Microsatellite Instability Colon Cancer Research Articles

Lymph Node Metastases and Associated Recurrence-Free Survival in Microsatellite Stable and Unstable Colon Cancer.

In contrast to microsatellite stable (MSS) colon cancer, predictors of lymph node metastases and their association with recurrence are not well-defined in microsatellite instability (MSI) colon cancer. A cohort of nonmetastatic colon cancer patients undergoing surgery between 2015 and 2021 were evaluated for predictors of lymph node metastases (LNMs) and their association with recurrence-free survival (RFS). Of 1466 patients included in the analyses, 361 (25 %) had MSI. Compared with MSS, MSI was associated with earlier stage, fewer LNMs in the patients with N1 or N2 disease, and fewer high-risk features. Compared with the T3-T4 MSS patients, the odds ratios for LNM were 0.52 (95% confidence interval [CI], 0.38-0.71) for the T3-T4 MSI patients, 0.27 (95% CI, 0.38-0.71) for the T1-T2 MSS patients, and 0.15 (95 % CI, 0.08-0.26) for the T1-T2 MSI patients. In both groups, LNMs were associated with T category, patient age, and venous, lymphatic, or perineural invasion. In the MSS patients, LNMs were additionally associated with patient sex and histologic grade. Compared with the MSS patients, the MSI patients with N0 and N1 disease had a better 3-year RFS. However, the MSI patients with N2 disease had a lower rate of 3-year RFS than the MSS patients (hazard ratio, 19.75 vs 4.49). In MSI colon cancer, LNMs are 50 % less prevalent, but the factors associated withLNM are like those in MSS colon cancer. The improved prognosis traditionally associated with early-stage MSI colon cancers dissipates with four or more LNMs. These findings should be taken into consideration by clinicians selecting the most appropriate course of treatment for MSI colon cancer.

Comprehensive analysis of the mutational status of early colon cancer: Real world data from the AIO ColoPredict registry study.

e15535 Background: The comprehensive analysis of the mutational status of colon cancer (CC) may identify targetable alterations also in early stages. This may open up potential beneficial treatment choices, e.g. immunotherapy in microsatellite instable (MSI) tumors. So far, there is few data regarding the distribution of molecular alterations in CC in a real world setting in Germany. Methods: We analyzed clinical and molecular data from our Colopredict Plus (CPP) registry trial including CC patients (pts.) stage I-III. Samples were tested for MS status by immunohistochemistry and for gene mutations (MT) using a 71 gene panel comprised for CC. Molecular data was correlated with clinicopathologic parameters. Disease free Survival (DFS) was estimated by the Kaplan-Meier method. Results: Data from 2746 pts. was included. Female: 1476/2746 (53,8 %), median age: 72 yrs (range: 22-100 yrs), right sided tumor: 1607/2746 (58,5 %), stage distribution I/II/III/NA: 414 (15,1 %)/1198 (43,6 %)/1076 (39,2 %)/58 (2,1 %). MSI/MSS: 544(19,8%)/2202/ (80,2%). A KRAS mutation was observed in 846/2746 (30,8 %). The most frequent hotspot MT were codon 12 KRAS MT with p.G12D (27%), p.G12V (19%), p.G12A (6%), and p.G12C (6%) followed by codon 13 MT with p.G13D (18%). The subtypes p.G12D, p.G12V, and p.Q61K were significantly more frequent in the right colon. BRAF MT was seen in 560/2746 (20,4%). Observed rare MT were: POLE (76/2746 (2,8 %), PTEN 188/2746 (6,8 %), and EGFR (21/2746; 0,8%). MSI CC were more frequent in the right colon and associated with BRAF MT (346/560; 61,8 %), and also MT in BRCA1 (35/53; 66 %), BRCA2 (119/171; 69,6 %), POLE (45/76 (59,2 %), PTEN 107/188 (56,9 %), and EGFR (18/21, 85,7%). In contrast, TP53 MT (1223/2746 (44,5 %)) were associated with MSS (1090/2746 (49,5 %)). 3 yrs DFS in MSI BRAF WT vs. MSS BRAF MT pts. was 0,75 (0,67 – 0,81 vs 0,52 (0,36 – 0,65) (p =0,11). DFS did not differ in specific KRAS MT variants. Conclusions: We here show a comprehensive analysis of genetic alterations in early CC in a prospective real world cohort. As expected, MSI was correlated with female sex, right sided primary tumor and BRAF MT. Interestingly, we also found a strong correlation of BRCA1 MT, BRCA2 MT, EGFR MT, POLE MT, and PTEN MT with presence of MSI while MSS was associated with TP53 MT. Broad molecular characterization of early CC may help individualize treatment strategies in the future.

