4 vs T1-T2 ) 2.03(1.08-3.80) 0.03 2.29(1.15-4.58) 0.02 N stage ( N1-N3 vs N0 ) 3.46(1.66-7.23) 0.00 2.54(1.17-5.56) 0.02 CEA ( >5ng/ml vs ≤5ng/ml ) 2.37(1.25-4.48) 0.01 1.56(0.70-3.48) 0.27

483 Background: Heterogeneity exists in recurrence and survival among patients with intestinal-type gastric cancer (IGC). Nucleotyping is the alterations in chromatin organization. We sought to identify the prognostic utility of nucleotyping, tumor stroma ratio (TSR) and DNA ploidy in these patients. Methods: Radical resection of gastric carcinoma (> 15 LNs removed) was performed in 283 patients (stage I, n=73; stage II, n=102; stage III, n=108) at our university hospital between 2010 and 2018. Automated image cytometry tools were used to quantitatively measure nucleotyping, TSR and DNA ploidy. Cutpoints of nucleotyping and TSR were 0.044 and 50%, respectively. Associations of these features with clinicopathological characteristics, time-to-recurrence (TTR) and cancer specific survival (CSS) were analyzed by univariate and multivariate analysis. Results: Among 283 patients, chromatin heterogeneity, high TSR and non-diploid were significantly associated with larger tumor size, higher T and N stage, and poorer TTR and CSS at 5 years. In a Cox multivariable analysis, nucleotyping and TSR were independently associated with TTR and CSS (P＜0.05) (Table). The top three contributors to TTR were N stage (33.9%), nucleotying (29.7%) and TSR (25.5%). The top three contributors to CSS were N stage (35.6%), nucleotying (29.7%) and TSR (18.0%). As a combined variable, the percent relative contribution of nucleotyping and TSR ranked first for TTR and CSS (56.0%, 50.0%), ahead of N stage (33.4%, 34.6%) and TILs (8.4%, 10.6%). Conclusions: Nucleotyping and TSR were each significantly and independently associated with TTR and CSS, and their combined variable was more strongly prognostic than either alone. Cox multivariable analysis of TTR and CSS. Features TTR HR (95%CI) TTR P value CSS HR (95%CI) CSS P value Tumor size (>3cm vs ≤3cm) 1.10 (0.48-2.54) 0.83 0.77 (0.35-1.70) 0.56 T stage (T3-T4 vs T1-T2) 1.07 (0.46-2.49) 0.87 1.41 (0.53-3.76) 0.49 N stage (N2N3 vs N0N1) 2.84 (1.39-5.78) <0.01 7.69 (3.37-17.55) <0.01 Adjuvant Therapy (with vs without) 1.32 (0.72-2.40) 0.37 0.85 (0.48-1.52) 0.59 TB (high vs low) 1.29 (0.62-2.69) 0.50 1.61 (0.85-3.07) 0.15 TILs（low vs high） 1.73 (0.96-3.13) 0.07 1.90 (1.06-3.40) 0.03 Nucleotyping (Chromatin heterogeneous vs Chromatin homogeneous) 2.19 (1.21-3.99) 0.01 3.44 (1.96-6.03) <0.01 TSR (high vs low) 2.12 (1.16-3.82) 0.01 2.51 (1.43-4.41) <0.01 DNA ploidy (Non-diploid vs Diploid) 1.20 (0.55-2.63) 0.65 0.98 (0.44-2.15) 0.95 1 TTR, time-to-recurrence; 2 CSS, cancer specific survival.

