MSH2 Mutations Research Articles

The importance of MLH1, MSH2, MSH6 and PMS2 in determining DNA damage response in digestive cancers

Introduction: Genomic instability is a common feature across human cancers, but presents variation across different types of cancer, patients and cells of from the same tumor.This can be caused either by changes in the DNA damage repair pathways, aberrant histone modifications or methylation. The aim of this study was to determine the common mutated genes between digestive cancers that present genomic instability and to determine the biological processes in which these are implicated. Material and Methods: Mutational profiles for patients presenting digestive cancers and mutations in MLH1, MSH2, MSH6 and PMS2 were downloaded from the MSK impact cohort via cBioPortal. PANTHER was used to determine the GO SLIM processes in which the mutated genes are involved. R 3.5.3 and the R package circlize were used to generate chord diagrams. Results and Discussions: Considering the number of mutated genes from different digestive cancers it could be observed that the deregulated processes are general cellular processes, while when taking into consideration the fold change in overrepresentation of certain processes, the main deregulated processes are represented by DNA damage repair pathways, showing their overrepresentation in the selected cohort based on a few mutated genes. Conclusion: MLH1,MSH2,MSH6 and PMS2 mutations dictate the alterations in DNA damage repair pathways in digestive cancers.

Germline and Tumor Sequencing as a Diagnostic Tool To Resolve Suspected Lynch Syndrome

Patients in whom mismatch repair (MMR)-deficient cancer develops in the absence of pathogenic variants of germline MMR genes or somatic hypermethylation of the MLH1 gene promoter are classified as having suspected Lynch syndrome (SLS). Germline whole-genome sequencing (WGS) and targeted and genome-wide tumor sequencing were applied to identify the underlying cause of tumor MMR deficiency in SLS. Germline WGS was performed on samples from 14 cancer-affected patients with SLS, including two sets of first-degree relatives. MMR genes were assessed for germline pathogenic variants, including complex structural rearrangements and noncoding variants. Tumor tissue was assessed for somatic MMR gene mutations using targeted, whole-exome sequencing or WGS. Germline WGS identified pathogenic MMR variants in 3 of the 14 cases (21.4%), including a 9.5-megabase inversion disrupting MSH2 in a mother and daughter. Excluding these 3 MMR carriers, tumor sequencing identified at least two somatic MMR gene mutations in 8 of 11 tumors tested (72.7%). In a second mother-daughter pair, a somatic cause of tumor MMR deficiency was supported by the presence of double somatic MSH2 mutations in their respective tumors. More than 70% of SLS cases had double somatic MMR mutations in the absence of germline pathogenic variants in the MMR or other DNA repair-related genes on WGS, and, therefore, were confidently assigned a noninherited cause of tumor MMR deficiency.

NFB-06. TREATMENT CHALLENGES IN PEDIATRIC GLIOBLASTOMA MULTIFORME WITH CONCURRENT SOMATIC AND GERMLINE NF1 MUTATIONS WITH GERMLINE MISMATCH REPAIR MUTATIONS: TWO UNIQUE CASES

INTRODUCTIONWe report the first known cases of pediatric glioblastoma (GBM) with prior clinical NF1 diagnoses, one with concurrent germline Lynch syndrome (LS) and NF1, and the other with somatic NF1 mutation and germline constitutional mismatch repair deficiency (CMMRD).METHODSTwo pediatric GBM cases with prior NF1 clinical diagnoses based on neurocutaneous criteria were reviewed. Next generation sequencing and immunohistochemical staining were used for somatic and germline NF1 and MMR gene mutation detection, and for MMR protein expression, respectively.RESULTSSixteen year old male with prior NF1 clinical diagnosis had resection of right frontal GBM revealing somatic mutations of POLE and PMS2, but not NF1. His father had confirmed LS with MSH2 mutation and no neurocutaneous stigmata. Patient’s germline testing revealed both pathogenic MSH2 plus NF1 mutations confirming LS and NF1. Treatment consisted of chemoradiation with temozolomide followed by adjuvant temozolomide with stable disease at 8 cycles. Nineteen year old male with former NF1 clinical diagnosis had 2 GBMs, first in left midbrain biopsied revealing somatic PMS2 and NF1 mutations underwent radiation then 7 cycles of temozolomide, then new left frontal GBM underwent resection followed by radiation and 5 cycles of pembrolizumab stable at 5th cycle.CONCLUSIONChildren with NF1 stigmata and GBM can have concurrent NF1 and LS, or CMMRD with NF1 somatic mutations. Our patients tolerated alkylating agents, despite risk for secondary malignancies as upfront therapy and at recurrence checkpoint inhibitors. Upfront therapy in GBM with mismatch repair syndrome with checkpoint inhibitors should be studied.

