Abstract 1712: Assessing tumor mutation load using an NGS-based, routine-friendly target gene panel

Molecular profiling of tumor mutations has expedited molecular targeted therapy for cancer patients. Additionally, tumor mutation load and DNA microsatellite instability (MSI) status can help predict patient’s response to immunotherapy. We analytically validated a gene test targeting clinically actionable cancer mutations of all 4 major types: single nucleotide variations (SNVs); insertion/deletions (INDELs); whole gene copy number variations (CNVs); and structural rearrangements (translocations) as well as the MSI status. This test interrogates all coding exons from 49 core cancer genes, introns for a subset of genes selected for detection of prevalent gene rearrangements, and the TERT (telomerase reverse transcriptase) promoter region. MSI status is determined from a set of 5 intronic mono-nucleotide repeats collectively associated with microsatellite instability. Targeted DNA regions are captured by in-solution hybridization with complementary biotinylated RNA baits and sequenced on the Illumina NextSeq®500 platform. Paired formalin-fixed, paraffin-embedded (FFPE) tumor samples and whole blood samples are analyzed simultaneously for the detection of SNV, INDEL, CNV, translocation and MSI status. FFPE samples can also be analyzed alone if the whole blood sample is not available, eliminating the reporting of MSI status. A minimum of 50 ng FFPE DNA and 100 ng of whole blood DNA are required for this test. A total of 123 FFPE, 2 FFPE FNA samples, 19 paired FFPE and whole blood paired samples covering lung cancer, colorectal cancer, melanoma and breast cancer were included in this validation study. Analytical validation of assay performance demonstrated that, on average, 700-fold read depth was achieved across all targeted regions with >95% of these regions covered by a minimum of 300 unique reads. Analytical sensitivity was ≥5% mutation frequency for SNV and INDEL and ≥20% for translocation and CNV. Thirty-eight unique variants were confirmed between this test and orthogonal methodologies: 22 SNVs, 6 INDELs, 5 translocations, and 5 CNV. Fifty-eight out of 59 (98% concordance) paired FFPE/blood samples achieved the same MSI status result with this targeted sequencing approach compared to the reference National Comprehensive Cancer Network (NCCN) Bethesda PCR test. MSI-positive samples, on average, were determined to have 5-fold higher mutation count compared to MSI-negative samples, consistent with the previously reported higher mutation burden. In conclusion, we have developed and analytically validated a core cancer gene test employing NGS technology with demonstrated high analytical sensitivity and specificity. Coupled with clinical interpretation, this test will facilitate clinical decision-making for molecular targeted therapy and immunotherapy. Citation Format: Lin Ma, Michael Hua, Steven Rivera, Anna Gerasimova, Quoclinh Nguyen, Sirisha Sunkara, Robert Lagier, Alla Smolgovsky, David Wolfson, Jared F. Taylor, Frederick Racke, Charles Strom, Andrew Grupe, Joseph Catanese, Feras Hantash. Validation of a clinically actionable cancer core gene test for solid tumors facilitating targeted molecular therapy and immunotherapy [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 5356. doi:10.1158/1538-7445.AM2017-5356

Background/Aims: Colorectal cancer (CRC) is mainly caused by chromosomal instability (CIN) and microsatellite instability (MSI). The RAS and RAF genes are essential components of the CIN pathway, and several studies have found that RAS and RAF mutations are associated with MSI status in CRC. Here, we examined these three factors in CRC in Northeast China and aimed to reveal new details of the relationship between these mutations and MSI status. Methods: This study involved 290 patients with CRC who had RAS or RAF gene mutation detected using fluorescence-based allele-specific polymerase chain reaction or Sanger sequencing. The majority of the identified patients were found to harbor MSI (MSI status). Accurate molecular detection was carried out using formalin-fixed paraffin-embedded tissue or blood samples. Results: The rates of RAS and RAF mutations were 58.5% and 4.1%, respectively. The prevalence of RAS mutation in CRC was clearly higher and that of RAF mutation was lower in Northeast China compared with previously reported cohorts in other locations. High MSI level (MSI-H status) was more complex, at around 10%. This was consistent with previous data from China. However, compared with data reported from other continents, MSI-H was higher than that of Japan or South Korea in Asia, and lower than that of Europe or the United States. Conclusion: RAS/RAF mutations and MSI status in CRC are closely associated with tumor location and ethnicity. Further studies investigating the relationship between these three factors can help in the development of treatment strategies for patients with CRC.