Pembrolizumab leading to complete resolution of non-small cell lung cancer and microsatellite instability stable colon adenocarcinoma; two birds one stone

Introduction: Staging of non-small cell lung cancer is crucial in predicting patient prognosis and more importantly, determining cancer management. In patients without driver mutations, PD-L1 tumor proportion score evaluation becomes vital in dictating treatment, as immunotherapy can be recommended. These agents have been shown to lead to excellent outcomes, even in patients with late-stage disease. Case Report: A 69-year-old male with a history of chronic obstructive pulmonary disease (COPD) presented with worsening dyspnea found to have lung collapse from a large hilar soft tissue mass causing obstruction of the left mainstem bronchus. After malignancy workup, the patient was diagnosed with non-small cell lung cancer clinically staged as IIIB. An incidental finding of microsatellite instability colon cancer was also found during workup. Pembrolizumab treatment was initiated and led to near resolution of both tumors. Conclusion: Stage IIIB non-small cell lung cancer has an overall poor prognosis. Biomarker testing in our case prior to starting concurrent chemoradiation revealed the malignancy to have a 100% tumor proportion score for PD-L1, the fundamental reason why our patient’s treatment was successful. Based on our findings, we advocate for all patients with non-small cell lung cancer regardless of stage to undergo biomarker testing prior to therapy initiation. Furthermore, the resolution of PD-L1 negative microsatellite instability stable colon cancer after pembrolizumab therapy supports further investigation of the utility and mechanism of PD-1/PD-L1-based therapy in PD-L1 negative colon cancer.

Label-free and automated approach to rapidly classify microsatellite instability (MSI) in early colon cancer (CC) analyzing the AIO ColoPredictPlus 2.0 (CPP) registry trial.

3616 Background: MSI due to mismatch repair defects accounts for 15-20% of all CC, has high prognostic and predictive value and is broadly utilized in treatment decisions. Artificial intelligence (AI) integrated, label-free quantum cascade laser (QCL) based infrared (IR) imaging resolves spatial and molecular alterations such as MSI in unstained cancer tissue sections. We aimed to evaluate the method for microsatellite instability/stability (MSI/MSS) classification in samples from the prospective multicenter AIO CPP registry trial. Methods: Paraffin-embedded unstained cancer tissue slides from patients (pts.) participating in CPP were measured (avg. 30 min/slide) and analyzed. The cohort was split into training (train), test (test), and validation (vali) sets. Cancer regions were first preselected based on a self-developed convolutional neural network (CNN) CompSegNet (Schuhmacher, medrxiv 2021). A VGG-16 CNN then classified MSI/MSS in these regions. Endpoints were area under receiver operating characteristic (AUROC) and area under precision recall curve (AUPRC). Results: 547 pts. (train n=331, test n=69, vali n=147) were analyzed. The baseline characteristics for the sub-cohorts are illustrated in the table. Mutation (MT) status: RAS MT: train 30% / test 30% / vali 37%; BRAF MT: train 27% / test 23% / vali 14%. The preselection of cancer regions reached a validation AUROC of 1.0. The subsequent MSI/MSS classifier reached a validation AUROC of 0.9 and AUPRC of 0.74 (sensitivity 85%, specificity 84%). Conclusions: Our multicenter approach using AI integrated label-free IR imaging provides an automated, fast, and reliable classification for MSI/MSS with an AUROC of 0.9 (sensitivity 85%, specificity 84%) almost comparable to the present gold standard immunohistochemistry. The method described here requires less samples for training when compared to other AI approaches which could facilitate the development of prognostic/predictive classifiers in the setting of randomized controlled trials. This novel technique may support further understanding of the increasingly important MSI CC cohort and support treatment decisions e.g. in specific subgroups such as targetable fusions. We expect our approach to be a broadly applicable diagnostic tool in the future.[Table: see text]

Medullary carcinoma of the colon: A comprehensive analysis of a large cancer database.