HGG-44. DEFECTS OF MISMATCH REPAIR PROTEINS IN PEDIATRIC HIGH GRADE GLIOMAS

Hetero- and homozygous germline mutations of the mismatch repair genes MLH1, PMS2, MSH2 and MSH6 cause Lynch and constitutional mismatch repair (CMMRD) cancer predisposition syndrome, respectively. Affected CMMRD individuals are at risk to develop a variety of neoplasms including CNS tumors, particularly high grade gliomas (HGG), during childhood. Currently, few data exist on the prevalence of CMMRD in children with pediatric HGG. We screened a consecutive series of 79 supratentorial HGGs. Tumor tissue was available in 42 patients, 5 were reclassified as non-HGGs. Immunohistochemistry with antibodies against MLH1, PMS2, MSH2 and MSH6 was performed in 37 tumors. Four patients (3 families) with known CMMRD were included. The evaluation of the slides was performed blinded to the CMMRD status. All four patients with known CMMRD (3 patients with PMS2, one with MSH6 mutation) were identified, showing loss of PMS2 and MSH2/MSH6, respectively. Additionally, we identified 6 patients with loss of MSH2/MSH6 staining in tumor cells, but retained staining in preexisting cells, indicating a pattern like in Lynch syndrome. NGS sequencing of these tumor tissues revealed in 2 patients MSH2 mutations and in one patient a hypermutator phenotype with MSH2 and MSH6 mutations. In 3/6 patients no mutations in the MMR genes were detectable. In summary, we found a low prevalence of CMMRD among HGGs, but identified also 2 patients with probable Lynch syndrome. Immunhistochemistry is an effective tool to screen for patients with MMR defects and should be performed in HGGs to optimize treatment and offer affected families genetic counseling.

RARE-14. DEVELOPMENT OF ANAPLASTIC ASTROCYTOMA AS A THIRD MALIGNANCY IN A PEDIATRIC PATIENT WITH CONSTITUTIONAL MISMATCH REPAIR DEFICIENCY (CMMRD): A CASE REPORT AND EVALUATION OF TUMOR GENOMICS IDENTIFYING BIALLELIC MSH6 MUTATIONS

Congenital mismatch repair deficiency (CMMRD) is a pediatric cancer predisposition syndrome secondary to biallelic mutations in mismatch repair genes including MLH1, MSH2, MSH6, and PMS2. Due to the resulting lack of repair mechanisms, these patients develop a high intracellular mutational burden and have a high risk of development of multiple malignancies at a young age. Similar to patients with Lynch Syndrome (monoallelic mutations in MMR genes), these patients are at risk for development of central nervous system (CNS) tumors including high grade gliomas. Forty-eight percent of patients with CMMRD are diagnosed with a CNS malignancy. In this interesting case, a patient developed three metachronous malignancies prior to the age of 13, including Burkitt lymphoma, T-Cell lymphoma and anaplastic astrocytoma. Genomic analysis revealed a high mutational burden in his initial tumors, with multiple oncogenic mutations, as well as a previously unreported germline compound heterozygous MSH6 E744fs*12 and R248fs*8 alteration. He received a gross total resection of the tumor which in previous studies has been shown to have the highest impact on survival. Surgery was followed by radiation and ongoing treatment with an immune checkpoint inhibitor with stable disease at 6 months. The purpose of this case report is to describe the interesting presentation of CMMRD and discuss the previously unreported biallelic MSH6 mutations.

Utility of a custom designed next generation DNA sequencing gene panel to molecularly classify endometrial cancers according to The Cancer Genome Atlas subgroups

BackgroundThe Cancer Genome Atlas identified four molecular subgroups of endometrial cancer with survival differences based on whole genome, transcriptomic, and proteomic characterization. Clinically accessible algorithms that reproduce this data are needed. Our aim was to determine if targeted sequencing alone allowed for molecular classification of endometrial cancer.MethodsUsing a custom-designed 156 gene panel, we analyzed 47 endometrial cancers and matching non-tumor tissue. Variants were annotated for pathogenicity and medical records were reviewed for the clinicopathologic variables. Using molecular characteristics, tumors were classified into four subgroups. Group 1 included patients with > 570 unfiltered somatic variants, > 9 cytosine to adenine nucleotide substitutions per sample, and < 1 cytosine to guanine nucleotide substitution per sample. Group 2 included patients with any somatic mutation in MSH2, MSH6, MLH1, PMS2. Group 3 included patients with TP53 mutations without mutation in mismatch repair genes. Remaining patients were classified as group 4. Analyses were performed using SAS 9.4 (SAS Institute Inc., Cary, North Carolina, USA).ResultsEndometrioid endometrial cancers had more candidate variants of potential pathogenic interest (median 6 IQR 4.13 vs. 2 IQR 2.3; p < 0.01) than uterine serous cancers. PTEN (82% vs. 15%, p < 0.01) and PIK3CA (74% vs. 23%, p < 0.01) mutations were more frequent in endometrioid than serous carcinomas. TP53 (18% vs. 77%, p < 0.01) mutations were more frequent in serous carcinomas. Visual inspection of the number of unfiltered somatic variants per sample identified six grade 3 endometrioid samples with high tumor mutational burden, all of which demonstrated POLE mutations, most commonly P286R and V411L. Of the grade 3 endometrioid carcinomas, those with POLE mutations were less likely to have risk factors necessitating adjuvant treatment than those with low tumor mutational burden. Targeted sequencing was unable to assign samples to microsatellite unstable, copy number low, and copy number high subgroups.ConclusionsTargeted sequencing can predict the presence of POLE mutations based on the tumor mutational burden. However, targeted sequencing alone is inadequate to classify endometrial cancers into molecular subgroups identified by The Cancer Genome Atlas.

