MLH1 Research Articles

Quantitative analysis of MMR deficiency in dMMR/MSI high CRC and levels of instability: Implications for ICI therapy of dMMR tumors.

e14650 Background: Mismatch repair deficiency (dMMR) is observed in ~15% of newly diagnosed colorectal cancer (CRC) tumors. It is routinely scored as a qualitative trait (positive or negative) and, if present, is considered a positive predictive marker for immune checkpoint inhibitors (ICI) therapy not only in patients with metastatic CRC (mCRC), but also in patients with other dMMR solid tumors. However, a significant proportion of these patients experience failure of ICI treatment and no predictive biomarkers exist that can identify such patients. Methods: A total of 42 patients with dMMR tumors were selected for analysis, of which 26 had a hereditary Lynch Syndrome (LS) with known pathogenic variants in the MMR genes (MLH1, MSH2, MSH6, PMS2) and 16 had sporadic dMMR CRC due to epigenetic silencing of the MLH1 gene. All tumors were also analyzed for the presence of the RAS/RAF pathway mutations. MMR deficiency was initially evaluated by two standard methods: immunohistochemistry (IHC) and by microsatellite instability (MSI)-PCR on DNA isolated from FFPE tissues with > 50% tumor content. Subsequently, whole exome sequencing (WES) with a mean depth of >150 reads was performed on the same DNA samples using the NovaSeq 6000 platform. NGS data were analyzed using the MSI sensor package which evaluates >2000 sites with mono- to pentanucleotide repeats. Each site was scored as NGS-MSI unstable if it contained at least 20% unstable reads. The percent of unstable loci was referred to as NGS-MSI level. All tumors with >3.5% of unstable loci were classified as NGS-MSI positive. Results: A considerable variability in the NGS-MSI levels ranging from 1.5 – 76% was observed in patients that were initially classified as dMMR/PCR-MSI high. CRC tumors of LS patients with deleterious MMR gene mutations and the majority of patients with sporadic CRC due to MLH1 gene promotor methylation had >50% unstable loci. In contrast, more than 1/3 of patients with isolated loss of MSH6 and PMS2 detected by IHC had substantially lower NGS-MSI levels of 1.5 to 12%. These patients had a negative PCR-MSI result and their molecular defects were primarily missense mutations in MSH6 and PMS2 genes. It is interesting to note that a low NGS-MSI score (<10%) was also detected in patients with sporadic CRC that had MLH1 gene promotor methylation but also the BRAF V600E mutation. Conclusions: Our results indicate that dMMR/MSI-H tumors exhibit a high variability in the percentage of unstable loci that correlates with particular molecular backgrounds. A larger study on different types of tumors is warranted, especially in the context of the efficacy of ICI therapy in patients with MMR tumors classified by qualitative MMR/MSI assays.

Molecular and immune profiling of TP53-mutated ovarian cancers with non-BRCA1/2 homologous recombination gene alterations.

5579 Background: Molecular alterations in homologous recombination (HR) pathway genes lead to HR deficiency (HRD) in ~50% of high grade serous ovarian carcinomas (HGSOC). The immune microenvironment of BRCA1/2-mutated HGSOC has been extensively characterized, in contrast to tumors with mutations in other HR genes. We evaluated a cohort of epithelial ovarian cancers (EOC) enriched for HGSOC and compared molecular and immune signatures of tumors with BRCA1/2 mutations ( BRCAm) to those with non-BRCA1/2 HR gene mutations (NBHRD). Methods: We characterized 173 EOC by comprehensive genomic and immune profiling (CGIP) using panel-based next-generation DNA (523 genes) and RNA sequencing (384 genes), respectively. We enriched for HGSOC by selecting for TP53 mutations and excluding tumors with non-serous histology or any mutation in POLE, MLH1, PMS2, MSH2, MSH6, or nucleotide excision repair genes. Tumors lacking BRCA1/2-mutations were classified as NBHRD if a reportable mutation was present in canonical HR genes. Mutational burden (TMB, mut/Mb) was estimated from detected SNV/indels. We calculated immune-related expression signatures, including tumor immunogenicity score (TIGS), cellular proliferation (CP), and cancer testis antigen burden (CTAB). PD-L1 expression was assessed by tumor proportion score (TPS) following 22C3 immunohistochemistry. Differences in TMB, TIGS, CP, CTAB, PD-L1 and gene expression rank were tested for using Wilcoxon rank-sum test. Results: Of 173 EOC, 84 cases had complete CGIP, including 11 (13%) BRCAm, 7 (8%) NBHRD (Table), and 66 (79%) lacking HR gene alterations. On average, NBHRD tumors harbored lower TMB (4.1±2.3 vs. 6.6±2.3 vs. p=0.03) and elevated TIGS (59.1±16.2 vs. 41.7±16.2 p=0.044) compared to BRCAm tumors. We found no difference in PD-L1 (p=0.21), CP (p=0.26), or CTAB (p=0.48). Expression changes were identified in 32 immune genes, of which 31 (97%) were substantially up-regulated (1.3-3.3 fold) in NBHRD compared to BRCAm tumors. Conclusions: Our findings showed our cohort of NBHRD tumors were more immunogenic but less mutagenic than BRCAm tumors, implying enhanced immunogenicity unrelated to neoantigen load. Given that immune checkpoint inhibitors have shown limited efficacy in ovarian cancer, our data suggest NBHRD tumors may have stronger and possibly distinct immune escape mechanisms compared to BRCAm tumors. [Table: see text]