180 Background: Medullary carcinoma (MC) of the colon was described first in 1999 and is characterized by undifferentiated sheets of epithelial cells in a solid growth pattern with pushing borders and prominent lymphoplasmacytic infiltrates. It frequently shows microsatellite instability (MSI). However, the clinicopathological and survival outcomes between MC and the more common adenocarcinoma of the colon (AC) are not well defined in a large database. Therefore, we used a large national registry to explore the association of survival characteristics with sociodemographic, geographic, and disease variables in this cohort of patients. Methods: We sampled the National Cancer Database (NCDB) for colon cancer patients diagnosed with MC from 2004-2018. Multivariate cox regression models were used to compare hazard ratios for demographic, geographic, and disease characteristics. In addition, Kaplan-Meier survival plots were utilized to assess survival rate differences between MC and AC tumors located in the ascending, transverse, or descending colon. A p-value<0.05 was considered statistically significant. Results: Only 2,709 (0.29%) of the 922,667 patients with AC had MC. MC was seen in older patients than AC (76 vs. 69 years). MC was also more common in females than AC (72.5 vs. 49%). The most striking finding in our analysis was the predominance of poorly differentiated (63%) and undifferentiated (21.7%) pathologies in MC. This was in sharp contrast to AC, where 15.8% of patients were poorly differentiated, and only 2% were undifferentiated. Furthermore, undifferentiated and poorly differentiated MC tumors had a poor prognosis (HR 1.45, 95% CI 1.26-1.68, p<0.001 and HR 1.44, 95% CI 1.29-1.62, p<0.001 respectively) as compared to the other histological grades. Another notable finding in our analysis was the high prevalence of MSI in MC vs. AC (82.4% vs. 25.9%). Cecal MC (HR 1) had the worst prognosis of any colonic site. Left-sided MC tumors also had an inferior prognosis compared to AC (63.21 vs. 87.72 months), although not statistically significant (p=0.217). Conclusions: Colon cancer presenting as undifferentiated or poorly differentiated with associated MSI should raise the possibility of a medullary carcinoma diagnosis. However, given the rarity and difficulty in its pathological delineation, MC may remain underdiagnosed. In the modern treatment era, with the approval of immunotherapy for metastatic MSI colon cancer patients, survival and outcomes for metastatic MC may be improved.

Mapping Proteome Changes in Microsatellite Stable, Recurrent Colon Cancer Reveals a Significant Immune System Signature.

Better stratification of the risk of relapse will help select the right patients for adjuvant treatment and improve targeted therapies for patients with colon cancer. To understand why a subset of tumors relapse, we compared the proteome of two groups of patients with colon cancer with similar stage, stratified based on the presence or absence of recurrence. Using tumor biopsies from the primary operation, we identified dissimilarity between recurrent and nonrecurrent mismatch satellite stable colon cancer and found that signaling related to immune activation and inflammation was associated with relapse. Immune modulation may have an effect on mismatch satellite stable colon cancer. At present, immune therapy is offered primarily to microsatellite instable colon cancer. Hopefully, immune therapy in mismatch satellite stable colon cancer beyond PD-1 and PD-L1 inhibitors can be implemented.