Metachronous cutaneous squamous cell carcinoma in a young patient as the only presenting symptom to uncover Lynch syndrome with MLH1 Germline mutation

BackgroundCutaneous malignancies are rare complications of Lynch syndrome and can include Muir-Torre and Turcot syndromes that are associated with sebaceous gland tumors and keratoacanthomas. The incidence and clinical course of cutaneous squamous cell carcinoma have not been well documented in Lynch syndrome due to its rarity.Case presentationA 49-year male presented with an enlarging groin skin lesion that was biopsed and demonstrated cutaneous squamous cell carcinoma for which he underwent a surgical resection. The patient experienced later a recurrence of cutaneous squamous cell carcinoma around the resected margins. Next generation sequencing of tumor tissue revealed mutations in MSH6 and MLH1, in addition to high microsatellite instability. The patient underwent pembrolizumab treatment with complete resolution of the cutaneous lesion in the groin, but subsequently developed a new mass in the right antecubital fossa shortly after discontinuation of pembrolizumab. Repeat biopsy of the antecubital fossa lesion revealed a recurrence of cutaneous squamous cell carcinoma. Germline mutation testing revealed MLH1 mutation, compatible with Lynch syndrome, and the patient restarted pembrolizumab which was associated with a complete response. The patient was referred for genetic counseling and cancer screening.ConclusionsCutaneous squamous cell carcinoma, although rare, can be the initial presenting symptom in patients with Lynch syndrome. This association has been described in patients with germline mutations in MLH1. Lynch syndrome should be considered when evaluating young patients presenting with recurrent cutaneous squamous cell carcinoma with positive family history of malignancy and/ or without any identifiable risk factors for skin cancers, including those with a durable and rapid response to immunotherapy.

TMOD-35. DEVELOPING RECURRENT GBM PDX, IN VIVO, FROM TREATMENT NAÏVE SOURCES

Abstract PURPOSE Post-therapy recurrent glioblastoma (GBM) patient-derived xenografts (PDX), developed from corresponding treatment-naïve PDX, could serve as useful resources for identifying therapeutics with activity against recurrent GBM. The goal of this study was to determine whether treatment-naïve intracranial GBM PDX, in mice receiving radiotherapy (RT) and/or temozolomide (TMZ), acquire the same mutations that occur in post-RT+TMZ GBMs from patients. METHODS Luciferase-modified, treatment-naïve GBM PDX were engrafted in the brains of athymic nude mice, followed by treatment with RT only (2 Gy/day x 5), TMZ only (10 mg/kg/day x 5), or RT+TMZ. Bioluminescence imaging was used to monitor intracranial tumor growth, response to treatment, and recurrence from treatment. Some mice with recurrent tumors received additional TMZ treatment. When mice became symptomatic, intracranial tumors were resected and engrafted subcutaneously in a new mouse host, then sequentially propagated subcutaneously into additional host mice. After the third passage, whole-exome sequencing (WES) was done, comparing post-therapy with treatment-naïve PDX sequence variants. RESULTS Analysis of PDX WES showed the following: 1) TMZ consistently caused more genes to incur coding sequence mutations than RT, as much as 13x more; 2) TMZ-treated tumor mutations were mostly G-C to A-T transitions (71-92%), consistent with the known mutagenic effect of TMZ; and 3) post-therapy PDX acquire similar mutations as do recurrent GBMs in patients, for example involving DNA mismatch repair gene MSH6. One of the derivative PDX with MSH6 mutation has been retested for response to RT and TMZ, with results showing its having become TMZ, but not RT resistant. CONCLUSIONS The mutation profiles of RT+TMZ-treated PDX are similar to those reported for GBMs that recur after RT+TMZ in patients. The new PDX resources described here may prove useful for identifying effective treatments against recurrent GBM.