Prevalence and clinical correlations of Lynch syndrome (LS) in northeast Brazil.

e22547 Background: LS is an autosomal dominant inherited condition with high risk for colorectal (CRC) and endometrial cancer, mostly. The LS is determined by a deficient Mismacht Repair (dMMR) system following germline mutations of MSH2, MLH1, MSH6, PMS2 and EPCAM genes. Identification of LS patients promotes adequate prevention and possible reduction of mortality for these population. The present study objetives to determine the frequencies of LS in a mixed race population from Northeast of Brazil, usually not represented in literature and characterize its genotype-fenotype feature. Methods: Our study prospectively screened 150 cancer patients (mostly CRC) with risk for LS according to NCCN guideline v2020 and performed germline mutational testing with a 33-multigene panel by NGS. Results: Median age was 48years and mainly female gender (63,3%). CRC was the most frequent tumor (n = 121, 80.7%), followed by breast cancer (11.3%), endometrium cancer (9.3%) and others (3,3% each). CCR patients presented MMR deficiency (dMMR) detected by IHC in a frequency of 29.3%. It was identified 32.7% of PV or PPV (according to ACMG criteria) with 20.7% for LS genes (mMMR). Two patients had 2 genes mutated. For CRC patients there was association of stage I-II tumors (p = 0,009) and right location (p < 0,001) with mutational status. There is no significant correlation of mutational status and other clinical caractheristics. MSH2 was the most frequently mutated (11) followed by MSH6 (10), heterozygous MUTYH (5), homozygous MUTHY (3), PMS2 (7), APC (4) and MLH1 (3) and others. MSH2 most frequent variants were c.388 389del (4) and c.2581C > T (2). Of the NCCN criteria for LS, personal history of CRC or endometrial cancer under 50 years of age was the most prevalent (67.3%) followed by tumoral dMMR (37.3%). dMMR tumor was the criteria with the best accuracy (sensibility 80.0 – 100.0% and specificity 76.6 – 93.5%) for LS detection. Patients with more than three criteria presented high sensibility and specificity values (80.6 and 90.3%, respectively). There were more than 60% of variants of uncertain significance variants (VUS). Conclusions: Our study detected a lower frequency of PV of LS genes (20,7%) than others even from Brazil, however it differs from these on criteria for LS (more inclusive), sequencing method (NGS multigene painel) and the population (derived from mixed caucasians, africans and native Indians). LS genotype correlates with CRC stages I-II and from right colon location. The most frequent mutated gene was MSH2 (c.388 389del and c.2581C > T). MLH1 was the less frequent mutated gene in our cohort.

Ancestry-adjusted genomic analysis to identify known and novel associations with single and multiple cancer phenotypes.

10504 Background: Germline cancer risks are impacted by cancer type, stage, specific mutation, and ancestry. This study aims to determine the magnitude of genetic risks for commonly tested genes across patients with single and multiple primary cancers (MPC). Methods: We conducted a partially de-identified, retrospective cohort study of all patients consented to using the MSK-IMPACT, a matched tumor-normal sequencing assay, over a five-year period. Approximately 83% of IMPACT testing is derived from patients with metastatic disease. We collected data on demographics and clinical cancer features through cBioPortal and institutional databases. MPC were classified based on the IARC guidelines. Clinically actionable, pathogenic variants in cases and gnomAD non-cancer controls were identified using PathoMAN. Statistical methods incorporated ancestry adjusted, weighted generalized linear model, and ancestry specific methods to estimate risk. Biallelic losses were analyzed from paired tumors. Results: We analyzed 28,000 individuals across 35 cancer types, where germline variants were discovered within 100 cancer predisposition genes. 16% carried pathogenic, clinically actionable variants in known and novel cancer predisposition genes (15% in those with single primary and 21% in MPC). We discovered statistically significant novel associations of germline variants with several cancer phenotypes, including BRCA2 and biliary cancer (OR = 4.5, CI = 2.2-8); ATM and BRCA2 with mesothelioma (OR = 4.8, CI = 2.1-9.2; OR = 8, CI = 3.1-16.6, respectively); NF1 and breast cancer (OR = 5.8, CI = 2.4-12.1); NBN and lung cancer (OR = 4.2, CI = 2.3-6.9), bladder with ATR and others. We replicated known associations and discovered pleiotropy of BRCA1/2, NF1, ATM, TP53 with several cancer types. We failed to replicate reported associations such as PALB2 and ovarian cancer and CHEK2 and colon cancer. Amongst cancer pairs of multiple primaries, we observed mismatch repair genes MSH2, MSH6 and MLH1 to be associated with increased risks for stomach, bowel, and uterine cancer pairs, and TP53 in breast MPC compared to breast single primaries. Conclusions: An ancestry adjusted, weighted analysis of a large real-world case-cohort enriched with metastatic disease did not confirm some prior genotype-phenotype correlations, while suggesting novel associations with hereditary cancer types.