Abstract 2924: In vivo CRISPR/Cas9 models of microsatellite instable colon cancer reveal recurrent mutations in putative tumor supressor genes

Abstract Microsatellite instability is a hallmark of DNA mismatch repair (MMR)-deficient colorectal cancer (CRC), and is accompanied by genome-wide somatic hypermutation and enhanced immunogenicity. Clinical trials have demonstrated remarkable efficacy of immune checkpoint blockade (i.e. anti-PD-1) in treating MMR-deficient cancers, which is thought to be driven by an enhanced burden of mutation-derived tumor specific antigens (neoantigens). Despite these successes, tumor mutation burden is an imperfect clinical predictor of immunotherapy response, and major questions remain regarding the mechanistic role of neoantigens and other features of MMR-deficiency in the anti-tumor immune response. Notably, most genetically engineered mouse models (GEMMs) fail to recapitulate the mutation burden of their associated human cancer and lack prominent immune involvement, precluding careful study of these questions. To model microsatellite instability in colon cancer, we developed novel autochthonous GEMMs utilizing CRISPR/Cas9-based knockout of DNA MMR genes in the distal colon. Colonoscopy-guided submucosal injection was used to deliver lentivirus containing two single-guide RNAs (sgRNAs) against Apc (the initiating event in the majority of colon cancers) and MMR pathway genes (Msh2, Mlh1, Msh3, Msh6). Whole-exome sequencing of MMR-targeted colon tumors revealed a high mutation burden with signatures consistent with those found in MMR-deficient human cancer. In addition, sequencing revealed a number of recurrently mutated microsatellites, including those within putative tumor suppressor genes Asxl1, Acvr2a, and Acvr1b. To interrogate the functional relevance of these genes in MMR-deficient colon cancer, we developed a novel ex vivo organoid competition assay. Specifically, Apc null organoids expressing Cas9 were infected with lentivirus expressing the fluorophore mScarlet and sgRNAs targeting the putative tumor suppressor genes. Flow cytometry was used to longitudinally assess kinetics of the mScarlet positive fraction. Preliminary results suggest a selective advantage following deletion of Asxl1 and Acvr2a. Further assessment of tumorigenesis in vivo will be performed to confirm the role of these putative tumor suppressors. Altogether, these genetically manipulable systems can be used for rapid functional interrogation of tumor suppressor genes, facilitating downstream mechanistic studies and preclinical testing of therapies in these important subsets of CRC. Citation Format: Daniel D. Zhang, Peter M. Westcott, Olivia Smith, Nate Sacks, Haley Hauck, Mary C. Beytagh, Abbey Jin, Tyler Jacks. In vivo CRISPR/Cas9 models of microsatellite instable colon cancer reveal recurrent mutations in putative tumor supressor genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2924.

Framing the potential of public frameshift peptides as immunotherapy targets in colon cancer.

Approximately 15% of Colon Cancers are Microsatellite Instable (MSI). Frameshift Peptides (FPs) formed in MSI Colon Cancer are potential targets for immunotherapeutic strategies. Here we comprehensively characterize the mutational landscape of 71 MSI Colon Cancer patients from the cancer genome atlas (TCGA). We confirm that the mutations in MSI Colon Cancers are frequently frameshift deletions (23% in MSI; 1% in microsatellite stable), We find that these mutations cluster at specific locations in the genome which are mutated in up to 41% of the patients. We filter these for an adequate variant allele frequency, a sufficient mean mRNA level and the formation of a Super Neo Open Reading Frame (SNORF). Finally, we check the influence of Nonsense Mediated Decay (MMD) by comparing RNA and DNA sequencing results. Thereby we identify a set of 20 NMD-escaping Public FPs (PFPs) that cover over 90% of MSI Colon, 62.2% of MSI Endometrial and 58.8% of MSI Stomach cancer patients and 3 out of 4 Lynch patients in the TCGA-COAD. This underlines the potential for PFP directed immunotherapy, both in a therapeutic and a prophylactic setting in multiple types of MSI cancers.

Analysis of the molecular nature associated with microsatellite status in colon cancer identifies clinical implications for immunotherapy

BackgroundMicrosatellite instability in colon cancer implies favorable therapeutic outcomes after checkpoint blockade immunotherapy. However, the molecular nature of microsatellite instability is not well elucidated.MethodsWe examined the immune microenvironment of colon...

