Low Microsatellite Instability Research Articles

Immunogenic characteristics of microsatellite instability-low esophagogastric junction adenocarcinoma based on clinicopathological, molecular, immunological and survival analyses.

Microsatellite instability (MSI) is categorized by mutation frequency: high MSI (MSI-H), low MSI (MSI-L) and microsatellite stable (MSS). MSI-H tumors have a distinct immunogenic phenotype, with immunotherapies using checkpoint inhibitors already approved for the treatment of MSI-H gastroesophageal adenocarcinoma (GEA); this is not observed for MSI-L or MSS. Here, we tested the hypothesis that MSI-L tumors are also a distinct phenotype and potentially immunogenic. MSI-PCR assays (BAT25, BAT26, BAT40, D2S123, D5S346 and D17S250) were performed on 363 Epstein-Barr virus-negative, surgically resected esophagogastric junction (EGJ) adenocarcinoma samples. Tumors were characterized as MSI-H (≥2 markers), MSI-L (1 marker) or MSS (0 markers). CD8+ cell counts, PD-L1 and HER2 expression levels, TP53 mutations, epigenetic alterations and prognostic significance were also examined. All pathological and molecular experiments were conducted using serial, whole-tumor sections of chemo-naïve surgical specimens. MSI-H and MSI-L were assigned to 28 (7.7%) and 24 (6.6%) cases, respectively. Compared to MSS cases, MSI-L cases had significantly higher intratumoral CD8+ cell infiltration (P = .048) and favorable EGJ cancer-specific survival (multivariate hazard ratio = 0.35, 95% CI, 0.12-0.82; P = .012). MSI-L tumors were also significantly associated with TP53-truncating mutations as compared to MSI-H (P = .009) and MSS (P = .012) cases, and this trend was also observed in GEA data from The Cancer Genome Atlas (TCGA). Indel mutational burden among TCGA MSI-L tumors was significantly higher than that of MSS tumors (P = .016). These results suggest that MSI-L tumors may have a distinct tumor phenotype and be potentially immunogenic in EGJ adenocarcinoma.

Relationship between Microsatellite Status and Other Prognostic Factors in Patients with Colon Cancer

It is reported that 0.5-13 % of all colorectal cancers are hereditary. Many mutations that cause genomic instability have been described lately in this cancers; the most famous one is yet microsatellite instability pathway. Investigating the presence of these mutations is important in tailoring patients' treatment and predicting prognosis.
 Aims: We evaluated the association between micro satellite status and other pathologic prognostic factors like grade, tumor size, lymph node metastasis, lymphovascular invasion and perineural invasion in patients who underwent curative colon resection for colorectal cancers (CRC) in our clinic in the past five years.
 Study Design: A total of 205 sequential patients who were older than 18 and had curative colon resection for CRC in Ankara University Surgical Oncology Unit and been tested for microsatellite instability (MSI) were analyzed on behalf of the facultys’ database.
 Methodology: Pathology results had been determined and tumor localizations, lymph node metastasis status, grade, lymphovascular and perineural invasion status were evaluated. Information about MSI status and defected genes were obtained from detailed pathology reports. Patients were divided into two groups as MSI and MSS.
 Results: No significant difference was found between two the groups in the context of microsatellite instability status. Lymphovascular invasion had been seen higher in high frequency microsatellite instability (MSH-H) compared to low frequency microsatellite instability (MSH-L) group (76.4% vs 53.1%, P =.02). There was no statistical difference in perineural invasion between the two groups (P = 0.102). Signet ring cell status between the groups we found a higher rate of signet ring cells and consequently a higher grade in MSH-H group (17.6% vs 10.6%, P = 0.042).
 Conclusion: In conclusion, although many important points have been identified in our study, more studies are needed to compare the evaluation of MSI in colon cancer with other prognostic factors and to investigate its effect on the course of the disease.

Abstract 3114: Analytical validation of Illumina's TruSight Oncology 500 ctDNA assay