The impact of cause of mismatch repair deficiency and other molecular markers on clinical outcomes with the use of durvalumab in advanced endometrial cancer in the phase 2 PHAEDRA trial (ANZGOG1601).

5604 Background: In the single arm phase 2 PHAEDRA trial, MMR deficiency (dMMR) was predictive of response to durvalumab (1500mg IV Q4W), with an objective tumor response rate (OTR; defined by iRECIST) of 47% in dMMR compared with 3% in MMR-proficient (pMMR) advanced endometrial cancer (AEC). This substudy of the PHAEDRA trial investigates MMR molecular subtypes and other genomic tumor features and their correlation with treatment outcomes. Methods: Testing was performed to determine molecular subtypes of dMMR, including germline pathogenic variants in DNA MMR genes (Lynch syndrome), somatic biallelic MMR gene inactivation through somatic mutation and somatic hypermethylation of the MLH1 gene promoter. DNA from formalin-fixed paraffin-embedded tumor tissue and matched peripheral blood was available from 41/71 (25 dMMR, 16 pMMR) participants for testing on a custom capture-based 298-gene targeted panel (2.005Mb) including the MMR genes and key somatic AEC driver genes. The derived tumor genomic features included microsatellite instability (MSI), tumor mutational burden (TMB), COSMIC v.3.2 tumor mutational signatures and insertion/deletion (Indel) somatic mutation count. Results: Of the 71 patients recruited, 35 were dMMR and 36 were pMMR. Median follow-up was 44 vs 52 months in dMMR vs pMMR participants, respectively. The dMMR molecular subtypes were 4 (11.4%) Lynch syndrome, 4 (11.4%) somatic MMR mutation, 25 (71.4%) MLH1 methylated and 2 (5.7%) dMMR-uncategorised. The OTR rate was 100% (4/4; 95%CI 40-100%) for Lynch, 75% (3/4; 95%CI 22-99%) for somatic MMR mutations and 40% (10/25; 95%CI 22-61%) for MLH1 methylated groups. The median TMB (assessed in 41/71) was higher in those with a confirmed radiological response (37, IQR:26-50) vs non-responders (16, IQR:9-25; p < 0.001). Within the MLH1 methylated group, TMB was also higher in responders vs non-responders (40 v 21; p = 0.03). Somatic mutations in KRAS, PTEN, PIK3CA, ARID1A and TP53 were not associated with OTR rate. Conclusions: dMMR- MLH1 methylated AEC demonstrated greater heterogeneity in OTR to single agent durvalumab than the dMMR-Lynch and dMMR-somatic MMR mutation molecular subtypes. Higher TMB was seen in responders, and specifically within dMMR- MLH1 methylated responders, compared to non-responders. Clinical trial information: ANZGOG1601.

Metabolomics Analysis as a Tool to Measure Cobalt Neurotoxicity: An In Vitro Validation.

In this study, cobalt neurotoxicity was investigated in human astrocytoma and neuroblastoma (SH-SY5Y) cells using proliferation assays coupled with LC-MS-based metabolomics and transcriptomics techniques. Cells were treated with a range of cobalt concentrations between 0 and 200 µM. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed cobalt cytotoxicity and decreased cell metabolism in a dose and time-dependent manner was observed by metabolomics analysis, in both cell lines. Metabolomic analysis also revealed several altered metabolites particularly those related to DNA deamination and methylation pathways. One of the increased metabolites was uracil which can be generated from DNA deamination or fragmentation of RNA. To investigate the origin of uracil, genomic DNA was isolated and analyzed by LC-MS. Interestingly, the source of uracil, which is uridine, increased significantly in the DNA of both cell lines. Additionally, the results of the qRT-PCR showed an increase in the expression of five genes Mlh1, Sirt2, MeCP2, UNG, and TDG in both cell lines. These genes are related to DNA strand breakage, hypoxia, methylation, and base excision repair. Overall, metabolomic analysis helped reveal the changes induced by cobalt in human neuronal-derived cell lines. These findings could unravel the effect of cobalt on the human brain.

A conserved motif in the disordered linker of human MLH1 is vital for DNA mismatch repair and its function is diminished by a cancer family mutation.

DNA mismatch repair (MMR) is essential for correction of DNA replication errors. Germline mutations of the human MMR gene MLH1 are the major cause of Lynch syndrome, a heritable cancer predisposition. In the MLH1 protein, a non-conserved, intrinsically disordered region connects two conserved, catalytically active structured domains of MLH1. This region has as yet been regarded as a flexible spacer, and missense alterations in this region have been considered non-pathogenic. However, we have identified and investigated a small motif (ConMot) in this linker which is conserved in eukaryotes. Deletion of the ConMot or scrambling of the motif abolished mismatch repair activity. A mutation from a cancer family within the motif (p.Arg385Pro) also inactivated MMR, suggesting that ConMot alterations can be causative for Lynch syndrome. Intriguingly, the mismatch repair defect of the ConMot variants could be restored by addition of a ConMot peptide containing the deleted sequence. This is the first instance of a DNA mismatch repair defect conferred by a mutation that can be overcome by addition of a small molecule. Based on the experimental data and AlphaFold2 predictions, we suggest that the ConMot may bind close to the C-terminal MLH1-PMS2 endonuclease and modulate its activation during the MMR process.