PATH-23. GENOMIC LANDSCAPE OF IDH-MUTANT PRIMARY GLIOBLASTOMAS SHOWS DISTINCT CLINICAL AND MOLECULAR FEATURES AND THAT CDKN2A SHOULD BE SUPPLEMENTED WITH MGMTp AND G-CIMP FOR PRECISE PROGNOSTICATION

Abstract There have only been rare studies of IDH-mutant primary glioblastomas (IDH-mutant astrocytoma IV); there were one or two studies on secondary glioblastomas. In a cohort of 70 cases, we conducted clinical analysis, methylation profiling, RNA sequencing, targeted sequencing, and TERTp seqeuncing on available FFPE tissues. Median follow-up was 58.2 months (n= 60). IDH-mutant primary glioblastomas had longer median OS (30.4 months) and median PFS (25.9 months) than IDH-mutant secondary glioblastomas as in the literature or established databases. MGMTp methylated cases had better OS (p= 0.001) and it was an independent prognosticator. We previously showed G-CIMP to be an independent prognostic marker for IDH-mutant glioblastomas (NOA 2019). Although CDKN2A deletion was an independent prognostic marker for poorer OS (p= 0.036) and PFS (p= 0.005), MGMTp methylation had a trend of superseding CDKN2A deletion (p= 0.055) for prognostication and G-CIMP subgroups could similarly partially supersede CDKN2A deletion (p= 0.582). Hence, CDKN2A deletion should be supplemented with these two biomarkers for finer prognostication. Targeted sequencing (n= 55) showed that there were more ATRX (35/55, 64%), TP53 (31/55, 56%), KMT2D (18/55, 33%), POLE (11/55, 20%) and MSH6 (7/55, 13%) mutations, but fewer TERTp (3/69, 4%) and PTEN (1/55, 2%) mutations than IDH-wildtype glioblastomas as from literature and databases. CNVs revealed by methylomes (n= 53) and mutations (n= 55) showed that there were more PDGFRA (amplification: 9/53, 17%, mutation: 10/55, 18%) alterations, but fewer MET (amplification: 3/53, 6%, mutation: 4/55, 7%) alterations and hypermutated (6/55, 11%) cases than IDH-mutant secondary glioblastomas from literature. GISTIC analysis revealed amplifications of CCND2, CDK4, MYC, and PDGFRA, deletions of CDKN2A, RB1, and chromosome 10q to be significant CNVs (q&amp;lt; 0.05). There were few EGFR amplifications (2/53, 4%), which was different from regular glioblastomas. RNA sequencing (n= 42) showed few fusions (4/42, 10%), which was different from IDH-mutant secondary glioblastomas.

CTIM-30. EFFICACY OF PEMBROLIZUMAB IN PATIENTS WITH PITUITARY CARCINOMA: RESULTS FROM A PHASE II STUDY

Abstract Pituitary carcinoma is an aggressive tumor characterized by metastatic spread beyond the sellar region that leads to debilitating symptoms and poor survival. Pituitary carcinomas recur despite conventional multimodality treatments. Given the recent advances in the use of immune checkpoint inhibitors (CPIs) to treat various solid cancers, there is interest in exploring the role of immunotherapy for treating aggressive, refractory pituitary tumors. We treated four pituitary carcinoma patients with pembrolizumab as part of a phase II clinical trial (NCT02721732). Here, we present their clinical course and outcomes and correlate responses with available molecular data: hypermutation status, PD-L1 staining, tumor-infiltrating lymphocyte score, microsatellite status and tumor mutational burden. Patients 1 and 2, with heavily pretreated, refractory corticotroph pituitary carcinoma, had partial radiographic (60% and 32% per irRECIST, respectively) and hormonal responses. Patient 1’s response continues 42 months after initiation of pembrolizumab and his baseline tumor tissue obtained after treatment with temozolomide demonstrated a hypermutator phenotype with MSH2 and MSH6 gene mutations. Patient 2’s tumor was not sampled after exposure to temozolomide, but prior somatic mutational testing was negative. Patient 3 (non-functioning corticotroph tumor) had a best response of stable disease for four months. Patient 4 (prolactin-secreting carcinoma) had progressive disease. The latter two patients’ tumors did not demonstrate a hypermutator phenotype after treatment with temozolomide. PD-L1 staining was negative in all tumors. TIL score was 2 in Patients 1 and 4, negative in Patient 3 and not available in Patient 2. All patients tolerated the treatment well with mild adverse events. Our study generates the hypothesis that an alkylating agent-induced hypermutator phenotype may be an indicator of response to CPIs in pituitary carcinomas. The role of CPI in treating patients with pituitary carcinoma and mechanisms of hypermutation in this population require further study.