Background: Despite recent advances in prevention and therapy, colorectal cancer (CRC) continues to account for nearly 50,000 deaths per year in the US. TNM stage is the current CRC prognostic standard, yet within-stage prognostic heterogeneity is well documented. Microsatellite instability (MSI), CpG island methylation phenotype (CIMP), and BRAF- and KRAS-mutation status represent candidate prognostic markers. However, associations between these markers and CRC survival have not been described in a population-based setting. Methods: Data and tissue resources were obtained from the Iowa Women's Health Study (IWHS), which recruited 41,836 women, ages 55-69 years, at study entry (1986). Incident CRC cases were ascertained by annual linkage with the Iowa Cancer Registry. Archived, paraffin-embedded tissue specimens were collected from 732 representative CRC cases diagnosed through 12/31/2002. Established laboratory methods were used to characterize CRC cases: MSI-H or MSS/MSI-L (by instability in β3 vs. <3 out of 10 markers, respectively); CIMP+ or CIMP− (by promoter hypermethylation in β3 vs. 0-2 out of 5 genes, respectively); BRAFwt or BRAFmut (by fluorescent allele specific PCR to detect the V600E point mutation in exon 15); and KRASwt or KRASmut (by direct sequence analysis of codons 12 and 13 in exon 2). Cox regression models were fit to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for time to death of any cause adjusted for age, stage, grade, and treatment status. Results: CRC cases with informative molecular marker data ranged from 515/732 (70%) for KRAS to 548/732 (75%) for MSI status. Mean follow-up was 4.4 years for deceased individuals and 11.2 years for women alive at last contact. After multivariable adjustment, both MSS/MSI-L (p=0.004) and KRASmut (p=0.048) tumors were associated with poorer survival. Survival did not significantly differ across levels of CIMP and BRAF status. Conclusions: MSI and KRAS status may influence CRC survival among older women. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4492. doi:1538-7445.AM2012-4492

Checkpoint inhibitor (CPI) therapy demonstrates a remarkable clinical benefit in many cancer types. However, the ability to successfully select patients who will benefit from CPIs is still limited. Tumor mutational burden (TMB), a measure of the number of somatic mutations per coding area of tumor genome, is a putative biomarker of response showing great promise in CPI and immunotherapy combination trials. The ability to measure TMB from tumor biopsies or plasma samples will be important for clinical adoption of this biomarker. Herein we report on initial performance evaluation of Illumina’s TruSight™ Oncology 500 gene (TSO500) NGS assay for the analysis of TMB in FFPE tissue and plasma cell-free (cf)DNA specimens. Illumina’s TSO500 assay employs hybrid-capture based approach for target enrichment coupled with unique molecular indices to enable low frequency variant detection of single nucleotide variants and indels. This comprehensive cancer panel interrogates relevant cancer biomarkers in >500 cancer genes (~2 Mb) from as little as 40 ng of FFPE DNA or 30 ng of cfDNA. In addition to variant calls, Illumina’s analysis pipeline reports a TMB score and microsatellite instability (MSI) status. Results obtained with the TSO500 TMB assay were compared to our validated whole exome sequencing (WES) TMB assessment for FFPE tissue specimens, with and without matched normal. As previously reported, the WES TMB tumor-only pipeline uses somatic variant classifications determined using a random forest model to generate TMB scores from analysis of tumor FFPE specimens. TSO500 TMB and MSI statues were evaluated in a set of 50 tumor FFPE specimens obtained from late-stage (III+) colorectal and bladder cancer patients. TSO500 TMB results correlated well with the WES-derived TMB results, although some differences were observed in the magnitude of high TMB scores. TSO500 TMB levels correlated strongly with the MSI status, with 100% of MSI-high tumors demonstrating high TMB scores (> 20), and greater than 85% of MSI-low tumors characterized as TMB-low (<20%). Additionally, correlations between tissue and blood-based TMB assessments in matched FFPE and plasma specimens are reported. Overall, our results demonstrate a strong correlation between TSO500 panel-based and whole exome sequencing-based TMB results in tissue and report comparisons to TMB calling in liquid biopsies. Our data further confirm that tumor mutational load is strongly connected to DNA damage repair deficiency in colorectal and bladder cancer patients. Citation Format: Stephanie B. Hastings, Gunjan Hariani, Victor J. Weigman, Tingting Jiang, Chen Zhao, Rajiv Raja, Traci Pawlowski, Philip Brohawn, Patrick Hurban. Panel-based tumor mutational burden (TMB) analysis of matched tumor and plasma specimens using Illumina’s TruSight Oncology 500 next-generation sequencing assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1346.

