Suspected Lynch Syndrome Research Articles

Novel MLH1 nonsense variant in a patient with suspected Lynch syndrome

Loss-of-function germline variants of MLH1 cause Lynch syndrome. Here, we present the case of a 43-year-old male patient diagnosed with cecal and transverse colon adenocarcinomas. The characteristics of the case met the revised Bethesda guidelines, and the tumors demonstrated a high frequency of microsatellite instability. Genetic testing for mismatch repair genes (indicative of Lynch syndrome) revealed a novel heterozygous germline pathogenic variant, NM_000249.4:c.856A>T/NP_000240.1:p.(Lys286Ter), in MLH1.

Real-world outcomes of stage II and III colorectal cancers treated by postoperative adjuvant chemotherapy based on the mismatch repair status.

In Japan, immunohistochemistry for mismatch repair (MMR) proteins targeted at stage II and III colorectal cancers (CRCs) has been covered by national insurance since October, 2022. This study aimed to clarify the long-term outcomes of patients with stage II and III CRCs receiving postoperative adjuvant chemotherapy based on their MMR status. The outcomes of 640 patients who underwent radical surgery for stage II and III CRCs were analyzed retrospectively. Deficient MMR (dMMR) was diagnosed in 41 (13.3%) patients with stage II and 28 (9.1%) patients with stage III CRC. The overall survival and recurrence rates were not significantly different between the patients with stage II and those with stage III CRC. The risk factors for recurrence among those with stage II CRC were tumors on the left side, T4 disease, and the presence of BRAF wild type. The recurrence rates were lower in the stage II CRC patients with sporadic dMMR than in those with suspected Lynch syndrome (LS). The first site of recurrence was more frequently the peritoneum or distant lymph node in patients with dMMR. Stage IICRC patients with sporadic dMMR were found to have a very good prognosis. On the other hand, peritoneal dissemination or distant lymph node metastasis tended to develop in patients with dMMR.

Tumor analysis of MMR genes in Lynch-like syndrome: Challenges associated with results interpretation.

Up to 70% of suspected Lynch syndrome patients harboring MMR deficient tumors lack identifiable germline pathogenic variants in MMR genes, being referred to as Lynch-like syndrome (LLS). Previous studies have reported biallelic somatic MMR inactivation in a variable range of LLS-associated tumors. Moreover, translating tumor testing results into patient management remains controversial. Our aim is to assess the challenges associated with the implementation of tumoral MMR gene testing in routine workflows. Here, we present the clinical characterization of 229 LLS patients. MMR gene testing was performed in 39 available tumors, and results were analyzed using two variant allele frequency (VAF) thresholds (≥5% and ≥10%). More biallelic somatic events were identified at VAF ≥ 5% than ≥10% (35.9% vs. 25.6%), although the rate of nonconcordant results regarding immunohistochemical pattern increased (30.8% vs. 20.5%). Interpretation difficulties question the current utility of the identification of MMR somatic hits in the diagnostic algorithm of suspected LS cases.

Abstract 5615: Functional analysis of variants of uncertain significance in 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 and classification of these mutations is important in LS diagnosis for guiding 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). Laboratory functional analysis to determine whether these variants disrupt MMR function in human cells can provide important evidence for understanding the pathogenicity of VUS and reclassifying them. Half of known variants in the MMR gene MSH6 are VUS and, thus, the goal of my project is to provide evidence for a subset of these variants to help provide evidence for reclassification for these variants. We are calibrating and validating functional assays for MSH6 variants, using known MSH6 pathogenic and benign controls. We will then test MSH6 VUS identified in patients as reported in the InSiGHT database, ClinVar database, and current literature. We are using CRISPR gene editing to recreate these variants in the endogenous MSH6 loci in human embryonic stem cells (hESC). 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. 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, Alexander Radecki, Abhijit Rath, Christopher Heinen. Functional analysis of variants of uncertain significance in the MSH6 mismatch repair gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5615.

Clinicopathologic characteristics of patients with early-onset colorectal cancer.