Abstract While tissue biopsies and imaging techniques remain the current standard of care, minimally invasive liquid biopsy tests are emerging as compelling complements in the clinical management of cancer patients. The ability to accurately detect low frequency somatic mutations, tumor mutational burden (TMB) and microsatellite instability (MSI) biomarkers from a single plasma sample using a broad-based NGS panel is of high value for biomarker discovery, detection and monitoring. Herein we report the results of our analytical validation of Illumina's TruSight Oncology 500 (TSO500) ctDNA assay which employs a hybrid-capture approach for target enrichment of 523 clinically-relevant cancer genes with unique molecular indices (UMIs) to enable detection of low frequency variants, CNVs, DNA fusions, TMB and MSI analysis. To characterize performance of small variant detection (SNVs and indels) and TMB analysis using the TSO500 ctDNA assay, we utilized various contrived reference standards with >1,500 unique variants below 5% variant allele frequency (VAF) and over 50 uncharacterized diseased and 20 healthy plasma samples. Cell-free DNA (cfDNA) isolates from healthy donors were used to evaluate the false positive (FP) rate as well as to evaluate potential impacts of blood collection tube (BCT) type on variant calling. For the tube type comparison, we procured 5 matched sets of healthy donor blood in both EDTA and Streck cfDNA BCTs. Where possible, unexpected somatic variants detected in healthy donor samples were orthogonally confirmed using droplet digital PCR (ddPCR). Additionally, to evaluate robustness of input on variant detection and TMB precision, cfDNA isolates from late stage cancer patients were tested at varying input amounts (10, 20, 30ng) in the TSO500 ctDNA assay. Analytical sensitivity, specificity and positive predictive value (PPV) were evaluated using reference standards. We observed >99% sensitivity for detecting SNVs and >98% for indels at or above 0.5% VAF. Additionally, we observed very high specificity and PPV (>99%), with a false positive rate of < 2 FP per 1.3 Mb down to 0.2% VAF. Further support for high specificity was observed in healthy donor samples where 25/26 apparent false positive somatic variants (0.2-0.7% VAF) were confirmed using ddPCR. Robustness of input studies revealed comparable coverage depth and uniformity can be achieved with 20 - 30 ng of cfDNA. Precision of variant detection and TMB will be reported for a subset of the diseased plasma sample cohort. In conclusion, the TSO500 ctDNA assay enables robust detection of low frequency variants and other clinically relevant biomarkers in EDTA and Streck derived plasma samples with at least 20ng of cfDNA input. Citation Format: Kirsten C. Verhein, Gunjan Hariani, Stephanie B. Hastings, Patrick Hurban. Analytical validation of Illumina's TruSight Oncology 500 ctDNA assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3114.

Crosstalk Between the MSI Status and Tumor Microenvironment in Colorectal Cancer

Colorectal cancer (CRC) patients, especially those with deficient mismatch repair (dMMR)/microsatellite instability-high (MSI-H) tumors, whose sensitivity to immune checkpoint inhibitors (ICIs) is significantly higher than that of patients with microsatellite-stable (MSS)/microsatellite instability-low (MSI-L) tumors, have derived clinical benefits from immunotherapy. Most studies have not systematically evaluated the immune characteristics and immune microenvironments of MSI-H and MSS/MSI-L CRCs. We analyzed the relationship between the MSI status and prognosis of ICI treatment in an immunotherapy cohort. We further used mutation data for the immunotherapy and The Cancer Genome Atlas (TCGA)-CRC [colon adenocarcinoma (COAD) + rectum adenocarcinoma (READ)] cohorts. For mRNA expression, mutation data analysis of the immune microenvironment and immunogenicity under different MSI statuses was performed. Compared with CRC patients with MSS/MSI-L tumors, those with MSI-H tumors significantly benefited from ICI treatment. MSI-H CRC had more immune cell infiltration, higher expression of immune-related genes, and higher immunogenicity than MSS/MSI-L CRC. The MANTIS score, which is used to predict the MSI status, was positively correlated with immune cells, immune-related genes, and immunogenicity. In addition, subtype analysis showed that COAD and READ might have different immune microenvironments. MSI-H CRC may have an inflammatory tumor microenvironment and increased sensitivity to ICIs. Unlike those of MSI-H READ, the immune characteristics of MSI-H COAD may be consistent with those of MSI-H CRC.

Undifferentiated carcinoma of the transverse colon with rhabdoid features that developed during treatment of non-small cell lung carcinoma with pembrolizumab: a case report

BackgroundUndifferentiated carcinoma of the colon is rare, and its prognosis is very poor. We report a case of undifferentiated carcinoma of the colon with rhabdoid features developed during treatment of non-small lung carcinoma (NSCLC) with pembrolizumab.Case presentationA 58-year-old man was diagnosed with transverse colon cancer during chemotherapy with pembrolizumab for NSCLC. Laparoscopic right hemicolectomy was performed. The histopathological diagnosis was undifferentiated carcinoma with rhabdoid features and lymph node metastasis. Immunohistochemically, programmed death ligand 1 (PD-L1) showed positivity. The microsatellite instability (MSI) status was low. He continued to receive pembrolizumab for NSCLC, and there have been no signs of colon cancer recurrence and progression of NSCLC for 15 months.ConclusionWe present the case of an undifferentiated carcinoma of the transverse colon with rhabdoid features. The development of the tumor with the expression of PD-L1 during pembrolizumab might have been associated with the low MSI.

Impact of Tumor Localization and Molecular Subtypes on the Prognostic and Predictive Significance of p53 Expression in Gastric Cancer.