149 Background: Microsatellite instability (MSI) plays a crucial role as a cancer immunotherapy and prognosis biomarker, making accurate MSI detection essential. However, currently, there is still a lack of available data on the impact of neoadjuvant therapy on MSI status, as well as validated data on standardized methods for MSI testing in small biopsy samples. The study aimed to investigate the concordance of MSI status between paired biopsy and surgical samples, as well as the impact of neoadjuvant therapy on MSI status using a novel MSI next-generation sequencing (MSI-NGS) detection panel. Methods: A total of 137 colorectal cancer (CRC) patients were enrolled for this study, of whom 116 with paired biopsy and surgical samples were analyzed. A custom MSI-NGS panel was employed and its performance was compared to MSI polymerase chain reaction (MSI-PCR), which served as the gold standard. Results: Out of the 116 cases, 112 patients showed consistent MSI status between biopsy and surgical samples, with an overall accuracy of 97% regardless of the detection method used. In the cases with MSI discrepancy (6 cases), 83% (5/6) of the patients showed a transition from MSS to MSI-H from biopsy to surgery. Interestingly, all eight patients who received neoadjuvant chemotherapy maintained an unchanged MSI status. However, in one patient who received adjuvant treatment and underwent a repeat biopsy after surgery, the MSI status exhibited alterations. Further analysis of clonal evolution revealed that this heterogeneity stemmed from the disappearance of the original clones and the emergence of new clones. Additionally, the NGS panel exhibited strong concordance with MSI-PCR and high accuracy, sensitivity and specificity with an AUC of 0.942 after rigorous validation. Conclusions: The study revealed a high concordance in MSI status between paired biopsy and surgical samples by employing a custom MSI-NGS panel for MSI status detection, providing reliable basis for further research in this field. Moreover, neoadjuvant chemotherapy seemed to have no impact on MSI status in our study, whereas postoperative adjuvant chemotherapy may influence changes in MSI status, highlighting the necessity of reevaluating MSI status after surgery. The findings also underscore the potential of NGS-based MSI detection as a valuable tool for clinical decision-making.