NCMP-17. MISMATCH REPAIR MUTATIONS AND THE CENTRAL NERVOUS SYSTEM: A CASE SERIES OF GERMLINE MUTATIONS AND CNS MALIGNANCY

Abstract Our understanding of genetic predispositions for malignancy is continually evolving. One family of germline mutations well described in the literature is that of the DNA mismatch repair mechanism (MMR). Lynch syndrome (LS) is due to a loss of function mutation of several MMR genes- MSH2, MLH1, MSH6, and PMS2. Germline MMR mutations lead to microsatellite instability and loss of genomic integrity resulting in an increased risk for various cancers (colorectal, genitourinary, etc). LS may be as common as 1 in 400 people and some MMR mutations have been associated with gliomas. There is a paucity of information regarding frequency of glioma subtypes as well as tumor genetic and molecular characteristics which have important clinical implications. We describe a case series of 6 individuals with germline MMR mutations and brain tumors. Those with MSH2 and PMS2 mutations (n=3) developed glioblastomas at a mean age at diagnosis of 48 years. These tumors expressed MGMT hyper-methylation and high tumor mutational burden. Only one had IDH-1 mutation. Those with MLH1 mutations (n=3), did not develop gliomas. This raises the question of differential glioma subtype development based on MMR gene. It also highlights the possibility of Lynch-associated gliomas having more favorable treatment response due to MGMT methylation and potential response to immunotherapy based on high tumor mutational burden. Though the sample size is small, there appears to be a preponderance of women compared to men (5:1 respectively). Larger studies are needed to verify CNS involvement in germline MMR mutations. In doing so, we hope to identify factors that may influence clinical management and lead to a better understanding of treatment response and disease prognosis.

Genotype-phenotype associations in colorectal adenocarcinomas and their matched metastases

Although primary colorectal carcinomas (CRCs) frequently share genetic alterations with their metastases, morphologic surrogates reflecting the genotype contexture of metastases remain largely unknown. We investigated phenotype/genotype associations in paired primary and metastatic colorectal adenocarcinomas from 75 patients. Thirty-three (44%) metastatic lesions were synchronous and 42 (56%) were metachronous. Tumor budding, micronecrosis, and tumor-infiltrating lymphocyte (TIL) density were compared with matched next-generation sequencing genotypes. Micronecrosis in the primary were significantly associated with nodal status (P=0.0054) and with micronecrosis in metastatic sites (P=0.0216), particularly in metachronous metastases (P=0.0033). With a 57-gene panel, one or more mutations were identified in 64 (85.3%) cases. In metastases, high (brisk) TILs were associated with overall mutational burden (P=0.0058) and with mutations in EGF (P=0.0325), RAS genes (P=0.0043), and MMR genes (P=0.0069), whereas high-level micronecrosis correlated with mutations in APC (P=0.0004) and MSH6 (P=0.0385) genes. Genomic alterations were shared in 90.1% of primary/metastatic pairs, but clonality of the same mutation was shared in only 57.1% of paired lesions. Compared with synchronous, metachronous metastases had more private clonal alterations (P=0.0291); in this group, clonal alterations coincided with brisk TILs (P=0.0334) and high micronecrosis (P=0.0133). High TILs in metastatic lesions were predictive of favorable overall survival (log-rank P=0.044). The observed phenotype/genotype associations favor the clonal evolution model in CRC metastases that seems accompanied by intense host immune response. If the role of micronecrosis and brisk TILs in metachronous metastases is validated in larger studies, these histologic parameters will be worth adding in the armamentarium for the evaluation of metastatic CRC.

Additional Evidence of Mismatch Repair Pathway As Relapse-Specific Alteration in B-Cell Precursor Acute Lymphoblastic Leukemia: Discovery of Novel Somatic Mutation in MLH1 and Establishment of New Cell Line with MLH1 Mutation