Abstract 4966: The SUV39H1-H3K9me3 axis mediates colon carcinoma cell intrinsic apoptosis and immune evasion

Abstract Human colon cancer, except for the small subset of microsatellite instable (MSI) colon cancer, does not respond to anti-PD-L1/PD-1 immune checkpoint inhibitor (ICI) immunotherapy. A well-established notion is that MSI colon cancer harbors various mutations that serve as neoantigens to generate tumor-specific cytotoxic T lymphocytes (CTLs). Because CTLs suppress tumor through inducing tumor cell apoptosis, we hypothesized that colon cancer cell intrinsic apoptosis resistance is potentially another mechanism underlying microsatellite stable (MSS) colon cancer nonresponse to ICI immunotherapy. If tumor cells are not sensitive to apoptosis induction, then tumor cells cannot be killed by CTLs regardless of how potent the CTLs are. To test this hypothesis, we analyzed MSI and MSS human colon carcinoma specimens and developed a small molecule that targets H3K9me3-mediated cell death pathways as a sensitizer for colon cancer ICI immunotherapy. As expected, we observed high level of CTL infiltration in all eight MSI colon carcinoma specimens examined. However, five of the nine MSS colon carcinoma specimens also exhibited high level of CTL infiltration, suggesting that CTL level is not the sole mechanism underlying MSS colon cancer non-response to ICI immunotherapy. Analysis of the TCGA database revealed that SUV39H1, a histone methyltransferase that catalyzes H3K9me3, is significantly elevated in human colon carcinoma specimens compared to normal human colon tissues. A SUV39H1 structure-based virtual chemical library screening in combination with functional assays identified a small-molecule SUV39H1 inhibitor. Further analysis of the top forty-three hits identified a potent SUV39H1 inhibitor. Structure modifications were then performed and a chemical synthesis procedure was developed to synthesize a novel SUV39H1 inhibitor, termed F5446. F5446 has an EC50 of 0.496 µM against SUV39H1 enzymatic activity and suppresses human colon carcinoma cell growth in a dose-dependent manner in vitro. F5446 suppresses colon carcinoma growth through upregulating key cell cycle regulators to induce cell cycle arrest at the S phase. Furthermore, F5446 also decreased H3K9me3 at the FAS promoter to upregulate Fas expression. Consequently, a sublethal dose of F5446 effectively overcame human colon carcinoma cell resistance to apoptosis induced by FasL, a ligand on activated CTLs in vitro. Treatment of colon carcinoma-bearing mice with F5446 and anti-PD-1 both significantly suppress tumor growth in vivo. Our data determined that F5446 is a novel SUV39H1 inhibitor that has the potential to be further developed as a sensitizer to overcome colon carcinoma resistance to ICI immunotherapy through activating the Fas-FasL pathway. Citation Format: Chunwan Lu, Dafeng Yang, John D. Klement, Aaron H. Colby, Mark W. Grinstaff, Cedric Pearce, Nicholas H. Oberlies, Thomas Albers, Iryna Lebedyeva, Kebin Liu. The SUV39H1-H3K9me3 axis mediates colon carcinoma cell intrinsic apoptosis and immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4966.

Abstract NG03: Marine omega-3 polyunsaturated fatty acid and colorectal cancer prevention and treatment

Abstract NG03: Marine omega-3 polyunsaturated fatty acid and colorectal cancer prevention and treatment

Impact of MLH1 expression on tumor evolution after curative surgical tumor resection in a murine orthotopic xenograft model for human MSI colon cancer.

Colorectal cancers (CRCs) displaying microsatellite instability (MSI) most often result from MLH1 deficiency. The aim of this study was to assess the impact of MLH1 expression per se on tumor evolution after curative surgical resection using a xenograft tumor model. Transplantable tumors established with the human MLH1-deficient HCT116 cell line and its MLH1-complemented isogenic clone, mlh1-3, were implanted onto the caecum of NOD/SCID mice. Curative surgical resection was performed at day 10 in half of the animals. The HCT116-derived tumors were more voluminous compared to the mlh1-3 ones (P = .001). Lymph node metastases and peritoneal carcinomatosis occurred significantly more often in the group of mice grafted with HCT116 (P = .007 and P = .035, respectively). Mlh1-3-grafted mice did not develop peritoneal carcinomatosis or liver metastasis. After surgical resection, lymph node metastases only arose in the group of mice implanted with HCT116 and the rate of cure was significantly lower than in the mlh1-3 group (P = .047). The murine orthotopic xenograft model based on isogenic human CRC cell lines allowed us to reveal the impact of MLH1 expression on tumor evolution in mice who underwent curative surgical resection and in mice whose tumor was left in situ. Our data indicate that the behavior of MLH1-deficient CRC is not only governed by mutations arising in genes harboring microsatellite repeated sequences but also from their defect in MLH1 as such.