Abstract Background Tumor microenvironment (TME) characteristics including tumor stroma ratio (TSR), tumor budding (TB), and tumor-infiltrating lymphocytes (TILs) were examined in resected gastric cancer. These TME features have been shown to indicate metastatic potential in colon cancer, and intestinal-type gastric cancer (IGC) has pathological similarities with that malignancy. Methods TSR, TB, and TILs were quantified in routine histological sections from 493 patients with IGC who underwent radical resection at 2 university hospitals in China from 2010 to 2016. TME variables were dichotomized as follows: TSR (50%), TILs (median), TB per international guidelines (4 buds/0.785mm2), and platelet-lymphocyte ratio (PLR) per survival ROC. Association of TME features with patient clinicopathological characteristics, time-to-recurrence (TTR), and cancer-specific-survival (CSS) were examined using univariate and multivariate analysis, including a relative contribution analysis by Cox regression. Results Patients whose tumors showed high TSR or high TB or low TILs were each significantly associated with increased T and N stage, higher histological grade, and poorer TTR and CSS at 5 years. Only TSR and N stage were independently associated with TTR and CSS after adjustment for covariates. PLR was only independently associated with TTR after adjustment for covariates. Among the variables examined, only TSR was significantly associated with both TTR (HR 1.72, 95% CI, 1.14-2.60, P = .01) and CSS (HR 1.62, 95% CI, 1.05-2.51, P = .03) multivariately. Relative contribution to TTR revealed that the top 3 contributors were N stage (45.1%), TSR (22.5%), and PLR (12.9%), while the top 3 contributors to CSS were N stage (59.9%), TSR (14.7%), and PLR (10.9%). Conclusions Among the examined TME features, TSR was the most robust for prognostication and was significantly associated with both TTR and CSS. Furthermore, the relative contribution of TSR to patient TTR and CSS was second only to nodal status.

4055 Background: Tumor microenvironmental (TME) features including tumor stroma ratio (TSR), tumor infiltrating lymphocytes (TILs) and tumor budding (TB), may be associated with tumor metastatic potential. We sought to identify the association of these features with prognosis of patients with resectable intestinal-type gastric cancer. Methods: Radical resection of gastric carcinoma (> 15 LNs removed) was performed in 493 patients (stage I, n=225; stage II, n=119; stage III, n=149) at two university hospitals between 2010 and 2016. Cutpoints were as follows: TSR (50%), TILs (median), and TB (Bd0-Bd3). TME features and their association with clinicopathological characteristics, time-to-recurrence (TTR) and overall survival (OS) were analyzed by univariate and multivariate Cox methods.. Results: Among 493 patients, high TSR, low TILs and high TB (Bd1-Bd3) were significantly associated with higher histological grade, larger tumor size, and increased T and N stages. Each of these features was significantly associated with poorer TTR and OS at 5 years. In Cox multivariable analysis, N stage and TSR were the only variables that were significantly associated with TTR and OS (Table). The relative contribution (%) of TSR to TTR ranked second (17.5%) behind N stage (49.2%) and ahead of TILs (7.2%) and TB (0.1%). Conclusions: Among TME features, TSR was the most robust and was significantly and independently associated with TTR and OS. The relative contribution of TSR to TTR was second only to nodal status. [Table: see text]

Abstract Tumor mutational burden (TMB), defined as the number of somatic mutations per megabase of interrogated genomic sequence, varies across malignancies. Panel sequencing–based estimates of TMB have largely replaced whole-exome sequencing–derived TMB in the clinic. Retrospective evidence suggests that TMB can predict the efficacy of immune checkpoint inhibitors, and data from KEYNOTE-158 led to the recent FDA approval of pembrolizumab for the TMB-high tumor subgroup. Unmet needs include prospective validation of TMB cutoffs in relationship to tumor type and patient outcomes. Furthermore, standardization and harmonization of TMB measurement across test platforms are important to the successful implementation of TMB in clinical practice. Significance: Evaluation of TMB as a predictive biomarker creates the need to harmonize panel-based TMB estimation and standardize its reporting. TMB can improve the predictive accuracy for immunotherapy outcomes, and has the potential to expand the candidate pool of patients for treatment with immune checkpoint inhibitors.

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]