&amp;lt;Introduction&amp;gt;Extensive molecular analysis revealed genetic alterations related to relapse such as mutations of CREEB, MSH2, or NT5C2 in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Recently Li B, et al. have established relationship between relapse-specific somatic alterations and timing of relapse (Blood 2020;135:41-55). They identified close link between alterations of DNA mismatch repair genes, including MSH2, MSH6, and PMS2 and early relapse of BCP-ALL as a result of alteration of thiopurine response and resistance to treatment. However, it remains to be clarified which subtype of BCP-ALL is prone to acquisition of particular type of relapse-specific molecular alteration. To further elucidate mechanism of recurrence we have analyzed childhood BCP-ALL cases, particularly focusing on DNA mismatch repair pathway. &amp;lt;Procedure&amp;gt; We analyzed diagnosis-relapse pair samples of recurrent 16 BCP-ALL cases, who had been treated in our institution to find single nucleotide variant (SNV), small Indel, and copy number variation in the coding exons, particularly focusing on mismatch repair pathway using the data captured by Ion AmpliSeq Exome kit and Ion Proton (Thermo Fisher Scientific, MA, USA). The identified variants were confirmed by Sanger sequence. Additionally, we performed RNA-seq using SMART-Seq Ultra Low Input RNA Kit (Clontech Laboratories, Inc, CA, USA), Ion Plus Fragment Library Kit, and Ion Proton, and in vitro cell culture of the leukemic blasts for several cases. &amp;lt;Result&amp;gt; Of several DNA mismatch repair pathway genes, we have identified somatic SNV of MLH1 in a case. The index case, three years old male had diploid BCP-ALL with t(7;9) and PAX5 alteration at diagnosis, who developed early relapse (11 months from diagnosis) and died of the disease. From the sample at 1st and 2nd relapse we have identified somatic MLH1 variant (NM_000249.4:c.901C&amp;gt;T;p.Gln301*). This SNV was detected in small fraction of the diagnostic sample. Furthermore, we have established permanently growing cell line, ICH-BCP-1 from the sample obtained at 2nd relapse. The doubling time is approximately 37 hours and the karyotype was, 46,XY. The same MLH1 variant was identified in the cell line. Of note is that the number of detected SNV increased rapidly at 1st relapse and 2nd relapse as suggested by function of MLH1 product. No other alteration of DNA mismatch repair pathway was observed in the cohort. As previously discovered relapse-specific alterations, we identified somatic SNV of NT5C2 in one hyperdiploid BCP-ALL case and somatic SNV of CREBBP in one hyperdiploid BCP-ALL case. The former case with SNV of NT5C2 gained deletion of IKZF1 and formation of P2RY8-CRLF2 fusion gene at recurrence. Throughout the cohort, hyperdiploid BCP-ALL cases had tendency to have RAS pathway somatic SNVs (KRAS, NRAS, FLT3, and PTPN11). In addition, one low hypodiploid BCP-ALL case had germline small Indel of TP53 and somatic SNV of RB. &amp;lt;Discussion&amp;gt; We add MLH1 alteration to the list of DNA repair pathway relapse-specific somatic alterations, further supporting the particular significance of DNA repair pathway as mechanism of BCP-ALL recurrence, probably related to massive acquisition of complex genetic alteration as a result of loss of DNA repair. In our cohort, the prevalence of previously reported relapse-specific mutation is relatively low, which may be caused by detection method and different ethnicity. Our novel cell line is useful staff for investigation to identify the role of DNA mismatch repair pathway in BCP-ALL leukemogenesis. Disclosures No relevant conflicts of interest to declare.

Telomere Instability in Lynch Syndrome Families Leads to Some Shorter Telomeres in MSH2+/- Carriers.

Lynch syndrome (LS) is an inherited predisposition to early onset of various cancers, caused by mutation in a DNA mismatch repair (MMR) gene. In heterozygous MMR+/− carriers, somatic mutation, loss or silencing of the wild type allele increases the mutation rate, facilitating the initiation of MMR-defective cancers. These cancers are characterized by instability at short tandem repeats (STRs) and in telomeric DNA. We have investigated telomere length in saliva DNA from LS and control families, using single telomere analysis at XpYp and 12q and by qPCR to measure total telomeric DNA. Single telomere analysis showed a trend for shorter XpYp telomeres in MSH2+/− carriers compared to MLH1+/− carriers or controls, but this was masked in the comparative analysis of total telomeric DNA. Comparison of age-adjusted telomere length within families showed that neither MSH2+/− or MLH1+/− children had consistently shorter or longer telomeres than their MMR+/− parent, indicating the absence of an inter-generational effect on telomere length. Unexpectedly however, wildtype children in families with MSH2 mutations, had significantly longer XpYp telomeres than their MMR+/− parent. Altogether our data suggest that MMR insufficiency, particularly in MSH2+/− carriers, increases telomere instability and somatic cell turnover during the lifetime of LS mutation carriers but has minimal consequences for telomere length in the germline.

Genomic Profiling Comparison of Germline BRCA and Non-BRCA Carriers Reveals CCNE1 Amplification as a Risk Factor for Non-BRCA Carriers in Patients With Triple-Negative Breast Cancer.