Introduction 10%–15% of colorectal cancer (CRC) is due to Microsatellite Instability(MSI). The aim of the study was to determine the prognostic significance of detecting MSI in Stage II CRC and to understand the demographic and pathological characteristics of the disease in the Indian scenario. Methods This was an 8 year retrospective study (2010 to 2017) of 195 patients with Stage II colorectal adenocarcinoma who were categorised into Microsatellite stable (MSS) or MSI based on immunohistochemical expression of the DNA Mismatch Repair(dMMR) proteins. Various clinical and pathological factors as per the Revised Bethseda criteria were compared and analysed between the MSI and MSS groups by Chi- square test and T Test. Kaplan- Meier method was used to calculate the Disease Free Survival (DFS) and Overall-survival (OS) for the 2 groups. Log Rank test was applied to know the strength of association between the DFS and OS with each of the parameters. The data was analysed using IBM SPSS version 20 software. Results There were 53 (27%) patients in the MSI group. Younger age, and presence of synchronous or metachronous malignancies, right sided location of tumour, poorly differentiated adenocarcinoma, mucin production and presence of peritumoral Crohn’s like lymphocytic response showed statistically significant association with MSI. A definite relationship of MSI status with family history could not be established. The mean DFS in MSI group was 74.7±3.496 months as compared to 69.2±3.631 months in MSS group. Disease related death was seen in 2.8% and 15.7% of patients in MSI and MSS group respectively, p=0.042. Overall survival among the MSI patients was significantly higher (76.6±4.149 months) than the MSS patients (65.05±3.555) p=0.04. MSI patients did not show improved survival with adjuvant therapy. Conclusions Early stage MSI related CRC has good prognosis even without adjuvant chemotherapy. Knowledge of the MSI status in CRC is useful in management decisions and prognostication. In addition it can help to detect those with Lynch Syndrome who may not fulfil the Revised Bethesda criteria.

Colorectal cancer (CRC) is one of the major causes of cancer-related morbidity and mortality worldwide. Despite of recent advances of variable therapeutic techniques in CRC, cancer progression and metastasis remain the major contributors to cancer-related morbidity and mortality.1 Cancer progression and metastasis are complex processes and arise through the accumulation of multiple genetic and epigenetic alterations.2 Therefore, advances in our understanding of these molecular alterations of CRC enable us to predict outcomes, personalize medicine and improve the care of CRC patients. Over the past years, meticulous morphologic and molecular features of CRC have led to the development of three major pathways including chromosomal instability (CIN), microsatellite instability (MSI), and cytosine-phospho-guanine (CpG) island methylator phenotype (CIMP).3,4 Most CRCs develop from conventional adenoma through the traditional adenoma-carcinoma pathway characterized by CIN involving KRAS, C-MYC, APC, and p53 mutations.3,4 About 15% to 20% of CRCs develop through the MSI arising from defects in the DNA mismatch repair (MMR) system to correct errors that often occur during DNA replication, and associated with Lynch syndrome. MSI is controlled by MMR genes including MLH1, MSH2, MSH6, and PMS2.3,4 CIMP, characterized by the hypermethylation of CpG islands has been identified as an important third major pathway, which accounts for almost 40% of CRCs. Especially, hypermethylation of CpG islands located in the promoter region leads to transcriptional silencing of tumor suppressor genes and other tumor-related genes.4–6 Therefore, CIMP status can influence the prognosis of CRC patients. Previous studies investigated clinicopathological and molecular features of CRC according to the CIMP status, and focused on the association between CIMP status and CRC prognosis. CIMP-high CRCs present with distinct clinicopathological and molecular features such as older age, female gender, proximal tumor location, poor differentiation, and high rate of MSI, KRAS and BRAF mutation, compared to CIMP-low/negative CRCs.5–7 However, the association between CIMP status and CRC prognosis are inconsistent in previous published reports.5–7 In this issue of Gut and Liver, Kang et al.8, investigated the MSI and CIMP status in Korean colon cancer patients except many rectal cancer patients, and examined their correlation with clinicopathological features including survival. The authors showed that regardless of the MSI status, CIMP-high colon cancer was associated with a poor survival outcome, and colon cancer with CIMP-high/MSI-negative in subgroup analysis showed a poor survival outcome compared to CIMP-low and negative/MSI-negative colon cancers. However, as the authors described, this study population was relatively small and had low incidence of MSI-high or CIMP-high cancers, which might influence some results, especially the association of CIMP status for CRC prognosis.8 Other Korean studies also showed that CIMP-high CRCs were significantly associated with female gender, proximal tumor location, poor differentiation, nodal metastasis, more advanced cancer, BRAF mutations, MSI, and poor prognosis.9,10 According to the combination of CIMP and MSI status, the CIMP-high/MSI-negative subtype showed the worst clinical outcome due to BRAF mutation.9,10 The clinicopathological and molecular features of CIMP-high CRCs in Korean studies8–10 were similar with those of previous published reports.5–7 However, although CIMP-high CRC patients were related to poor prognosis in many studies including Korean studies, other studies reported no association between CIMP-high CRC patient and prognosis or even noted a better survival benefit from adjuvant chemotherapy.5–7 There are several explanations for this discrepancy. First, results are inconsistent, perhaps due to variable factors including MSI, KRAS and BRAF mutations, variations in use of adjuvant chemotherapy, the ambiguous pathophysiolgy of CIMP, methodologic differences such as differences in the selected methylation markers and definitions of CIMP.5,6 Actually, specific methylated loci using for definition of CIMP and prevalence of CIMP status are different among studies. It is the major concern in studying CIMP status in variable cancers. Second, CIMP is an event that predominantly occurs in early stage of carcinogenesis and may less involve to cancer progression. Therefore, although much effort has been made to identify the impact of CIMP status as prognostic biomarkers in clinical practice, it still cannot be concluded whether association between CIMP status and CRC prognosis exist. In future, large-scale well-designed studies should be considered in order to clarify the impact of CIMP on the prognostic significance in CRC patients.