We investigated the prognostic and predictive impact of p53 expression for gastric cancer (GC) patients treated without or with preoperative chemotherapy (CTx) and its relationship with specific molecular GC subtypes. Specimens from 694 GC patients (562 surgical resection specimens without or after CTx, 132 biopsies before CTx) were analyzed by p53 immunohistochemistry. High (H) and low (L) microsatellite instability (MSI) and Epstein–Barr virus positivity were determined previously. Our results show that aberrant p53 expression was a negative prognostic factor in uni- and multivariable analysis in the resection specimens cohort (each p < 0.01). Subgroup analysis showed the strongest prognostic effect for patients with distally located tumors or no CTx treatment. In the biopsy cohort before CTx, p53 did not predict response or survival. p53 expression was significantly different among the molecular subtypes in surgical resection and bioptic specimens with strong association of altered p53 with MSI-L. Patients with MSI-H and aberrant p53 showed the worst survival in the biopsy cohort. In conclusion, the prognostic impact of p53 in GC differs according to tumor localization and CTx. Altered p53 is characteristic for MSI-L, and the p53 status in biopsies before CTx delineates MSI-H subtypes with inverse prognostic impact.

Crosstalk between the MSI status and tumor microenvironment in colorectal cancer.

e15236 Background: In recent years, cancer immunotherapy has been extensively studied, and colorectal cancer (CRC) patients have also derived clinical benefits from immunotherapy, especially CRC patients with mismatch repair deficiency (dMMR)/microsatellite instability-high (MSI-H), whose sensitivity to immune checkpoint inhibitors (ICIs) is significantly higher than that of patients with microsatellite-stable (MSS)/microsatellite instability-low (MSI-L) disease. This study suggests that patients with MSI-H CRC have a higher mutational burden and more immune cell infiltration than those with MSS/MSI-L disease. However, most studies have not systematically evaluated the immune characteristics and immune microenvironments of MSI-H and MSS/MSI-L CRC. Methods: A published CRC cohort with mutation and immunotherapy-related prognostic data was collected. We analyzed the relationship between the MSI status and prognosis of ICI treatment in an immunotherapy cohort. We then further used mutation data for the immunotherapy and The Cancer Genome Atlas (TCGA)-CRC (colon adenocarcinoma (COAD) + rectum adenocarcinoma (READ) cohorts. For mRNA expression, mutation data analysis of the immune microenvironment and immunogenicity under different MSI status was performed. Results: Compared with MSS/MSI-L CRC patients, patients with MSI-H CRC significantly benefited from ICI treatment. We found that MSI-H CRC had more immune cell infiltration, higher expression of immune-related genes and higher immunogenicity than MSS/MSI-L disease. The MANTIS score used to predict the MSI status was positively correlated with immune cells, immune-related genes, and immunogenicity. In addition, subtype analysis showed that COAD and READ might have different tumor immune microenvironments. Conclusions: MSI-H CRC may have an inflammatory tumor microenvironment and increased sensitivity to ICIs. Unlike those of MSI-H READ, the immune characteristics of MSI-H COAD may be consistent with those of MSI-H CRC. Furthermore, the possible mechanism underlying the prognostic differences among CRC patients receiving ICIs in relation to the immune microenvironment were elucidated to provide theoretical guidance for further improving the curative effect of ICIs treatment on MSI-H CRC patients in the future and solve the problems underlying why MSS/MSI-L CRC patients do not benefit from ICIs treatment.

An Explorative Analysis of ABCG2/TOP-1 mRNA Expression as a Biomarker Test for FOLFIRI Treatment in Stage III Colon Cancer Patients: Results from Retrospective Analyses of the PETACC-3 Trial.

Biomarker-guided treatment for patients with colon cancer is needed. We tested ABCG2 and topoisomerase 1 (TOP1) mRNA expression as predictive biomarkers for irinotecan benefit in the PETACC-3 patient cohort. The present study included 580 patients with mRNA expression data from Stage III colon cancer samples from the PETACC-3 study, which randomized the patients to Fluorouracil/leucovorin (5FUL) +/− irinotecan. The primary end-points were recurrence free survival (RFS) and overall survival (OS). Patients were divided into one group with high ABCG2 expression (above median) and low TOP-1 expression (below 75 percentile) (“resistant”) (n = 216) and another group including all other combinations of these two genes (“sensitive”) (n = 364). The rationale for the cut-offs were based on the distribution of expression levels in the PETACC-3 Stage II set of patients, where ABCG2 was unimodal and TOP1 was bimodal with a high expression level mode in the top quarter of the patients. Cox proportional hazards regression was used to estimate the hazard ratios and the association between variables and end-points and log-rank tests to assess the statistical significance of differences in survival between groups. Kaplan-Meier estimates of the survival functions were used for visualization and estimation of survival rates at specific time points. Significant differences were found for both RFS (Hazard ratio (HR): 0.63 (0.44–0.92); p = 0.016) and OS (HR: 0.60 (0.39–0.93); p = 0.02) between the two biomarker groups when the patients received FOLFIRI (5FUL+irinotecan). Considering only the Microsatellite Stable (MSS) and Microsatellite Instability-Low (MSI-L) patients (n = 470), the differences were even more pronounced. In contrast, no significant differences were observed between the groups when patients received 5FUL alone. This study shows that the combination of ABCG2 and TOP1 gene expression significantly divided the Stage III colon cancer patients into two groups regarding benefit from adjuvant treatment with FOLFIRI but not 5FUL.