T cell therapy targeting a public neoantigen in microsatellite instable colon cancer reduces in vivo tumor growth

ABSTRACTT-cell receptor (TCR) transfer is an attractive strategy to increase the number of cancer-specific T cells in adoptive cell therapy. However, recent clinical and pre-clinical findings indicate that careful consideration of the target antigen is required to limit the risk of off-target toxicity. Directing T cells against mutated proteins such as frequently occurring frameshift mutations may thus be a safer alternative to tumor-associated self-antigens. Furthermore, such frameshift mutations result in novel polypeptides allowing selection of TCRs from the non-tolerant T-cell repertoire circumventing the problem of low affinity TCRs due to central tolerance. The transforming growth factor β Receptor II frameshift mutation (TGFβRIImut) is found in Lynch syndrome cancer patients and in approximately 15% of sporadic colorectal and gastric cancers displaying microsatellite instability (MSI). The -1A mutation within a stretch of 10 adenine bases (nucleotides 709–718) of the TGFβRII gene gives rise to immunogenic peptides previously used for vaccination of MSI+ colorectal cancer patients in a Phase I clinical trial. From a clinically responding patient, we isolated a cytotoxic T lymphocyte (CTL) clone showing a restriction for HLA-A2 in complex with TGFβRIImut peptide. Its TCR was identified and shown to redirect T cells against colon carcinoma cell lines harboring the frameshift mutation. Finally, T cells transduced with the HLA-A2-restricted TGFβRIImut-specific TCR were demonstrated to significantly reduce the growth of colorectal cancer and enhance survival in a NOD/SCID xenograft mouse model.

Loss of KCNQ1 expression in stage II and stage III colon cancer is a strong prognostic factor for disease recurrence.

Background:Colorectal cancer (CRC) is the third most common cancer worldwide. Accurately identifying stage II CRC patients at risk for recurrence is an unmet clinical need. KCNQ1 was previously identified as a tumour suppressor gene and loss of expression was associated with poor survival in patients with CRC liver metastases. In this study the prognostic value of KCNQ1 in stage II and stage III colon cancer patients was examined.Methods:KCNQ1 mRNA expression was assessed in 90 stage II colon cancer patients (AMC-AJCCII-90) using microarray gene expression data. Subsequently, KCNQ1 protein expression was evaluated in an independent cohort of 386 stage II and stage III colon cancer patients by immunohistochemistry of tissue microarrays.Results:Low KCNQ1 mRNA expression in stage II microsatellite stable (MSS) colon cancers was associated with poor disease-free survival (DFS) (P=0.025). Loss of KCNQ1 protein expression from epithelial cells was strongly associated with poor DFS in stage II MSS (P<0.0001), stage III MSS (P=0.0001) and stage III microsatellite instable colon cancers (P=0.041). KCNQ1 seemed an independent prognostic value in addition to other high-risk parameters like angio-invasion, nodal stage and microsatellite instability-status.Conclusions:We conclude that KCNQ1 is a promising biomarker for prediction of disease recurrence and may aid stratification of patients with stage II MSS colon cancer for adjuvant chemotherapy.