Cancer morbidity and mortality can be dramatically reduced by colonoscopic screening of individuals with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome, creating a need to identify HNPCC. We studied how HNPCC identification should be carried out on a large scale in a sensitive and efficient manner. Colorectal cancer specimens from consecutive newly diagnosed patients were studied for microsatellite instability (MSI). Germline mutations in the MLH1 and MSH2 genes were searched for in MSI(+) individuals. Among 535 colorectal cancer patients, 66 (12%) were MSI(+). Among these, 18 (3.4% of the total) had disease-causing germline mutations in MLH1 or MSH2. Among these 18 patients, five were less than 50 years old, seven had a previous or synchronous colorectal or endometrial cancer, and 15 had at least one first-degree relative with colorectal or endometrial cancer. Notably, 17 (94%) of 18 patients had at least one of these three features, which were present in 22% of all 535 patients. Combining these data with a previous study of 509 patients, mutation-positive HNPCC accounts for 28 (2.7%) of 1,044 cases of colorectal cancer, predicting a greater than one in 740 incidence of mutation-positive individuals in this population. Large-scale molecular screening for HNPCC can be done by the described two-stage procedure of MSI determination followed by mutation analysis. Efficiency can be greatly improved by using three high-risk features to select 22% of all patients for MSI analysis, whereby only 6% need to have mutation analysis. Sensitivity is only slightly impaired by this procedure.

TPS808 Background: The current analysis for the determination of Microsatellite Instability (MSI) status in tumor requires matched normal DNA as a reference. The Promega MSI panel included five monomorphic markers (NR-21, BAT-26, BAT-25, NR-24, and MONO-27) which were known to have few variant alleles in Caucasian as well as Japanese. Therefore, these markers might be able to determine the MSI status without matched normal DNA, although it is conceivable that Quasi-Monomorphic Variation Range (QMVR) in each ethnic group could be different. In our pilot study, the QMVR in 149 Japanese healthy individuals were 98.4-104.4 base pair (bp) in NR-21, 111.4-117.4 bp in BAT-26, 121.0-127.0 bp in BAT-25, 129.5-135.5 bp in NR-24, and 149.9-155.9 bp in MONO-27, respectively. Methods: This clinical evaluation study is to establish the QMVR in Japanese colorectal cancer (CRC) patients (pts) and to evaluate the clinical utility of the QMVR on the determination of MSI status without matched normal DNA. The primary endpoint is the concordance of MSI status between the standard method using DNA from tumor plus matched normal samples and testing method using DNA from only tumor samples. The new MSI kits including the Promega MSI panel were manufactured under the Quality Management System (QMS) for in vitro diagnostics (IVDs). As the decision algorithm, tumors exhibiting 2 or more markers outside the QMVR were classified as MSI-High, cases with 1 marker outside the QMVR were classified as MSI-Low, and those without any marker outside the QMVRs were classified as microsatellite stable (MSS). Planned enrollment will be 400 pts. Clinical trial information: UMIN000024144.