Differential microRNA expression profiles associated with microsatellite status reveal possible epigenetic regulation of microsatellite instability in gastric adenocarcinoma.

BackgroundAlthough microsatellite instability (MSI) is a powerful predictive biomarker for the efficacy of immunotherapy, the mechanism of MSI in sporadic gastrointestinal cancer is not fully understood. However, epigenetics, particularly microRNAs, has been suggested as one of the main regulators that contribute to the MSI formation.MethodsWe used microRNA expression data of 386 gastric adenocarcinoma samples from The Cancer Genome Atlas (TCGA) database to identify differential microRNA expression profiles by different MSI status. We also obtained putative common target genes of the top differential microRNAs with miRanda online tools, and we analyzed these data by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment (KEGG).ResultsWe found that 56 and 67 gastric adenocarcinoma samples were positive for low and high MSI, respectively, and that a high MSI status was associated with age, sex and subregion (P=0.049, 0.014 and 0.007, respectively). In the 67 samples with a high MSI status, expression levels of 14 microRNAs were upregulated but five microRNAs were downregulated as assessed by the fold change (FC), compared with that of the 56 samples with a low MSI status (P<0.05, |FC| >2). Further analysis suggested that the expression of miR-210-3p, miR-582-3p, miR-30a-3p and miR-105-5p predicted a high MSI status (P=4.93×10−10, 5.63×10−10, 3.23×10−9 and 7.64×10−4, respectively). Regulation of the transcription pathways ranked the top of lists from both GO and KEGG analyses, and these microRNAs might regulate DNA damage-repair genes that were also associated with a high MSI status.ConclusionsMiR-30a-3p and miR-105-5p are potential biomarkers for the MSI-H gastric adenocarcinoma, possibly by altering expression of DNA damage-repair genes.

Microsatellite instability and its associations with the clinicopathologic characteristics of diffuse large B-cell lymphoma.

Microsatellite instability (MSI) has been investigated as a prognostic and predictive factor for chemotherapy in colorectal cancer and has recently been demonstrated to be predictive of the PD‐1/PD‐L1 checkpoint blockade response in various solid tumors. However, MSI status in diffuse large B‐cell lymphomas (DLBCLs) has not been thoroughly explored. This study investigated MSI status in DLBCLs and analyzed the associations between MSI and clinicopathologic characteristics and clinical outcomes. Ninety‐two cases of primary DLBCLs treated with R‐CHOP/CHOP chemotherapy between 2009 and 2017 were collected. MSI detection was performed by the Promega MSI Analysis System. The protein expression of MLH1, MSH2, MSH6, and PMS2 was detected by immunohistochemistry. The associations of MSI‐H and MSI‐L with progression‐free survival (PFS) and overall survival (OS) were assessed by COX models and Kaplan–Meier curves. The correlations of complete response (CR) after R‐CHOP/CHOP chemotherapy with MSI‐H and MSI‐L were examined by univariate and multivariate logistic regression analyses, respectively. 3 of 92 cases (3.2%) were high MSI (MSI‐H), and 9 cases (9/92, 9.8%) exhibited low MSI (MSI‐L). One case with MSI‐H showed negative expression of MSH2 and MSH6. Univariate analysis indicated that MSI‐L was correlated with poor response to R‐CHOP/CHOP chemotherapy in DLBCLs (OR, 0.178; 95% CI, 0.041‐0.776; P = .022). Multivariate analysis showed that MSI‐L was an independent predictive factor for non‐CR to R‐CHOP/CHOP chemotherapy (OR, 0.144; 95% CI, 0.027‐0.761; P = .023). Kaplan‐Meier curves showed that there was a trend that MSI‐H patients had favorable PFS (P = .36) and OS (P = .48), which did not have statistical significance and MSI‐L was not significantly correlated with PFS (P = .24) and OS (P = .52).Our study indicated that there existed MSI‐H and MSI‐L in DLBCLs. MSI‐L could be an independent predictive factor for the chemotherapy response in DLBCLs.

Clinical significance of MLH1/MSH2 for stage II/III sporadic colorectal cancer.