Genetic mutations in HER2-positive breast cancer: possible association with response to trastuzumab therapy

BackgroundHER2-positive breast cancer occurs in 15–20% of breast cancer patients and is characterized by poor prognosis. Trastuzumab is considered the key drug for treatment of HER2-positive breast cancer patients. It improves patient survival; however, resistance to trastuzumab remains a challenge in HER2-positive breast cancer patients. Therefore, the prediction of response to trastuzumab is crucial to choose optimal treatment regimens. The aim of the study was to identify genetic variants that could predict response to anti-HER2-targeted therapy (trastuzumab) using next-generation sequencing.MethodGenetic variants in the hotspot regions of 17 genes were studied in 24 Formalin-Fixed Paraffin-Embedded (FFPE) samples using Ion S5 next-generation sequencing system. FFPE samples were collected from HER2‑positive breast cancer patients previously treated with anti‑HER2‑targeted treatment (Trastuzumab). Patients were divided into two groups; trastuzumab-sensitive group and trastuzumab-resistant group based on their response to targeted therapy.ResultsWe identified 29 genetic variants in nine genes that only occurred in trastuzumab-resistant patients and could be associated with resistance to targeted therapy including TP53, ATM, RB1, MLH1, SMARCB1, SMO, GNAS, CDH1, and VHL. Four variants out of these 29 variants were repeated in more than one patient; two variants in TP53, one variant in ATM gene, and the last variant in RB1 gene. In addition, three genes were found to be mutated only in resistant patients; MLH1, SMARCB1 and SMO genes. Moreover, one novel allele (c.407A > G, p. Gln136Arg) was detected within exon 4 of TP53 gene in one resistant patient.ConclusionNGS sequencing is a useful tool to detect genetic variants that could predict response to trastuzumab therapy.

Identification of Genomic Alterations in Thai Patients With Colorectal Cancer Using Next-Generation Sequencing-Based Multigene Cancer Panel.

Introduction Colorectal cancer (CRC) is one of the leading causes of death and illness in the general population. Although the incidence of CRC is steadily decreasing worldwide, it is being diagnosed more in individuals under 50 years of age. Multiple disease-causing variants have been reported to be involved in the development of CRC. This study aimed to investigate the molecular and clinical characteristics of Thai patients with CRC. Methods NGS-based multigene cancer panel testing was performed on 21 unrelated patients. Target enrichment was performed using a custom-designed Ion AmpliSeq on-demand panel. Thirty-six genes associated with CRC and other cancer were analyzed for variant detection. Results Sixteen variants (five nonsense, eight missense, two deletions, and one duplication) in nine genes were identified in 12 patients. Eight (66.7%) patients harboring disease-causing deleterious variants in genes APC, ATM, BRCA2, MSH2, and MUTYH. One of the eight patients also carried additional heterozygous variants in genes ATM, BMPR1A, and MUTYH. In addition, four patients carried variants of uncertain significance in genes APC, MLH1, MSH2, STK11, and TP53. Among all detected genes, APC was the most frequent causative gene observed in CRC patients, which is consistent with previous reports. Conclusion This study demonstrated the comprehensive molecular and clinical characterization of CRC patients. These findings showed the benefits of using multigene cancer panel sequencing for pathogenic gene detection and showed the prevalence of genetic aberrations in Thai patients with CRC.

AB039. SOH23ABS_198. MLH1-deficient colorectal carcinoma with wild type BRAF and MLH1 promoter hypermethylation—an understudied molecular phenotype

Background: Up to 15% of colorectal cancers (CRCs) display microsatellite instability (MSI). MSI is reflective of a deficient mismatch repair (MMR) system (dMMR). dMMR CRC represents a heterogeneous group of diseases with separate tumorigenic pathways. The majority of dMMR CRC is sporadic (>90%) and occurs due to acquired methylation of the MLH1 gene promoter. Sporadic MLH1-deficient CRC is thought to originate in lesions that arise from the serrated neoplasia pathway. Although the presence of the BRAF V600E mutation is characteristic of a sporadic cancer, as many as half of CRCs with MLH1 promoter hypermethylation will lack a BRAF mutation.

Spontaneous regression of advanced transverse colon cancer with deficient mismatch repair: a case report

BackgroundSpontaneous regression (SR) of cancer occurs in 1 in 60,000–100,000 patients. This phenomenon has been reported in almost all cancer types, most commonly neuroblastoma, renal cell carcinoma, malignant melanoma, and lymphoma/leukemia. However, SR in colorectal cancer (CRC) is extremely rare, particularly in advanced cases. Hence, this report describes a very rare case of spontaneous regression of advanced transverse colon cancer.Case presentationA 76-year-old female with anemia was diagnosed with a type II well-differentiated adenocarcinoma in the middle transverse colon. Two months later, a second colonoscopy examination was performed for preoperative marking, and it revealed tumor shrinkage and a shift to type 0–IIc morphology. Endoscopic tattooing was then performed, followed by a laparoscopic partial resection of the transverse colon with D3 lymph node dissection. However, the resected specimen contained no tumor, and colonoscopy showed no tumor remnants in the remaining colon. Histopathological examination revealed mucosal regeneration and a mucus nodule in between the submucosal and muscular layers, with no cancer cells detected. Immunohistochemical analysis revealed the loss of MutL homolog 1 (MLH1) and postmeiotic segregation increased 2 (PMS2) expression in the cancer cells of biopsied specimens, suggesting deficient mismatch repair (dMMR). The patient continues to be followed up until 6 years postoperatively, and no recurrence has been observed. In this study, we also reviewed similar reported cases of spontaneous regression of cancer involving dMMR.ConclusionThis study presents a rare case of spontaneous regression of advanced transverse colon cancer wherein dMMR is strongly involved. However, further accumulation of similar cases is needed to elucidate this phenomenon and to develop new treatment strategies for CRC.