Background: Differences in genomic profiling and immunity-associated parameters between germline BRCA and non-BRCA carriers in TNBC with high tumor burden remain unexplored. This study aimed to compare the differences and explore potential prognostic predictors and therapeutic targets.Methods: The study cohort included 21 consecutive TNBC cases with germline BRCA1/2 mutations and 54 non-BRCA carriers with a tumor size ≥ 2 cm and/or ≥1 affected lymph nodes. Differences in clinicopathological characteristics and genomic profiles were analyzed through next-generation sequencing. Univariate Kaplan–Meier analysis and Cox regression model were applied to survival analysis. Immunohistochemistry was used to confirm the consistency between CCNE1 amplification and cyclin E1 protein overexpression.Results: The cohort included 16 and five patients with germline BRCA1 and BRCA2 mutations, respectively. Patients with germline BRCA1/2 mutations were diagnosed at a significantly younger age and were more likely to have a family history of breast and/or ovarian cancer. Six non-BRCA carriers (11.11%) carried germline mutations in other cancer susceptibility genes, including five mutations in five homologous recombination repair (HRR) pathway genes (9.26%) and one mutation in MSH3 (1.85%). Somatic mutations in HRR pathway genes were found in 22.22 and 14.29% of the non-BRCA and BRCA carriers, respectively. PIK3CA missense mutation (p = 0.046) and CCNE1 amplification (p = 0.2) were found only in the non-BRCA carriers. The median tumor mutation burden (TMB) was 4.1 Muts/Mb, whereas none of the cases had high microsatellite instability (MSI). BRCA status did not affect disease-free survival (DFS, p = 0.15) or overall survival (OS, p = 0.52). CCNE1 amplification was an independent risk factor for DFS in non-BRCA carriers with TNBC (HR 13.07, 95% CI 2.47–69.24, p = 0.003). Consistency between CCNE1 amplification and cyclin E1 protein overexpression was confirmed with an AUC of 0.967 for cyclin E1 signal intensity.Conclusions: We found differences in genetic alterations between germline BRCA and non-BRCA carriers with TNBC and a high tumor burden. TMB and MSI may not be suitable predictors of TNBC for immune checkpoint inhibitors. Notably, CCNE1 amplification is a novel potential prognostic marker and therapeutic target for non-BRCA carriers with TNBC. Cyclin E1 may be used instead of CCNE1 to improve clinical applicability.

Value of upper gastrointestinal endoscopy for gastric cancer surveillance in patients with Lynch syndrome.

In our study, we evaluated the effectiveness of upper gastrointestinal (GI) endoscopy as an instrument for early gastric cancer (GC) detection in Lynch syndrome (LS) patients by analyzing data from the registry of the German Consortium for Familial Intestinal Cancer. In a prospective, multicenter cohort study, 1128 out of 2009 registered individuals with confirmed LS underwent 5176 upper GI endoscopies. Compliance was good since 77.6% of upper GI endoscopies were completed within the recommended interval of 1 to 3 years. Forty-nine GC events were observed in 47 patients. MLH1 (n = 21) and MSH2 (n = 24) mutations were the most prevalent. GCs in patients undergoing regular surveillance were diagnosed significantly more often in an early-stage disease (UICC I) than GCs detected through symptoms (83% vs 25%; P = .0231). Thirty-two (68%) patients had a negative family history of GC. The median age at diagnosis was 51 years (range 28-66). Of all GC patients, 13 were diagnosed at an age younger than 45. Our study supports the recommendation of regular upper GI endoscopy surveillance for LS patients beginning no later than at the age of 30.

Complete Loss of EPCAM Immunoexpression Identifies EPCAM Deletion Carriers in MSH2-Negative Colorectal Neoplasia.

Simple SummaryColorectal carcinomas from patients with Lynch syndrome (LS) due to EPCAM deletions show loss of MSH2 expression. The aim of our study was to evaluate the usefulness of EPCAM expression in identifying carriers of EPCAM deletion among patients with MSH2-negative lesions. MSH2 and EPCAM immunohistochemistry was performed in a large series of lesions (190) composed of malignant and benign neoplasms as well as precursor lesions of different organs from 71 patients with suspected LS due to MSH2 alterations. Germ-line analysis confirmed LS in 68 patients due to MSH2 mutations (53) and EPCAM deletions (15). Among colorectal lesions with lack of MSH2 expression, only 17 were EPCAM-negative and belonged to patients with EPCAM deletions. We confirm that loss of EPCAM expression identifies EPCAM deletion carriers with 100% specificity and we recommend adding EPCAM IHC to the algorithm of MSH2-negative colorectal neoplasia.The use of epithelial cell adhesion molecule (EPCAM) immunohistochemistry (IHC) is not included in the colorectal cancer (CRC) screening algorithm to detect Lynch syndrome (LS) patients. The aim of the present study was to demonstrate that EPCAM IHC is a useful tool to guide the LS germ-line analysis when a loss of MSH2 expression was present. We retrospectively studied MSH2 and EPCAM IHC in a large series of 190 lesions composed of malignant neoplasms (102), precursor lesions of gastrointestinal (71) and extra-gastrointestinal origin (9), and benign neoplasms (8) from different organs of 71 patients suspicious of being LS due to MSH2 alterations. LS was confirmed in 68 patients, 53 with MSH2 mutations and 15 with EPCAM 3′-end deletions. Tissue microarrays were constructed with human normal tissues and their malignant counterparts to assist in the evaluation of EPCAM staining. Among 154 MSH2-negative lesions, 17 were EPCAM-negative, including 10 CRC and 7 colorectal polyps, and 5 of them showed only isolated negative glands. All lesions showing a lack of EPCAM expression belonged to patients with EPCAM 3′-end deletions. EPCAM IHC is a useful screening tool, with 100% specificity to identify LS patients due to EPCAM 3′-end deletions in MSH2-negative CRC and MSH2-negative colorectal polyps.