BACKGROUNDThe development of colorectal cancer (CRC) is a complicated multistep process that involves an accumulation of mutations in tumor suppressor genes and oncogenes. In the process of DNA replication, base mismatch often occurs due to various factors leading to abnormal expression of mismatch repair genes (MMR), among which MLH1 and MSH2 are the most important. Recently, numerous studies indicated that MLH1/MSH2 phenotype is associated with CRC. We wanted to elucidate the role of MLH1/MSH2 in the prediction and prognosis of CRC through long-term clinical observation.AIMTo evaluate the prognostic and predictive significance of MLH1/MSH2 in patients with stage II-III CRC using immunohistochemical analysis and GeneScan.METHODSSpecimens from 681 patients with CRC (395 stage II and 286 stage III, 387 males and 294 females) who underwent curative surgical resection from 2013 to 2016 were tested. Immunohistochemistry was used to analyze MMR status and the microsatellite status of 133 patients was determined by GeneScan analysis.RESULTSFive hundred and fifty (80.76%) patients were MLH1/MSH2 positive and 131 (19.24%) were negative by immunohistochemistry. MLH1/MSH2-positive tumors were significantly more frequent in the colon than in the rectum, and had poor differentiation and less mucin production (P < 0.05). Patients of different groups did not differ in terms of age, gender, tumor size, tumor stage, lymphocytic infiltration, or circumscribed margin. MLH1/MSH2-negative patients had a more favorable OS than MLH1/MSH2-positive patients (P < 0.001). Univariate and multivariate analyses demonstrated MLH1/MSH2 expression as an independent prognostic and predictive factor for stage II/III CRC. MLH1/MSH2 expression was a strong prognostic factor in all patients [P < 0.001, hazard ratio (HR) = 4.064, 95%CI: 2.241–7.369]. Adjuvant chemotherapy had a greater correlation with survival advantage in MLH1/MSH2-negative patients with stage III disease (P < 0.001, HR = 7.660, 95%CI: 2.974–15.883). However, patients with stage II disease or MLH1/MSH2-positive patients with stage III disease did not benefit from adjuvant chemotherapy. GeneScan analysis demonstrated that among 133 patients, 105 (78.95%) were microsatellite stable, and 28 (21.05%) had microsatellite instability (MSI), including 18 (13.53%) with high MSI and 10 (7.52%) with low MSI. This is consistent with the immunohistochemical results.CONCLUSIONMLH1/MSH2 phenotype constitutes a pathologically and clinically distinct subtype of sporadic CRC. MLH1/MSH2 is an independent prognostic and predictive factor for outcome of stage II-III CRC.

Genomic alterations in signet ring and mucinous patterned colorectal carcinoma

BackgroundThe genetic alterations (GAs) in two specific histological subtypes of colorectal cancer (CRC), signet ring cell colorectal carcinoma (SRC) and mucinous colorectal carcinoma (MC), are not well known. In the present study, we employed next-generation sequencing to perform genetic profiling of SRC and MC, and compared the spectrum of GAs with the alterations found in conventional type colorectal cancer (CON). Materials and methodsWe selected 46 CRCs comprising 17 SRCs and mucinous carcinoma with signet ring cell component (SRCCs), 17 MCs, and 12 CONs with microsatellite stability or microsatellite instability-low. Deep sequencing was performed using a targeted cancer panel composed of 171 cancer-related genes. SMAD4 protein expression was evaluated by immunohistochemical staining. ResultsWe detected 108 mutations in 18 different genes. Overall, 2.34 GAs were detected per tumor (range, 0–14). The overall frequency of GA and alteration in targetable genes was less prevalent in SRC/SRCC compared to the frequency of alteration in MC/CON (p = 0.040 and p < 0.001, respectively). The GA profile of SRC/SRCC included TP53 (8/17, 47.1%), SMAD4 (5/17, 29.4%), KRAS (4/17.23.5%), APC (4/17.23.5%), PIK3CA, ATM, BRAF, and PIK3R1 (1/17, 5.9%, each). KRAS mutation was significantly less prevalent in SRC/SRCC compared to the number of KRAS mutations in MC (12/17, 70.6%) and CON (9/12, 75.0%) (p = 0.015 and 0.01, respectively). Compared to the 152 non-hypermutated CONs from TCGA database, SMAD4 alteration was predominant in SRC/SRCC (p = 0.045) with aberrant loss of SMAD4 expression (13/17, 76.5%) compared to the SMAD4 alterations in CON (5/15, 33.3%) (p = 0.031). Accordingly, KRAS (12/17, 70.6%), APC (6/17, 35.3%), SMAD4, TP53 (4/17, 23.5%, each), PIK3CA (3/17, 17.6%), AKT1, ATM, BRAF, EGFR, and EZH2 (1/17, 5.9%, each) were altered in MC. APC and TP53 mutations were less frequent in MC compared to those in TCGA-CON (p < 0.001 and 0.003, respectively) whereas KRAS mutation was prevalent (p = 0.041). ConclusionAlterations of known cancer associated genes and targetable genes in SRC/SRCC are infrequent. The profile of GAs in SRC/SRCC and MC differs from the GA profile of CON. Specifically, SMAD4 mutation and loss of SMAD4 expression is frequently found in SRC/SRCC. The genetic profiles revealed in the present study may aid in developing precision medicine for CRC treatment based on histological subtype.

Prognostic implication of molecular subtypes and response to neoadjuvant chemotherapy in 760 gastric carcinomas: role of Epstein-Barr virus infection and high- and low-microsatellite instability.