Lynch syndrome-associated chordoma with high tumor mutational burden and significant response to immune checkpoint inhibitors

Chordoma is a rare malignant bone tumor arising from notochordal tissue. Conventional treatments, such as radical resection and high-dose irradiation, frequently fail to control the tumor, resulting in recurrence and re-growth. In this study, genetic analysis of the tumor in a 72-year-old male patient with refractory conventional chordoma of the skull base revealed a high tumor mutational burden (TMB) and mutations in the MSH6 and MLH1 genes, which are found in Lynch syndrome. The patient and his family had a dense cancer history, and subsequent germline genetic testing revealed Lynch syndrome. This is the first report of a chordoma that has been genetically proven to be Lynch syndrome. Chordomas usually have low TMB; however, this is an unusual case, because the TMB was high, and immune checkpoint inhibitors effectively controlled the tumor. This case provides a basis for determining the indications for immunotherapy of chordoma based on the genetic analysis. Therefore, further extensive genetic analysis in the future will help to stratify the treatment of chordoma.

Predictive biomarkers of immunotherapy response with pharmacological applications in solid tumors.

Immune-checkpoint inhibitors show promising effects in the treatment of multiple tumor types. Biomarkers are biological indicators used to select patients for a systemic anticancer treatment, but there are only a few clinically useful biomarkers such as PD-L1 expression and tumor mutational burden, which can be used to predict immunotherapy response. In this study, we established a database consisting of both gene expression and clinical data to identify biomarkers of response to anti-PD-1, anti-PD-L1, and anti-CTLA-4 immunotherapies. A GEO screening was executed to identify datasets with simultaneously available clinical response and transcriptomic data regardless of cancer type. The screening was restricted to the studies involving administration of anti-PD-1 (nivolumab, pembrolizumab), anti-PD-L1 (atezolizumab, durvalumab) or anti-CTLA-4 (ipilimumab) agents. Receiver operating characteristic (ROC) analysis and Mann-Whitney test were executed across all genes to identify features related to therapy response. The database consisted of 1434 tumor tissue samples from 19 datasets with esophageal, gastric, head and neck, lung, and urothelial cancers, plus melanoma. The strongest druggable gene candidates linked to anti-PD-1 resistance were SPIN1 (AUC = 0.682, P = 9.1E-12), SRC (AUC = 0.667, P = 5.9E-10), SETD7 (AUC = 0.663, P = 1.0E-09), FGFR3 (AUC = 0.657, P = 3.7E-09), YAP1 (AUC = 0.655, P = 6.0E-09), TEAD3 (AUC = 0.649, P = 4.1E-08) and BCL2 (AUC = 0.634, P = 9.7E-08). In the anti-CTLA-4 treatment cohort, BLCAP (AUC = 0.735, P = 2.1E-06) was the most promising gene candidate. No therapeutically relevant target was found to be predictive in the anti-PD-L1 cohort. In the anti-PD-1 group, we were able to confirm the significant correlation with survival for the mismatch-repair genes MLH1 and MSH6. A web platform for further analysis and validation of new biomarker candidates was set up and available at https://www.rocplot.com/immune . In summary, a database and a web platform were established to investigate biomarkers of immunotherapy response in a large cohort of solid tumor samples. Our results could help to identify new patient cohorts eligible for immunotherapy.

Methylation study of tumor suppressor genes in human aberrant crypt foci, colorectal carcinomas, and normal colon.

Aberrant crypt foci (ACF) are the earliest preneoplastic lesions in human colon, identifiable on chromoendoscopic screening. Our objective was to evaluate the %methylation of APC, CDKN2A, MLH1, RASSF1, MGMT, and WIF1 tumor suppressor genes (TSG) in ACF, corresponding colorectal carcinomas (CRC), and normal colonic mucosal controls. In this study, macroscopically normal-appearing mucosal flaps were sampled 5-10 cm away from the tumor mass from 302 fresh colectomy specimens to identify ACF-like lesions. Thirty-five cases with multiple ACFs were selected (n 35) as the main study group, with corresponding sections from CRC (n 35) as disease controls, and mucosal tissue blocks from 20 colectomy specimens (normal controls), operated for non-neoplastic pathologies. Genomic DNA was extracted, and methylation-specific polymerase chain reaction (PCR) was performed on a customized methylation array model. %Methylation data were compared among the groups and with clinicopathological parameters. Selected target mRNA and protein expression studies were performed. %Methylation of TSGs in ACF was intermediate between normal colon and CRC, although a statistically significant difference was observed only for the WIF1 gene (P < 0.01). Also, there was increased nuclear β-catenin expression and upregulation of CD44-positive cancer-stem cells in ACF and CRCs than in controls. Right-sided ACFs and dysplastic ACFs had a higher %methylation of CDKN2A (P < 0.01), whereas hyperplastic ACFs had a higher %methylation of RASSF1 (P 0.04). The topographic characteristics of ACFs did not correlate with TSG %methylation. Early epigenetic methylation of WIF1 gene is one of the mechanisms for ACF development in human colon.