3564 Background: Lethal-7 (let-7) microRNA has been shown to inhibit colon cancer cell proliferation by inducing KRAS downregulation after binding to specific sites in the 3’- UTR. Recent studies identified a KRAS 3’-UTR polymorphism in the let-7 complimentary site (LCS6) that has been shown to weaken let-7 binding and alter KRAS expression. Carriers of the LCS6 G-allele showed worse survival in metastatic colorectal cancer from prior studies of limited sample size. In the current study, we evaluated the LCS6 variant in 2,834 Stage III colon cancer patients and its association with disease-free survival (DFS), KRAS mutation status, and other clinicopathological features. Methods: In the NCCTG phase III trial (N0147), the LCS6 variant was assayed in germline DNA extracted from whole blood using RT-PCR. Extracted tumor DNA was analyzed for KRAS exon 2 mutations. Chi-squared and two-sample t test were used to compare baseline factors and KRAS mutation status between patients defined by LCS6 variant status. Log-rank tests and multivariate Cox models assessed the unadjusted and adjusted associations between LCS6 status and DFS, respectively. Results: We identified 432 (15.2%) patients heterozygous or homozygous for the LCS6 G-allele and 2402 (84.8%) patients homozygous for the LCS6 T-allele. G-allele carriers were significantly more frequent in Caucasians than other races (Chi-Squared Test, p<0.0001). No associations were found between the LCS6 genotype and T stage, positive lymph node number, tumor differentiation, or primary tumor location (all p>0.2). Moreover, the LCS6 genotype was not associated with DFS (hazard ratio = 0.93, p=0.49) even after combining LCS6 genotype with KRAS mutation status. Conclusions: We report the largest association study investigating the LCS6 polymorphism and colon cancer outcome. There is no significant evidence that the germline LCS6 genotype was associated with DFS in stage III colon cancer patients. Additional studies are needed to determine if LCS6 genotype differs between tumor and germline DNA which may further clarify its prognostic value.

AbstractPurpose: A let-7 microRNA-complementary site (LCS6) polymorphism in the 3′ untranslated region of the KRAS gene has been shown to disrupt let-7 binding and upregulate KRAS expression. We evaluated the LCS6 genotype and its association with KRAS mutation status, clinicopathologic features, and disease-free survival (DFS) in patients with stage III colon cancer who enrolled in a phase III clinical trial (NCCTG N0147).Experimental Design: The LCS6 genotype was assayed by real-time PCR in DNA extracted from whole blood (n = 2,834) and compared with paired tumor tissue (n = 977). χ2 and two-sample t tests were used to compare baseline factors and KRAS mutation status between patients defined by LCS6 variant status. Log-rank tests and multivariate Cox models assessed associations between LCS6 status and DFS, respectively.Results: We identified 432 (15.2%) blood samples and 143 (14.6%) tumor samples heterozygous or homozygous for the LCS6 G-allele, and 2,402 of 2,834 (84.8%) blood samples and 834 of 977 (85.4%) tumor samples homozygous for the LCS6 T-allele. Genotype results were highly concordant (99.8%) in cases with paired blood and tumor tissue (n = 977). G-allele carriers were significantly more frequent in Caucasians versus other races (χ2 test, P &lt; 0.0001). The LCS6 genotype was not associated with KRAS mutation status, clinicopathologic features (all P &gt; 0.2), or DFS (log-rank P = 0.49; HR, 0.929; 95% confidence interval, 0.76–1.14), even after combining LCS6 genotype with KRAS mutation status.Conclusions: In the largest association study investigating the LCS6 polymorphism in colon cancers, the germline LCS6 genotype was not associated with KRAS mutation status or with clinical outcome in patients with stage III tumors. Clin Cancer Res; 20(12); 3319–27. ©2014 AACR.

Abstract Abundant epidemiologic evidence indicates that regular and long-term use of aspirin is associated with a significant reduction in the incidence of colorectal cancer. The long duration of aspirin needed to prevent colorectal cancer is believed to be due to inhibition of precursor lesions known as adenomas, the recurrence of which is inhibited by aspirin in randomized trials. Aspirin intake has also been associated with a statistically significant improvement in patient survival after curative resection of colorectal cancer in large observational studies. In these cohorts, the survival benefit of aspirin was shown to depend upon the level of COX-2 expression in the primary colorectal cancer. More recent analysis of patient tumors from these observational cohorts suggests that the benefit of aspirin may be limited to specific molecular subtypes. Aspirin intake following colorectal cancer resection was associated with a significant improvement of survival in patients whose tumors carried mutant, but not wild-type, copies of the phosphoinositide 3-kinase (PI3KCA) gene, especially tumors that overexpressed COX-2. A mechanistic explanation is suggested by the finding that inhibition of COX-mediated prostaglandin E2 synthesis by aspirin attenuates PI3K signaling activity that is known to regulate cancer cell proliferation and survival. Aspirin has also been shown to reduce the incidence of colorectal cancers bearing wild-type, but not mutant alleles of the BRAFV600E oncogene. Although provocative, the potential utility of these molecular markers for predicting aspirin efficacy awaits prospective evaluation in clinical trials. If validated, these findings may support a personalized approach to using aspirin for the therapy of colorectal cancer. Clin Cancer Res; 20(5); 1087–94. ©2013 AACR.