Epstein–Barr virus positivity (EBV(+)) and high‐microsatellite instability (MSI‐H) have been identified as molecular subgroups in gastric carcinoma. The aim of our study was to determine the prognostic and predictive relevance of these subgroups in the context of platinum/5‐fluorouracil (5‐FU) based preoperative chemotherapy (CTx). Additionally, we investigated the clinical relevance of the low‐MSI (MSI‐L) phenotype. We analysed 760 adenocarcinomas of the stomach or the gastro‐oesophageal junction encompassing 143 biopsies before CTx and 617 resected tumours (291 without and 326 after CTx). EBV was determined by PCR and in situ hybridisation for selected cases. MSI was analysed by PCR using five microsatellite markers and classified as MSI‐H and MSI‐L. Frequencies of EBV(+), MSI‐H and MSI‐L in the biopsies before CTx were 4.2, 10.5 and 4.9% respectively. EBV(+) or MSI‐H did not correlate with response, but MSI‐L was associated with better response (p = 0.011). In the resected tumours, frequencies of EBV(+), MSI‐H and MSI‐L were 3.9, 9.6 and 4.5% respectively. Overall survival (OS) was significantly different in the non‐CTx group (p = 0.014). Patients with EBV(+) tumours showed the best OS, followed by MSI‐H. MSI‐L was significantly associated with worse OS (hazard ratio [HR], 2.21; 95% confidence interval [CI], 1.21–4.04, p = 0.01). In the resected tumours after CTx, MSI‐H was also associated with increased OS (HR, 0.54; 95% CI, 0.26–1.09, p = 0.085). In multivariable analysis, molecular classification was an independent prognostic factor in the completely resected (R0) non‐CTx group (p = 0.035). In conclusion, MSI‐H and EBV(+) are not predictive of response to neoadjuvant platinum/5‐FU based CTx, but they are indicative of a good prognosis. In particular, MSI‐H indicates a favourable prognosis irrespective of treatment with CTx. MSI‐L predicts good response to CTx and its negative prognostic effect for patients treated with surgery alone suggests that MSI‐L might help to identify patients with potentially high‐benefit from preoperative CTx.

Immunotherapy in colorectal cancer: rationale, challenges and potential.

Following initial successes in melanoma treatment, immunotherapy has rapidly become established as a major treatment modality for multiple types of solid cancers, including a subset of colorectal cancers (CRCs). Two programmed cell death 1 (PD1)-blocking antibodies, pembrolizumab and nivolumab, have shown efficacy in patients with metastatic CRC that is mismatch-repair-deficient and microsatellite instability-high (dMMR-MSI-H), and have been granted accelerated FDA approval. In contrast to most other treatments for metastatic cancer, immunotherapy achieves long-term durable remission in a subset of patients, highlighting the tremendous promise of immunotherapy in treating dMMR-MSI-H metastatic CRC. Here, we review the clinical development of immune checkpoint inhibition in CRC leading to regulatory approvals for the treatment of dMMR-MSI-H CRC. We focus on new advances in expanding the efficacy of immunotherapy to early-stage CRC and CRC that is mismatch-repair-proficient and has low microsatellite instability (pMMR-MSI-L) and discuss emerging approaches for targeting the immune microenvironment, which might complement immune checkpoint inhibition.

Phase Ib trial of cabozantinib (C) in combination with atezolizumab (A) in patients (pts) with advanced hepatocellular carcinoma (HCC), gastric or gastroesophageal junction cancer (GC/GEJC), or colorectal cancer (CRC).

TPS478 Background: C is an inhibitor of tyrosine kinases involved in tumor growth, angiogenesis, and immune regulation, including MET, VEGFR, and TAM family kinases (Tyro3, AXL, MER). Preclinical and clinical studies suggest that C promotes an immune-permissive tumor environment which may enhance response to checkpoint inhibitors such as the anti-PD-L1 mAb A. C significantly improved overall survival vs placebo in previously treated advanced HCC, and the combination of A and bevacizumab has shown synergistic activity in advanced HCC. Checkpoint inhibitors have shown clinical activity in advanced CRC primarily in pts with high microsatellite instability (MSI); combination with immune-modulating agents may enhance activity in pts with stable or low MSI. Likewise, combination therapy may improve response in pts with GC/GEJC resistant to standard chemotherapy. Here, we present the study design of an ongoing phase 1b trial which includes cohorts with advanced HCC, GC/GEJC, or CRC. Methods: This global, phase 1b, open-label trial will assess the safety, tolerability, preliminary efficacy, and pharmacokinetics of C in combination with A (NCT03170960). The study consists of a dose-escalation stage and an expansion stage. In the dose-escalation stage (3+3 design), a recommended C dose for combination with a standard dose of A will be established. In the expansion stage, 18 cohorts will be enrolled at the recommended dose of C + A including 3 cohorts with gastrointestinal cancers: (1) advanced HCC not previously treated with systemic therapy; (2) advanced GC/GEJC after progression on platinum- or fluoropyrimidine-containing therapy; and (3) advanced CRC after progression on fluoropyrimidine combined with oxaliplatin or irinotecan. Pts will continue treatment as long as they experience clinical benefit as judged by the investigator or until unacceptable toxicity. The primary objective of the expansion stage is the objective response rate for each cohort. Exploratory objectives include correlation of tumor and plasma biomarkers, immune cell profiles, and MSI status with clinical outcome. Clinical trial information: NCT03170960.