Abstract 5219: Telomere length distribution, metabolic trait association, and genome-wide association study among Singaporean cohorts

Abstract Leukocyte telomere length (LTL) is known as a heritable marker of cellular aging and is a risk factor for several chronic diseases. LTL distribution and association studies are essential in providing insights into telomere homeostasis, its relationship with different metabolic traits, and age-related conditions. Such studies have been underperformed in Asian population groups. Here we estimated LTL using whole-genome sequencing data in 8,045 Singaporean samples across three major ethnic groups - Chinese, Indian, and Malay divided into six cohorts: GUSTO, HELIOS, MEC, PRISM, SEED, and TTSH. LTLs were measured using TelSeq, with normalized LTL as phenotype in subsequent analyses. We demonstrated that average LTL was robustly longer in the childhood cohort (GUSTO) compared to the adult cohorts (P&amp;lt;1.19 × 10−195). We observed ethnicity differences in LTL where adult Singaporean Chinese samples had the longest LTL (5.08kb) as compared to Malay (4.87kb) and Indian (4.65kb) samples in our datasets (P&amp;lt;4.98 × 10−12). Our genetic association analyses replicated multiple variants previously reported to be associated with LTL (binomial P&amp;lt;0.00001) in Asian population groups, providing added confidence in the methodologies used. At the same time, we identified a novel East-Asian-specific novel variant beyond genome-wide association levels (meta P=2.05 × 10−08) at the COL28A1/MIOS gene loci to be associated with LTL in our study. Individual analysis of the GUSTO childhood dataset highlighted a second novel genome-wide hit at RAD17 gene locus to be associated with LTL in our study and indicated that the previously reported variant at the TINF2 locus may have significantly opposing effects (compared to adults) during early developmental stages. Gene-level burden analysis for rare variants (MAF &amp;lt;5%) in adult Chinese cohorts revealed potential cancer association with RET gene (SKAT, P=5.22 × 10−03) and MLH1 gene (SKAT, P=0.037), as well as telomere biology condition association with RECQL4 gene (CMC, P=0.0399, and VT, P=0.0379). Our findings have broadened the understanding of LTL distribution and the underlying associated genetic characteristics in both childhood and adult Singaporean populations. Citation Format: Trang Nguyen, Penny Chan, Ives Lim, Brian Kennedy, Neerja Karnani, Rajkumar Dorajoo, Joanne Ngeow. Telomere length distribution, metabolic trait association, and genome-wide association study among Singaporean cohorts. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5219.

Abstract 4177: Genetic testing for hereditary colorectal cancer syndromes in Algerian patients: A multicenter study

Abstract Background To date, 5% to 6 % of all colorectal cancers (CRCs) are associated with germline pathogenic variants in cancer predisposition genes that confer inherited predisposition to CRC. The use of genetic testing to identify individuals at risk for hereditary CRC syndromes can help to prevent the development of cancer and in the clinical management of the colorectal patients in the areas of both prevention and treatment. We report here the experience of our research laboratory of genetic testing for hereditary polyposis syndromes and Lynch syndrome (LS), respectively, in 126 patients. Methods Fifty four (54) severe familial adenomatous polyposis (FAP) patients and 72-suspected Lynch syndrome (LS) patients, respectively, were selected from 2851 consecutive colorectal patients at five public hospitals during 2012-2019. Family histories of cancer were obtained from interviews, pedigrees and medical records of patients. Index cases and relatives diagnosed with FAP syndrome or Lynch syndrome have been tested for germline variants in APC, MLH1, MSH2, MSH6 and PMS2 genes, respectively, using PCR-Sanger Sequencing or by NGS using a cancer panel of 30 hereditary cancer genes (Color Genomics). Results We detected 13 germline pathogenic variants in APC gene in 17 unrelated families, one germline pathogenic variant in BMPRA1 gene in juvenile polyposis syndrome (JPS) patient; seven (7) germline pathogenic variants and 2 variants of uncertain clinical significance (VUS) in MMR genes. Interestingly, 4 novel germline pathogenic variants in APC gene and 3 novel germline pathogenic variants in MMR genes, respectively, have been detected in our study. The most occurring germline pathogenic variants in APC gene were c.3927_3931del and c.4728dup that were identified in four and two index cases in 6 unrelated FAP families, respectively. In Lynch syndrome patients, the rare germline pathogenic variant MLH1 c.1546C&amp;gt;T has been found in 21 individuals from 9 LS families, 6 of them related, with two large kindreds. In addition, the recurrent germline pathogenic variant MSH2 c.942+3A&amp;gt;T has been detected in five unrelated index cases with a strong family history of LS syndrome. Moreover, the rare germline VUS PMS2 c.989-107_989-106insA has been detected in 14 unrelated LS patients and could be reclassified as likely benign. Interestingly, our NGS analysis detected the novel BMPR1A pathogenic variant c.1474-1G&amp;gt;C in young JPS patient that has been misdiagnosed as FAP. The in-silico analysis for this novel variant showed an alteration of the wild type acceptor site and an activation of a cryptic acceptor site, respectively, most probably affecting splicing. Conclusions Our current study will contribute to the molecular genetics characterization of hereditary colorectal cancer syndromes in Algerian population that is relevant for clinical management in the areas of genetic testing, early diagnosis, treatment and prevention. Citation Format: Farid Cherbal, Asma-Lamia Boumehdi, Feriel Khider, Karima Landelouci, Abdelwahab Zemam, Sarah Sabri, Adam.Walid Damache, Mohammed Oukkal, Hassen Mahfouf, Ferhat Zebboudj, Mustapha Maaoui. Genetic testing for hereditary colorectal cancer syndromes in Algerian patients: A multicenter study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4177.