Performance of Next-Generation Sequencing for the Detection of Microsatellite Instability in Colorectal Cancer With Deficient DNA Mismatch Repair

Microsatellite instability (MSI) implies the deletion of mismatch repair genes caused by DNA methylation or gene mutation. MSI is a good predictor for efficacy of 5-fluorouracil (FU)-based chemotherapy in the treatment of colorectal cancer. Some gastric cancer studies have reported that MSI has no apparent impact on prognosis after patients receive 5-FU-based adjuvant chemotherapy. However, other studies suggest that high-frequency MSI (MSI-H) status reduced survival in patients receiving 5-FU-based adjuvant chemotherapy. Thus, the correlation between MSI status and efficacy of 5-FU-based adjuvant chemotherapy for gastric cancer remains controversial. We performed a PubMed, Embase, and Cochrane search to retrieve studies that explore the correlation between MSI status and 5-FU-based adjuvant chemotherapy efficacy in gastric cancer. After extracting 65 potentially eligible studies, four were ultimately included in this meta-analysis using Stata software (ver. 12.0). For each study, we estimated the hazard ratio (HR) value for overall survival (OS), and HR was extracted per the survival curve in the studies. Heterogeneity was estimated using the random-effects model. Overall, 1174 patients after operation were included: 84 patients were classed as MSI-H and 1090 as microsatellite stable (MSS)/low-frequency MSI (MSI-L). For the four studies, the overall estimate of HR for OS between MSI-H and MSS/MSI-L groups was 1.90 (95% confidence interval: 0.91-3.93; p = 0.08). We found no correlation to exist between MSI status and efficacy of 5-FU-based adjuvant chemotherapy for gastric cancer. Although MSI can effectively predict efficacy of 5-FU-based chemotherapy in patients with colorectal cancer, the correlation between MSI status and efficacy of 5-FU-based adjuvant chemotherapy for gastric cancer remains controversial. This meta-analysis suggests that MSI status is unrelated to efficacy of 5-FU-based adjuvant chemotherapy in gastric cancer, and more prospective clinical studies are needed to further investigate predictive value of MSI status in patients with gastric cancer who, after operation, receive 5-FU-based adjuvant chemotherapy.

ObjectiveTo investigate the relationship among 18 heavy metals, microsatellite instability (MSI) status, ERCC1, XRCC1 (rs25487), BRAF V600E and 5 tumor markers and their role in the development of colorectal cancer (CRC).MethodsA total of 101 CRC patients and 60 healthy controls were recruited in the present study. The levels of 18 heavy metals were measured by ICP-MS. MSI status and the genetic polymorphism were determined by PCR (FP205-02, Tiangen Biochemical Technology Co., Ltd., Beijing, China) and Sanger sequencing. Spearman’s rank correlation was used to analyze the relationship among various factors.ResultsThe level of selenium (Se) was lower in the CRC group compared with the control group (p < 0.01), while vanadium (V), arsenic (As), tin (Sn), barium (Ba) and lead (Pb) were higher (p < 0.05), chromium (Cr) and copper (Cu) were significantly higher (p < 0.0001) in the CRC group than those in the control group. Multivariate logistic regression analysis indicated that Cr, Cu, As and Ba were the risk factors for CRC. In addition, CRC was positively correlated with V, Cr, Cu, As, Sn, Ba and Pb, but negatively correlated with Se. MSI was positively correlated with BRAF V600E, but negatively correlated with ERCC1. BRAF V600E was positively correlated with antimony (Sb), thallium (Tl), CA19-9, NSE, AFP and CK19. XRCC1 (rs25487) was found to be positively correlated with Se but negatively correlated with Co. The levels of Sb and Tl were significantly higher in the BRAF V600E positive group compared to the negative group. The mRNA expression level of ERCC1 was significantly higher (P = 0.035) in MSS compared to MSI. And there was a significant correlation between XRCC1 (rs25487) polymorphism and MSI status (P<0.05).ConclusionThe results showed that low level of Se and high levels of V, As, Sn, Ba, Pb, Cr, and Cu increased the risk of CRC. Sb and Tl may cause BRAF V600E mutations, leading to MSI. XRCC1 (rs25487) was positively correlated with Se but negatively correlated with Co. The expression of ERCC1 may be related to MSS, while the XRCC1 (rs25487) polymorphism is related to MSI.