Comparative Genomic Profiling of Refractory/Metastatic Penile and non-Penile Cutaneous Squamous Cell Carcinoma: Implications for Selection of Systemic Therapy

BackgroundMetastatic penile squamous cell carcinoma (mPSCC) is an aggressive malignancy with limited treatment options. We compared the potential therapy impacting genomic alterations (GA) between mPSCC and non-penile cutaneous metastatic squamous cell carcinoma (mCSCC). MethodsDNA was extracted from 40 microns of FFPE samples from 78 cases of mPSCC and 338 mCSCC. Comprehensive genomic profiling (CGP), was performed using a hybrid-capture, adaptor ligation based next generation sequencing assay to a mean coverage depth of >500X. Tumor mutational burden (TMB) was determined on 1.1 Mbp of sequenced DNA and microsatellite instability (MSI) was determined on 114 loci. ResultsPotential targeted therapy opportunities in mPSCC included alterations in MTOR pathway (NF1 GA in 7% and PTEN GA in 4%) and DNA repair pathway (BRCA2 and ATM GA, each in 7%) and tyrosine kinase (EGFR GA in 6%; FGFR3 and ERBB2 GA each in 4%). TMB was significantly higher in the predominantly UV light exposed mSCC than mPSCC, making mSCC potentially more responsive to immunotherapies than mPSCC. MSI-High status was extremely rare for both mPSCC and mCSCC. CD274 (PD-L1) amplification was also rare in both tumor types. ConclusionsmPSCC is a unique subtype of SCC with distinctive genomic features that contrast with those identified in mCSCC of non-penile UV light exposed skin. Although not rich in predictors for response to immunotherapy (low TMB and MSI low), more than one quarter of mPSCC may potentially benefit from existing and available therapies targeting MTOR, DNA repair, and Tyrosine Kinase pathways.

Independent Mechanisms Lead to Genomic Instability in Hodgkin Lymphoma: Microsatellite or Chromosomal Instability †.

Background: Microsatellite and chromosomal instability have been investigated in Hodgkin lymphoma (HL). Materials and Methods: We studied seven HL cell lines (five Nodular Sclerosis (NS) and two Mixed Cellularity (MC)) and patient peripheral blood lymphocytes (100 NS-HL and 23 MC-HL). Microsatellite instability (MSI) was assessed by PCR. Chromosomal instability and telomere dysfunction were investigated by FISH. DNA repair mechanisms were studied by transcriptomic and molecular approaches. Results: In the cell lines, we observed high MSI in L428 (4/5), KMH2, and HDLM2 (3/5), low MSI in L540, L591, and SUP-HD1, and none in L1236. NS-HL cell lines showed telomere shortening, associated with alterations of nuclear shape. Small cells were characterized by telomere loss and deletion, leading to chromosomal fusion, large nucleoplasmic bridges, and breakage/fusion/bridge (B/F/B) cycles, leading to chromosomal instability. The MC-HL cell lines showed substantial heterogeneity of telomere length. Intrachromosmal double strand breaks induced dicentric chromosome formation, high levels of micronucleus formation, and small nucleoplasmic bridges. B/F/B cycles induced complex chromosomal rearrangements. We observed a similar pattern in circulating lymphocytes of NS-HL and MC-HL patients. Transcriptome analysis confirmed the differences in the DNA repair pathways between the NS and MC cell lines. In addition, the NS-HL cell lines were radiosensitive and the MC-cell lines resistant to apoptosis after radiation exposure. Conclusions: In mononuclear NS-HL cells, loss of telomere integrity may present the first step in the ongoing process of chromosomal instability. Here, we identified, MSI as an additional mechanism for genomic instability in HL.

Low-dose decitabine enhances the effect of PD-1 blockade in colorectal cancer with microsatellite stability by re-modulating the tumor microenvironment

PD-1 blockade has demonstrated impressive clinical outcomes in colorectal cancers that have high microsatellite instability. However, the therapeutic efficacy for patients with tumors with low microsatellite instability or stable microsatellites needs further improvement. Here, we have demonstrated that low-dose decitabine could increase the expression of immune-related genes such as major histocompatibility complex genes and cytokine-related genes as well as the number of lymphocytes at the tumor site in CT26 colorectal cancer-bearing mice. A more significant inhibition of tumor growth and a prolongation of survival were observed in the CT26 mouse model after treatment with a combination of PD-1 blockade and decitabine than in mice treated with decitabine or PD-1 blockade alone. The anti-tumor effect of the PD-1 blockade was enhanced by low-dose decitabine. The results of RNA sequencing and whole-genome bisulfite sequencing of decitabine-treated CT26 cells and tumor samples with microsatellite stability from the patient tumor-derived xenograft model have shown that many immune-related genes, including antigen-processing and antigen-presenting genes, were upregulated, whereas the promoter demethylation was downregulated after decitabine exposure. Therefore, decitabine-based tumor microenvironment re-modulation could improve the effect of the PD-1 blockade. The application of decitabine in PD-1 blockade-based immunotherapy may elicit more potent immune responses, which can provide clinical benefits to the colorectal cancer patients with low microsatellite instability or stable microsatellites.