Microsatellite instability is associated with reduced disease specific survival in stage III colon cancer

BackgroundUp to 15% of colorectal cancers exhibit microsatellite instability (MSI), where errors in replication go unchecked due to defects in the mismatch repair system. This study aimed to determine survival in a large single-centre series of 1250 consecutive colorectal cancers subjected to universal MSI testing. MethodsClinical and pathological features of patients with colorectal cancer identified on prospectively maintained colorectal and pathology databases at St. Vincent's University Hospital from 2004 to May 2012 were examined. Mismatch repair (MMR) status was determined by immunohistochemistry. Kaplan–Meier curves, the log-rank test and Cox regression were used to associate survival with clinical and pathological characteristics. ResultsOf the 1250 colorectal cancers in the study period, 11% exhibited MSI (n = 138). Patients with MSI tumours had significantly lower rates of lymph node and distant metastases (MSI N+ rate: 24.8% compared with MSS N+ rate: 46.2%, p < 0.001). For Stage I and II disease MSI was associated with improved disease free survival (DSS) compared with MSS colon cancer. However, patients with Stage III MSI colon cancers had a worse DSS than those with MSS tumours. Stage III MSI tumours exhibited higher rates of lymphovascular invasion and perineural invasion than Stage I/II MSI tumours. ConclusionMSI is associated with a reduced risk of nodal and distant metastases, with an improved DSS in Stage I/II colon cancer. However, when MSI tumours progress to Stage III these patients had worse outcomes and pathological features. New strategies for this cohort of patients may be required to improve outcomes.

Abstract B24: Identification of immunosuppressive networks in colon cancer

Abstract Despite important improvements over the past two decades in preventive screening and targeted therapies, colorectal cancer remains the third major cause of cancer-related deaths in the U.S. The majority (~85%) of colon cancer tumors are non-immunogenic, i.e. they lack a significant number of infiltrating cytotoxic T cells, and are typically unresponsive to the new immune-checkpoint inhibitor based therapies that have dramatically changed the way we treat many cancer patients. These non-immunogenic colon cancer tumors are also characterized by chromosome instability and are microsatellite stable (MSS). Microsatellite instable (MSI) tumors present a greater variety of antigens and are significantly more immunogenic. We analyzed the paired mRNA and microRNA RNA-Seq expression profiles contained in The Cancer Genome Atlas (TCGA), representing MSS colon cancer, MSI colon cancer, and normal colon tissues. These expression profiles reflect the heterogeneity of the tumor samples, with MSI samples showing distinct immune cell signatures. Integrative analysis of mRNA and microRNA expression patterns revealed signaling networks that may represent, in part, the molecular mechanisms by which cancer cells modulate the host response to create an immune privileged microenvironment. These findings could result in a novel therapeutic avenue, targeting a tumor's ability to suppress the immune response, addressing a critical barrier to treatment for non-immunogenic tumors. Citation Format: Anne E. Sarver, Aaron L. Sarver, Subbaya Subramanian. Identification of immunosuppressive networks in colon cancer. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B24.

Abstract A141: The vigorous immune microenvironment of microsatellite instable colon cancer isbalanced by multiple counter-inhibitory checkpoints

Abstract We examined the immune microenvironment of primary colorectal cancer (CRC) using immunohistochemistry, laser capture microdissection/qRT-PCR, flow cytometry and functional analysis of tumor infiltrating lymphocytes. A subset of CRC displayed high infiltration with activated CD8+ CTL as well as activated Th1 cells characterized by IFN-gamma production and the Th1 transcription factor Tbet. Parallel analysis of tumor genotypes revealed that virtually all of the tumors with this active Th1/CTL microenvironment had defects in mismatch repair, as evidenced by microsatellite instability (MSI). Counterbalancing this active Th1/CTL microenvironment, MSI tumors selectively demonstrated highly up-regulated expression of multiple immune checkpoints, including five - PD-1, PD-L1, CTLA-4, LAG-3 and IDO - currently being targeted clinically with inhibitors. These findings link tumor genotype with the immune microenvironment, and explain why MSI tumors are not naturally eliminated despite a hostile Th1/CTL microenvironment. They further suggest that blockade of specific checkpoints may be selectively efficacious in the MSI subset of CRC. Our findings are the first to demonstrate a link between a genetically defined subtype of cancer and its corresponding expression of immune checkpoints in the tumor microenvironment. The mismatch repair defective subset of CRC selectively up-regulates at least 5 checkpoint molecules that are targets of inhibitors currently being clinically tested. Furthermore, our results were clinically validated in a phase 2 study at Hopkins which showed mismatch-repair status as a predictor of clinical benefit to immune checkpoint blockade with pembrolizumab. Citation Format: Nicolas J. Llosa, Franck Housseau, Elizabeth Wick, Hechenbleikner Lizzy, Michael Cruise, Robert Anders, Cynthia Sears, Drew M. Pardoll, Hongni Fan, Nicholas Siegel, Lee Blosser, Ada Tam, Hao Wang. The vigorous immune microenvironment of microsatellite instable colon cancer isbalanced by multiple counter-inhibitory checkpoints. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A141.