Purpose: The most frequent cause of hereditary colorectal cancer (CRC) is Lynch syndrome, accounting for 3% of all CRC cases. The underlying germline predisposition is a mutation in any of the four mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS2), while microsatellite instability (MSI) is a hallmark of these tumors. However, CRC cases with MSI, no MLH1 somatic hypermethylation and absence of MMR germline mutation account for up to 60% of cases of MMR-defective tumors. They have been termed Lynch-like syndrome (LLS) and treatment management decisions are complicated due to unconfirmed suspicions of hereditary cancer. The aim of the present study was to investigate whether LLS cases in patients under the age of 40 carry potentially pathogenic germline variants in new genes for CRC predisposition causing a MMR-defective tumor phenotype. Methods: We performed whole-exome sequencing (WES) in the germline and tumoral DNA of 16 early-onset LLS patients. We filtered for recessive germline variants in genes involved in DNA repair. An exhaustive functional evaluation for 2 MCM8 genetic variants detected in one patient was performed. The CRISPR/Cas9 system was used to generate MCM8ko clones in a cell model (DLD-1). Site-directed mutagenesis produced the genetic variants for ectopic expression in the MCM8ko model. Functional characterization included the comet assay, which detected DNA damage induced by oxaliplatin in the transfected cells. MCM8ko cells were evaluated for MSI after 30, 60 and 90 days of culture, and mutational signatures were assessed with SigProfiler. Results: The MCM8 variants c.351_354delAAAG (p.Lys118GlufsTer5, p.K118fs) and c.414A&amp;gt;G (p.Ile138Met, p.I138M) were identified in one early-onset LLS patient with biallelic somatic inactivation of MLH1. Manual evaluation of WES data showed that germline MCM8 variants were in trans. In the comet assay, both MCM8 variants revealed higher sensitivity to oxaliplatin and more DNA damage compared to the wild-type control. One of the MCM8KO clones showed MSI after 30 days of sub-culturing. After 120 days, MCM8KO clones had acquired a significant contribution of the SBS20 mutational signature, which is associated with defective MMR DNA. Conclusions: We postulate that MCM8 is a new germline CRC predisposing gene in early-onset LLS, which may be involved in both MMR and double-strand break repair. We further provide evidence that in some LLS CRC cases, especially in young patients, the biallelic somatic MMR inactivation may be caused by new CRC germline predisposing genes. Additional mutational screening is warranted for such cases. Citation Format: Mariano Golubicki, Laia Bonjoch, José G. Acuña-Ochoa, Marcos Diaz-Gay, Jennifer Muñoz, Miriam Cuatrecasas, Teresa Ocaña, Juan Robbio, Enrique L. Roca, Antoni Castells, Fracesc Balaguer, Marina Antelo, Sergi Castellvi-Bel. Germline biallelic mutations in MCM8 are associated with early-onset Lynch-like syndrome [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 2366.

