Sinicrope, Frank A.

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.

270 Background: While immune checkpoint blockade (ICB) is used to treat metastatic deficient mismatch repair (dMMR) colorectal cancers (CRCs), such tumors are heterogeneous and resistance to ICB is frequent. To date, biomarkers that can consistently predict patient response to ICB are lacking. We determined whether biomarkers that integrate tumor and immune-related molecular mechanisms could predict ICB response. Methods: Consecutive patients with metastatic dMMR CRCs (N=32) who had been treated with anti-PD-1 therapy were profiled using a validated immune-enhanced exome and transcriptome platform (ImmunoID NeXT, Personalis) using tumor and paired normal tissue. This platform provides antigen-specificity of T or B cells, indicated by the TCR or BCR respectively, neoantigen prediction, tumor mutation burden (TMB), immune profile, HLA allele-specific loss of heterogeneity (LOH) and a Neoantigen Presentation Score (NEOPS) that combines a neoantigen prediction tool with mechanisms of immune evasion. Tumor variables were analyzed in relationship to objective tumor response, and a Cox proportional hazards model was fit to predict progression-free survival (PFS) or overall survival (OS). Results: In CRCs, TCR and BCR repertoire diversity were each significantly increased in tumors from responders versus those with stable or progressive disease after anti-PD-1 treatment (p=0.015 and p=0.048, respectively). Neoantigen burden score, TMB, and HLA allele-specific LOH (reflects loss of antigen presentation machinery) were each unable to distinguish responders from nonresponders. An immune checkpoint gene expression signature was increased in nonresponders relative to responders (P= 0.005). Neoantigen presentation score (NEOPS) was prognostic when adjusted for tumor grade only for OS (p=0.048). Conclusions: The T-cell and B-cell receptor (TCR/BCR) repertoire, as well as NEOPS, were each significantly predictive of favorable response to anti-PD-1 therapy in dMMR tumors. In addition, an immune checkpoint gene expression signature was predictive of resistance to ICB.

251 Background: The development of colorectal liver metastases (CRLM) is associated with poor prognosis, and recent data suggest that metastasis to the liver is associated with resistance to immunotherapy. We characterized the microenvironment of primary colorectal carcinomas (CRCs) relative to their synchronous CRLM using a validated segmentation algorithm that quantifies 15 distinct morphologic tumor features. Methods: Adult CRC patients with synchronous CRLM (N=57) at Mayo Clinic were identified from the electronic health record using Epic Slicer Dicer and from an internal database. Tumor H&E sections were digitized and reviewed for quality control (RKP). QuantCRC (Aiforia, Inc) was applied to digitized images to extract 15 GI pathologist pre-defined morphological features. Tumor features were compared between primaries and CRLM using the Kruskal–Wallis test. The project was approved by the Mayo Clinic Institutional Review Board. Results: The study included 57 patients (median age 59 years [IQR 50, 73], 51% female) with CRC primaries and synchronous CRLM. Among primaries, 20 (35%) were right-sided and 37 (65%) were left-sided. QuantCRC identified 6 of 15 morphological features that differed significantly between primaries and their CRLM, including reduced stroma, more high-grade and necrosis, and a higher tumor: stromal ratio (TSR) in CRLM (Table). The increase in TIL density in CRLM vs the primary tumor was of borderline significance (p =0.053). Among patients with left-sided primary tumors, their CRLM had significantly higher TSR, percent high-grade, percent necrosis, and TIL density compared to the primary (all p values ≤ 0.02). Among right sided primaries, CRLM had a significantly reduced percent mature stroma (p=0.034) whereas percent necrosis was increased (p =0.01). Conclusions: Using deep learning, we identified tumor morphological features that differed significantly between primary CRC and their synchronous CRLM. This included higher TSR in CRLMs compared to primaries that is associated with epithelial-mesenchymal transition and has been shown to contribute to treatment resistance. Analysis of tumor morphological features with patient prognosis is ongoing. Deep learning-derived morphological features of primary CRC and CRLM. Morphological feature (Median) Primary tumor Liver metastasis P-value Tumor-Stroma Ratio 0.78 1.50 0.008 % High-grade 23.08 31.72 0.005 TIL Count 44.36 60.20 0.053 % Necrosis 6.35 19.32 <0.001 %Signet Ring Cells 0.25 0.13 0.088 TB/PDC 0.89 1.13 0.484 % Stroma 54.89 40.98 <0.001 % Immature Stroma 46.76 34.13 <0.001 % Mature Stroma 6.27 5.08 0.030