Abstract 5236: Functional analysis of variants of uncertain significance of the MSH6 mismatch repair gene

Abstract Lynch syndrome (LS) is a hereditary condition that increases patients’ lifetime risk of cancer, primarily colorectal cancer. LS is caused by germline mutations in mismatch repair (MMR) genes, MLH1, MSH2, MSH6, and PMS2. Identification of these mutations is important in LS diagnosis in order to guide treatment plans and preventative care for patients’ and their families. However, the consequences of some variants in these genes are not immediately obvious, granting them classification as variants of uncertain significance (VUS). Half of known variants in the MMR gene MSH6 are VUS and, thus, the goal of my project is to provide evidence to help better determine their pathogenic significance. Laboratory functional analysis to determine whether these variants disrupt MMR function in human cells can provide such evidence toward this goal. We have previously used CRISPR gene editing to recreate variants in the endogenous loci for the MMR genes MSH2 and MLH1 in human embryonic stem cells (hESC) in order to test their effects on MMR function in a cellular environment. We will now use test whether this approach can be used to study MSH6 VUS. Using these edited hESC lines we are examining the impact of the variants on RNA and protein stability, repair of DNA microsatellites, which is a hallmark of normal MMR function, and induction of the MMR-dependent DNA damage response. Using a panel of known MSH6 pathogenic and benign controls, we will calibrate these functional assays in order to associate functional performance with likelihood of pathogenicity. We will next test a separate panel of known controls to validate these assays. Finally, we will examine up to 35 VUS. With this compilation of data, we will convert the results into a quantitative Odds of Pathogenicity value. This Odds of Pathogenicity score can be used by expert variant interpretation committees to help readily reclassify these variants, and provide a clearer diagnosis for these suspected Lynch syndrome patients. Citation Format: Elizabeth Szabo, Emily Blackburn, Patrick Pagano, Abhijit Rath, Christopher Heinen. Functional analysis of variants of uncertain significance of the MSH6 mismatch repair gene. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5236.

Abstract 1938: Germline mutations in breast cancer genes are associated with early age of diagnosis and triple negative disease in Guatemalan and US Hispanic women

Abstract Abstract Background: Mutations in hereditary breast cancer genes play an essential role in cancer risk. Little is known of the type and frequency of mutations in Hispanic populations in the United States and Central American countries, including Guatemala. Methods: We used exome sequencing to identify mutations in blood DNA from unselected Hispanic breast cancer cases from community recruitment and from two hospitals each in Texas and Guatemala. Data from a structured questionnaire was used to compare mutation carriers of medium and high penetrance genes. Variants were annotated with ClinVar and VarSome. Results: We recruited 262 Hispanic US women, 37 (14%) from community-based recruitment and 225 (86%) from hospitals in Texas. In addition, we ascertained 633 patients from two hospitals in Guatemala City. A total of 91 out of 895 subjects (10%) had a variant classified as pathogenic in a gene with known high or medium penetrance for inherited breast cancer. The most frequently mutated genes were the high penetrance BRCA1 (44/895, 4.9%) followed by BRCA2 (23/895, 2.6%), PALB2 (5/895, 0.6%), CHEK2 (5/895 0.6%), ATM (6/895, 0.7%) and TP53 (6/895, 0.7%). Pathogenic variants were also detected in the moderate penetrance genes BARD1 and MSH6, and rare pathogenic variants detected in the low penetrance genes AXIN2, FH, MLH1, MSH2, MUTYH, NF1, and SDHB. The high ratio of BRCA1/BRCA2 mutations is due to the presence of two potential founder mutations, BRCA1 c.212+1G&amp;gt;A splice mutation (18 cases) and BRCA1 c.799delT (9 cases) in Guatemala. Compared to all others, cases with pathogenic mutations had a significantly earlier age at diagnosis (45 vs. 52 years, P&amp;lt;0.001) and were more likely to have a diagnosis before menopause. A higher percentage of mutation carriers had a relative with any cancer (51% vs. 37%, P=0.038) or breast cancer (33% vs. 15%, P&amp;lt;0.001). Patients with pathogenic mutations had a significantly higher percentage of triple-negative disease (60% vs. 13%, P=0.00046). Finally, mammography usage was less frequent in women with a lower socioeconomic status, indicating that this group is less likely to be screened for breast cancer (P&amp;lt;0.001). Conclusions: Guatemalan women have rates of hereditary breast cancer mutations like other populations, and these women are more likely to have early age at diagnosis, triple-negative disease, and family history. Patient recruitment was higher using hospital-based versus community enrollment. This data supports genetic testing in breast cancer patients and those at high risk as part of a strategy to reduce breast cancer mortality in Hispanic women. Citation Format: Jesica Godinez Paredes, Isabel Rodriguez, Megan Ren, Anali Orozco, Jeremy Ortiz, Anaseidy Albanez, Catherine Jones, Zeina Nahleh, Lilian Barreda, Lisa Garland, Dongjing Wu, Jiahui Wang, Victor Argueta, Roberto Orozco, Eduardo Gharzouzi, Michael Dean. Germline mutations in breast cancer genes are associated with early age of diagnosis and triple negative disease in Guatemalan and US Hispanic women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1938.