In addition to chromosomal instability and microsatellite instability (MSI), a third pathway, epigenetic instability, has been implicated in progression to colorectal carcinogenesis. CpG island methylator phenotype (CIMP) refers to a subset of colorectal cancers (CRCs) that occur through the epigenetic instability pathway and that are characterized by widespread hypermethylation of promoter CpG island loci, resulting in the inactivation of several tumor suppressor genes or tumor-related genes. Colorectal cancers can be classified into 4 molecular subtypes according to their CIMP and MSI statuses: CIMP+/MSI+, CIMP+/MSI-, CIMP-/MSI+, and CIMP-/MSI-. There are differences between Western (United States and European Union) and Eastern (Korea and China) populations in the number of CRCs that are MSI+, and in the number of MSI+ CRCs that are CIMP+. To review the clinicopathologic and molecular features of the 4 molecular subtypes of CRCs and their precursor lesions, and to emphasize geographic differences in CRCs between Eastern and Western populations. This article is based on the author's own experimental data and a literature review of relevant articles indexed in PubMed (US National Library of Medicine). The 4 molecular subtypes of CRC that are defined by their CIMP and MSI statuses are characterized by their own distinct clinicopathologic and molecular features and precursor lesions. In particular, the clinicopathologic features of MSI+ CRCs differ depending on the CIMP status. Further understanding of the heterogeneity in CRC molecular pathways may help to explain the diverse morphologic features of CRCs.

Objective: Fusobacterium nucleatum (F. nucleatum) is a microbial pathogen that contributes to the initiation and progression of colorectal cancer. Although evidence points to its immunosuppressive effects in colorectal cancer, F. nucleatum has been associated with high-level microsatellite instability (MSI) which is characterized by intense immune response. We hypothesized that the association of F. nucleatum with local immunosuppression might be stronger in tumors with high-level MSI or a high neoantigen load, which is a direct consequence of MSI that influences tumor-immune interactions. Design: We utilized molecular pathological epidemiology database of 1,027 colon and rectal cancer cases in the Nurses’ Health Study and the Health Professionals Follow-up Study. We assessed the association of F. nucleatum (negative, low, or high) with tumor immunity status in strata of MSl status. We measured F. nucleatum DNA in tumor tissue using quantitative PCR. As immunity status, we evaluated hystopathological lymphocytic reaction (tumor-infiltrating lymphocytes [TIL], intratumoral periglandular reaction, peritumoral lymphocytic reaction, or Crohn's-like lymphoid reaction) and measured T cell densities (CD3+, CD8+, CD45RO [PTPRC]+ or FOXP3+ cells) using immunohistochemistry and computer-assisted image analysis. Logistic regression model was used to adjust for potential confounders, including CpG island methylator phenotype, LINE-1 methylation level, and KRAS, BRAF, and PIK3CA mutations. Using whole-exome sequencing data (N = 586), we conducted secondary analyses to assess the interaction of F. nucleatum with a neoantigen load. Results: The association between F. nucleatum and TIL differed by MSI status (P for interaction = 0.0017; with the adjusted α level of 0.006). Compared with F. nucleaum-negative cases, F. nucleatum-positive cases were associated with low-level TIL in MSI-high tumors (odds ratio, 0.40 [95% confidence interval, 0.14-1.08] and 0.40 [95% confidence interval, 0.17-0.97] for F. nucleaum low and high cases, respectively), but not in microsatellite stable/MSI-low tumors. We did not find any interaction between F. nucleatum and MSI status in T cell subsets. The association between F. nucleatum and TIL did not differ significantly by levels of a neoantigen load (P for interaction = 0.015). Conclusions: The association of F. nucleatum with the immunosuppressive microenvironment may be stronger in colorectal cancer with high-level MSI. Our findings suggest the interplay between F. nucleatum and MSI status in host immune response to the tumor. Citation Format: Kosuke Mima, Tsuyoshi Hamada, Reiko Nishihara, Jonathan Nowak, Zhi Rong Qian, Keisuke Kosumi, Marios Giannakis, Susan Bullman, Hideo Baba, Curtis Huttenhower, Wendy Garrett, Andrew Chan, Matthew Meyerson, Charles Fuchs, Shuji Ogino. Fusobacterium nucleatum and tumor immunity status according to microsatellite instability in colorectal cancer [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 4560. doi:10.1158/1538-7445.AM2017-4560