Role of androgen receptor splice variant-7 (AR-V7) in prostate cancer resistance to 2nd-generation androgen receptor signaling inhibitors.

The role of truncated androgen receptor splice variant-7 (AR-V7) in prostate cancer biology is an unresolved question. Is it simply a marker of resistance to 2nd generation androgen receptor signaling inhibitors (ARSi) like Abiraterone Acetate (Abi) and Enzalutamide (Enza) or a functional driver of lethal resistance via its ligand-independent transcriptional activity? To resolve this question, the correlation between resistance to ARSi and genetic chances and expression of full length AR (AR-FL) vs. AR-V7 were evaluated in a series of independent patient-derived xenografts (PDXs). While all PDXs lack PTEN expression, there is no consistent requirement for mutation in TP53, RB1, BRCA2, PIK3CA, or MSH2, or expression of SOX2 or ERG and ARSi-resistance. Elevated expression of AR-FL alone is sufficient for Abi- but not Enza-resistance, even if AR-FL is gain-of-function (GOF) mutated. Enza-resistance is consistently correlated with enhanced AR-V7 expression. In vitro and in vivo growth responses of Abi-/Enza-resistant LNCaP-95 cells in which CRISPR-Cas9 was used to knockout AR-FL or AR-V7 alone or in combination were evaluated. Combining these growth responses with RNAseq analysis demonstrates that both AR-FL and AR-V7 dependent transcriptional complementation are needed for Abi/Enza resistance.

Risk factors of ovarian cancer: family history, obesity and oral contraceptive use

Introduction and purpose: Ovarian cancer (OC) takes the 7th place among most common female malignancies worldwide. There are almost 240 thousand of new cases diagnosed each year and the number is constantly growing. Defining risk factors of OC is essential to provide good education among women and predict this malignancy better. The aim of this review is to analyze the current data on risk factors of OC concentrating on family history, obesity andoral contraceptives use. Material and method: This review was based on articles from 2010 to 2020 collected in PubMed and concerning the impact of several risk factors on developing OC. Results: The best described risk factor of OC is genetics and family history. 75% or hereditary OCs are associated to BRCA genes mutations, especially BRCA1. Other mutations increasing the risk of OC are MSH2 and MLH1 genes mutations. Diverse risk factors correlate with different histological subtypes of OC. Obesity increases the levels of estrogens, androgens and leptin, which may promote the proliferation of ovarian epithelial cells. Using oral contraceptives is related to 20% decreased risk of OC for each 5 years of use. Conclusions: BRCA1/2, MSH2 and MLH1 mutations and obesity are well-defined risk factors of ovarian cancer. The first-choice risk-reducing procedure among BRCA carriers is bilateral salpingo-oophorectomy. On the contrary, using oral contraceptives results in decreased risk of OC. Promoting knowledge about OC risk factors among women and healthcare professionals is crucial in order to reduce the number of ovarian cancer.

Germline biallelic Mcm8 variants are associated with early-onset Lynch-like syndrome.

Lynch syndrome is the most common cause of hereditary colorectal cancer (CRC), and it is characterized by DNA mismatch repair (MMR) deficiency. The term Lynch-like syndrome (LLS) is used for patients with MMR-deficient tumors and neither germline mutation in MLH1, MSH2, MSH6, PMS2, or EPCAM nor MLH1 somatic methylation. Biallelic somatic inactivation or cryptic germline MMR variants undetected during genetic testing have been proposed to be involved. Sixteen patients with early-onset LLS CRC were selected for germline and tumor whole-exome sequencing. Two potentially pathogenic germline MCM8 variants were detected in a male patient with LLS with fertility problems. A knockout cellular model for MCM8 was generated by CRISPR/Cas9 and detected genetic variants were produced by mutagenesis. DNA damage, microsatellite instability, and mutational signatures were monitored. DNA damage was evident for MCM8KO cells and the analyzed genetic variants. Microsatellite instability and mutational signatures in MCM8KO cells were compatible with the involvement of MCM8 in MMR. Replication in an independent familial cancer cohort detected additional carriers. Unexplained MMR-deficient CRC cases, even showing somatic biallelic MMR inactivation, may be caused by underlying germline defects in genes different than MMR genes. We suggest MCM8 as a gene involved in CRC germline predisposition with a recessive pattern of inheritance.