XRCC3 Thr241Met and TYMS variable number tandem repeat polymorphisms are associated with time-to-metastasis in colorectal cancer.

BackgroundMetastasis is a major cause of mortality in cancer. Identifying prognostic factors that distinguish patients who will experience metastasis in the short-term and those that will be free of metastasis in the long-term is of particular interest in current medical research. The objective of this study was to examine if select genetic polymorphisms can differentiate colorectal cancer patients based on timing and long-term risk of metastasis.MethodsThe patient cohort consisted of 402 stage I-III colorectal cancer patients with microsatellite instability (MSI)-low (MSI-L) or microsatellite stable (MSS) tumors. We applied multivariable mixture cure model, which is the proper model when there is a substantial group of patients who remain free of metastasis in the long-term, to 26 polymorphisms. Time-dependent receiver operator characteristic (ROC) curve analysis was performed to determine the change in discriminatory accuracy of the models when the significant SNPs were included.ResultsAfter adjusting for significant baseline characteristics, two polymorphisms were significantly associated with time-to-metastasis: TT and TC genotypes of the XRCC3 Thr241Met (p = 0.042) and the 3R/3R genotype of TYMS 5’-UTR variable number tandem repeat (VNTR) (p = 0.009) were associated with decreased time-to-metastasis. ROC curves showed that the discriminatory accuracy of the model is increased slightly when these polymorphisms were added to the significant baseline characteristics.ConclusionsOur results indicate XRCC3 Thr241Met and TYMS 5’-UTR VNTR polymorphisms are associated with time-to-metastasis, and may have potential biological roles in expediting the metastatic process. Once replicated, these associations could contribute to the development of precision medicine for colorectal cancer patients.

Abstract 1759: Description of molecular marker disparities and KRAS mutational spectrum of colorectal tumors from Puerto Rican Hispanics

Abstract Colorectal cancer (CRC) causes 27% of all cancer deaths among Puerto Rican Hispanics (PRH). Most CRC cases are due to sporadic genetic or epigenetic events that lead to the development of the tumor. These events aggregate to cause CRC carcinogenesis and involve genes that regulate cell growth and differentiation. Improving understanding of the molecular events that lead to CRC carcinogenesis has the potential to reduce CRC mortality by risk stratifying individuals as well as developing prevention strategies. Currently, there are a set of molecular markers that are used to characterize CRC tumors and inform prognosis and treatment decisions. These markers include: microsatellite instability (MSI), CpG island methylation and mutational analysis of the KRAS and BRAF oncogenes. The objective of this study was to characterize the molecular markers present in the CRC tumors of PRH and understand the disparities present at the molecular level in these tumors. The frequency of the MSI and CIMP were evaluated in 488 CRC tumors from PRH. Mutations in the KRAS gene and the BRAF V600E mutation were evaluated using SuperArray technology. Additionally, information regarding sociodemographic and clinicopathologic characteristics of the study population was obtained. Of the 488 evaluated CRC tumors 1.6% (n=2) showed MSI and 90.2% of the studied tumors had CIMP low phenotype (n=102). MSI-High tumors were more likely to be distal tumors compared to MSS tumors (p=0.02). The V600E mutation in the BRAF gene were found in 9.17% (n=11) of the studied tumors. KRAS mutations were found in 31.25% (n=40) of tumors. The most common KRAS mutations found in the CRC tumors of PRH were: c.35G&amp;gt;A (KRAS COSMIC521) (n=12) and c.38G&amp;gt;A (n=8) (KRAS COSMIC532). Furthermore, the traditional CRC carcinogenesis pathway was the predominant pathway observed for the development of the CRC tumors of our study population. The following study highlights a distinct molecular signature for the CRC tumors from PRH descent. The disparities observed in the prevalence of the molecular markers (low MSI, CIMP Low and mainly wild-type for KRAS and BRAF) suggest that the drivers for CRC in this population might differ from other populations. Additional studies are required to fully elucidate the CRC carcinogenesis pathway in PRH. Citation Format: Julyann Perez-Mayoral, Camila Rivera-Lynch, Marievelisse Soto-Salgado, Xavier Llor, Luis Tous-Lopez, Marla Torres-Torres, Jose S. Reyes, Carlos Torres, Ajay Goel, Segundo Rodriguez-Quillichini, Marcia Cruz-Correa. Description of molecular marker disparities and KRAS mutational spectrum of colorectal tumors from Puerto Rican Hispanics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1759. doi:10.1158/1538-7445.AM2017-1759