The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints

Meeting abstracts We examined the immune microenvironment of primary colorectal cancer (CRC) using immunohistochemistry, laser capture microdissection/qRT-PCR, flow cytometry and functional analysis of tumor infiltrating lymphocytes. A subset of CRC displayed high infiltration with activated CD8+

Prognostic value of BRAF and KRAS mutation status in stage II and III microsatellite instable colon cancers.

Microsatellite instability (MSI) has been associated with favourable survival in early stage colorectal cancer (CRC) compared to microsatellite stable (MSS) CRC. The BRAF V600E mutation has been associated with worse survival in MSS CRC. This mutation occurs in 40% of MSI CRC and it is unclear whether it confers worse survival in this setting. The prognostic value of KRAS mutations in both MSS and MSI CRC remains unclear. We examined the effect of BRAF and KRAS mutations on survival in stage II and III MSI colon cancer patients. BRAF exon 15 and KRAS exon 2-3 mutation status was assessed in 143 stage II (n = 85) and III (n = 58) MSI colon cancers by high resolution melting analysis and sequencing. The relation between mutation status and cancer-specific (CSS) and overall survival (OS) was analyzed using Kaplan-Meier and Cox regression analysis. BRAF V600E mutations were observed in 51% (n = 73) and KRAS mutations in 16% of cases (n = 23). Patients with double wild-type cancers (dWT; i.e., BRAF and KRAS wild-type) had a highly favourable survival with 5-year CSS of 93% (95% CI 84-100%), while patients with cancers harbouring mutations in either BRAF or KRAS, had 5-year CSS of 76% (95% CI 67-85%). In the subgroup of stage II patients with dWT cancers no cancer-specific deaths were observed. On multivariate analysis, mutation in either BRAF or KRAS vs. dWT remained significantly prognostic. Mutations in BRAF as well as KRAS should be analyzed when considering these genes as prognostic markers in MSI colon cancers.

Regulator of Chromosome Condensation 2 Identifies High-Risk Patients within Both Major Phenotypes of Colorectal Cancer.

Colorectal cancer has high incidence and mortality worldwide. Patients with microsatellite instable (MSI) tumors have significantly better prognosis than patients with microsatellite stable (MSS) tumors. Considerable variation in disease outcome remains a challenge within each subgroup, and our purpose was to identify biomarkers that improve prediction of colorectal cancer prognosis. Mutation analyses of 42 MSI target genes were performed in two independent MSI tumor series (n = 209). Markers that were significantly associated with prognosis in the test series were assessed in the validation series, followed by functional and genetic explorations. The clinical potential was further investigated by immunohistochemistry in a population-based colorectal cancer series (n = 903). We identified the cell-cycle gene regulator of chromosome condensation 2 (RCC2) as a cancer biomarker. We found a mutation in the 5' UTR region of RCC2 that in univariate and multivariate analyses was significantly associated with improved outcome in the MSI group. This mutation caused reduction of protein expression in dual luciferase gene reporter assays. siRNA knockdown in MSI colon cancer cells (HCT15) caused reduced cell proliferation, cell-cycle arrest, and increased apoptosis. Massive parallel sequencing revealed few RCC2 mutations in MSS tumors. However, weak RCC2 protein expression was significantly associated with poor prognosis, independent of clinical high-risk parameters, and stratifies clinically important patient subgroups with MSS tumors, including elderly patients (>75 years), stage II patients, and those with rectal cancer. Impaired RCC2 affects functional and clinical endpoints of colorectal cancer. High-risk patients with either MSI or MSS tumors can be identified with cost-effective routine RCC2 assays.