Abstract 29: Characterization of a murine intestinal cancer homograft model for therapeutic evaluation

Abstract Introduction: Intestinal cancers are one of the most lethal malignancies and are responsible for a mass of cancer-related deaths. The discovery of new drugs and therapeutic strategies is underway, and to complement this, the development of superior animal models is required for preclinical studies. Genetically engineered mouse models (GEMMs) mimic de novo human tumor growth within an intact tumor microenvironment and are potent immunocompetent models for studying pathogenesis and immuno-oncology of intestinal cancers. GEMMs with dysfunctional DNA mismatch repair (MMR) genes, like MHL1 and MSH2, manifest intestinal tumors related to microsatellite instability (MSI). However, the low incidence and distinct latency among individuals hamper the application of these GEMMs in preclinical research. In this study, a murine intestinal cancer homograft from GEMMs with MMR deficiency is established to enroll larger cohorts of mice for preclinical studies. Methods: MMR-deficient GEMMs were generated by knockout of Mlh1 gene with a CRISPR-Cas9 system. Intestinal cancer development was investigated in 16 GEMMs. The developed tumor in the intestine of GEMM was isolated by surgery and inoculated into C57BL/6 mice subcutaneously to establish a murine homograft model. The tumor homograft was passaged and expanded in vivo, and stable tumor growth was established over consecutive passages. Histopathological studies were conducted for both the original and passaged tumors. Genomic mutations and gene expression were analyzed by whole exome sequencing and RNA-sequencing, and tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry. Therapeutic efficacy of chemotherapies and immune checkpoint inhibitors were evaluated in vivo for tumor growth inhibition. Results: The intestinal tumors or lesions were observed in two of the sixteen Mlh1-KO mice with an incidence of 12.5%. A murine homograft model of small bowel adenocarcinoma was successfully established using the tumor located in jejunum of a Mlh1-KO mouse. This model was stably passaged in vivo with semblable pathological features as the original lesion in Mlh1-KO mice. The various degrees of TILs and response to treatments revealed the potential of this model for in vivo investigation of immune response. Conclusion: Our murine intestinal cancer homograft model presented rapid and stable subcutaneous growth in immunocompetent mice, providing an efficient approach for intestinal cancer researchers and a superior preclinical model for new drug discovery. Citation Format: Jinxi Wang, Leilei Chen, Likun Zhang, Jingqian Zhu, Ludovic Bourre, Jingjing Wang. Characterization of a murine intestinal cancer homograft model for therapeutic evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 29.

MMR markers correlate with clinical outcome in patients with esophageal squamous cell carcinoma.

The DNA mismatch repair system is one of the defense mechanisms in the body, and the inactivation of mismatch repair plays a pivotal role in secondary carcinogenesis and progression. However, the significance of mismatch repair in esophageal squamous cell carcinoma (ESCC) has not been established. In this study, we explored the diagnostic and prognostic significance of mismatch repair markers, mutL homologue 1 (MLH1), post-meiotic segregation increased 2 (PMS2), mutS homologue 2 (MSH2), and mutS homologue 6 (MSH6), in patients with ESCC. We used a notation based on the proportion of immunoreactivity/expression for immunohistochemistry (PRIME notation), which allows the comparison of mismatch repair expression by assigning a score to PRIME notation. MLH1, PMS2, MSH2, and MSH6 were examined immunohistochemically in 189 surgically resected ESCC specimens. A total of 100/189 patients with ESCC (53%) received preoperative chemotherapy. The rates of ESCC cases with decreased mismatch repair status were 13.2%, 15.3%, 24.8%, and 12.6% for MLH1, PMS2, MSH2, and MSH6, respectively. The decreased status of individual mismatch repair markers was significantly correlated with worse prognosis in patients with ESCC. Additionally, MSH2, MSH6, and PMS2 were significantly associated with response to preoperative chemotherapy. Multivariate analysis revealed that MLH1, PMS2, and MSH2 are independent prognostic factors. Our results suggest that mismatch repair is a prognostic biomarker for ESCC and could contribute to the selection of appropriate adjuvant therapy for patients with ESCC.