Background Immune checkpoint inhibitors (ICIs) are recommended to treat patients with deficient mismatch repair/microsatellite instability high (dMMR/MSI-H) metastatic colorectal cancer (mCRC). Pivotal trials have fixed a maximum ICI duration of 2 years, without a compelling rationale. A shorter treatment duration has the potential to improve patients' quality of life and reduce both toxicity and cost without compromising efficacy. Here we examine whether early treatment discontinuation (ETD) before 13 months in patients without progressive disease (PD) can lead to similar long-term disease control compared with a longer treatment duration (LTD). Methods To assess whether ETD is associated with similar outcomes compared with LTD, we assembled an international cohort of patients with dMMR/MSI-H mCRC treated with ICIs who stopped treatment for a reason other than PD within 395 days (ETD group) and compared them to those who continued for >395 days (LTD group). Outcomes were adjusted for patient/tumor characteristics. Primary endpoint was progression-free survival (PFS) and secondary endpoints were objective response rate (ORR), overall survival (OS) and safety. Results Of 976 patients, 137 and 394 were allocated to the ETD and LTD groups, respectively. In the ETD group, treatment was discontinued due to toxicity (n=56), objective response (n=43), surgery (n=28), patient decision (n=2) or other reasons (n=8). Baseline characteristics were well balanced between the two groups: 22% in both groups received both anti-programmed death-(ligand) 1 (anti-PD-(L)1) + anti-cytotoxic T-lymphocyte antigen-4 (anti-CTLA-4); all others received anti-PD-(L)1 monotherapy. ORR to ICIs was 81% in both groups. Median duration of treatment was ~7 months in the ETD and ~24 months in the LTD group. After a median follow-up of 44 months (IQR: 30–67), similar PFS (HR: 0.92, 95% CI: 0.60 to 1.40, p=0.69) and OS (HR: 1.15, 95% CI: 0.66 to 1.99, p=0.62) from the start of ICIs were observed in ETD and LTD patients. In the ETD group, 28 (20%) patients had a PFS event and 9 restarted ICIs with a disease control rate of 66%. Conclusions In our international series of dMMR/MSI-H mCRC, ETD of ICIs in the absence of PD did not seem detrimental in terms of PFS and OS compared with continuing treatment beyond 1 year. Randomized clinical trials to compare short and long treatment duration are now warranted.

Rates of adjuvant chemotherapy in patients with early-onset versus later-onset stage II colon cancer

Neoadjuvant immunotherapy effectively uses the in situ tumor as a reservoir of tumor antigens to promote systemic antitumor immunity. Studies indicate that intratumoral responses to immune checkpoint inhibitors (ICIs) are mediated by resident memory T cells cells that are sequestered in tumors and have specificity for a wide range of tumor antigens. ICI treatment produces de novo priming of CD8+ T cells in tumor and in tumor-draining lymph nodes, and can boost the antitumor immune response by blocking inhibitory checkpoint proteins that can turn off T cells within the tumor. Neoadjuvant ICI treatment can enhance both intratumoral and systemic antitumor immunity, including expansion of intratumoral T-cell clones which is strongly associated with pathological treatment response. Recent data have shown high rates of pathological response to neoadjuvant immunotherapy with prolongation of survival compared with adjuvant ICI therapy alone in patients with unresectable or advanced melanoma. These data suggest that removal of the reservoir of tumor-specific T cells in the tumor and draining nodes by surgical resection may remove a significant proportion of the patient’s antitumor immunity with the potential to compromise ICI outcomes.

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.

3520 Background: In patients with metastatic colorectal cancer (CRC) and deficient DNA mismatch repair (dMMR), we previously found transcriptomic signatures reflecting stromal compartment composition and proliferative capacity that can predict the efficacy of immune checkpoint blockade (ICB). To identify proteomic markers of response and/or resistance to ICB, we performed spatially resolved proteomic analysis of the tumor microenvironment. Methods: Patients with surgically resected, primary dMMR metastatic CRCs received pembrolizumab (Mayo Clinic, N=30). In FFPE tissues, Digital Spatial Profiling of 71 proteins was performed using panels covering immune cells, immune activation, cell death, PI3/AKT signaling, MAPK signaling, and pan-tumor markers (GeoMx nCounter). In ten regions of interest/slide (~600 µm2) at the tumor invasive margin, protein expression was determined in four cellular compartments: epithelium (panCK+), immune cells (CD45+; leucocyte common antigen), stroma (panCK-), and nonimmune stroma (panCK-/CD45-). After normalization, protein abundance per compartment was analyzed by response (RECIST 1.1) [OmicVerse Python]. Differentially expressed proteins (binary) were examined by progression-free survival (PFS) using multivariable Cox regression with covariate adjustment (Lifelines Python). Results: Pre-treatment panels including 71 proteins were analyzed by treatment response and PFS after PD-1 inhibition. Proteins found to be significantly associated with response (p< 0.01) and PFS are described. Across all compartments, responders showed upregulation of NK cells and T cell subsets (CD3+, CD4+, CD8+), and ICOS; strongest association was found for CD4+: HRadj: 0.16; 95%CI: 0.08-0.31; p< 0.001. Nonresponders showed higher phospho-JNK (HRadj: 2.09; 95%CI: 1.28-3.42; p=0.003) and SMA. In the epithelial compartment, upregulation of the Treg marker CD25 and reduced caspase-9 were observed in responders. Among responders, the stromal compartment was highly enriched in HLA-DR, dendritic cells (CD11c), PD-L1, immune activation markers (CD40, CD44, CD80, CD27), CD95/Fas, and GZMB. In the stromal compartment, downregulation of BCL6 and phospho-GSK3B was observed in responders. The immune compartment of responders showed increased beta2-microglobulin and Ki-67, while the non-immune stromal compartment showed increase in the macrophage marker CD68. Importantly, proteins found to be upregulated in responders were significantly and independently associated with longer PFS whereas the opposite was true for nonresponders. Conclusions: In metastatic dMMR CRCs, spatially defined protein markers were significantly associated with anti-PD-1 response, and independently associated with patient PFS (strongest predictive utility for CD4 in immune stroma and phospho-JNK in epithelium).