74 Background: Although the incidence of colorectal cancer has been declining in the United States in recent years, the incidence of young patients under 50 years of age has been reported to be increasing by several percent per year. Various studies have been conducted to characterize early-onset colorectal cancer (EO-CRC); however, it has been unclear whether it is a different entity from late-onset colorectal cancer (LO-CRC). In this study, we evaluated the clinicopathologic characteristics of EO-CRC in Japan. Methods: This study included 1,336 patients who underwent surgical resection for colorectal cancer at Tokyo Metropolitan Komagome Hospital from January 2015 to December 2019. We performed genetic testing for patients with suspected Lynch syndrome after providing genetic counseling and obtaining informed consent. The study protocol was approved by the Institutional Review Board. Results: Of the 1,336 colorectal cancer patients, 117 (8.9%) had EO-CRC with a median age at the diagnosis of 44 (17–49) years. Tumors were located at right-sided colon in 23 patients and left-sided colorectum in 92 patients with EO-CRC, respectively. The clinical stage of the tumor was I in 19, II in 23, III in 50, and IV in 23 patients with EO-CRC, respectively. Histologically, differentiated type, undifferentiated type, and mucinous carcinoma were 106, 5, and 3 patients, respectively. KRAS mutation was detected in 25.0% and BRAF V600E was detected in 4.3% of patients with EO-CRC. In the microsatellite status, MSI-High was detected in 6 (5.1%) patients with EO-CRC, of whom 3 (2.6%) were diagnosed with Lynch syndrome by genetic testing. Causative genes were MLH1 in 2 patients and MSH2 in one patient. In hereditary tumors, 2 patients with familial adenomatous polyposis were included in addition to Lynch syndrome. On the other hand, 1,219 (91.2%) had LO-CRC with 34.5% of KRAS mutation and 6.4% of BRAFV600E. MSI-High was detected in 69(5.7%) patients with LO-CRC，of whom 5 (0.4%) were diagnosed with Lynch syndrome. Lynch syndrome was significantly more common in EO-CRC (p=0.026). Conclusions: The alterations of KRAS and BRAF tended to be more frequent in EO-CRC. The frequency of hereditary CRC was more frequent in EO-CRC than in LO-CRC.

Rare single-nucleotide variants of MLH1 and MSH2 genes in patients with Lynch syndrome.

Approximately 5% of colorectal cancers (CRCs) are hereditary. Lynch syndrome (LS), also known as hereditary nonpolyposis colorectal cancer (HNPCC), is the most common form of recognized hereditary CRC. Although Iran, as a developing country, has a high incidence of CRC, the spectrum of variants has yet to be thoroughly investigated. This study aimed to investigate pathogenic and non-pathogenic variants in MLH1 and MSH2 genes in Iranian patients with suspected Lynch syndrome (sLS). In the present study, 25 peripheral blood samples were collected from patients with sLS and high microsatellite instability (MSI-H). After DNA extraction, all samples underwent polymerase chain reaction and Sanger sequencing to identify the variants in the exons of MLH1 and MSH2 genes. The identified variants were interpreted using prediction tools, and were finally reported under ACMG guidelines. In our study population, 13 variants were found in the MLH1 gene and 8 in the MSH2 gene. Interestingly, 7 of the 13 MLH1 variants and 3 of the 8 MSH2 variants were novel, whereas the remaining variants were previously reported or available in databases. In addition, some patients with sLS did not have variants in the exons of the MLH1 and MSH2 genes. The variants detected in the MLH1 and MSH2 genes had specific characteristics regarding the number, area of occurrence, and their relationship with demographic and clinicopathologic features. Overall, our results suggest that analysis of MLH1 and MSH2 genes alone is insufficient in the Iranian population, and more comprehensive tests are recommended for detecting LS.

A mosaic pathogenic variant in MSH6 causes MSH6-deficient colorectal and endometrial cancer in a patient classified as suspected Lynch syndrome: a case report

Germline pathogenic variants in the DNA mismatch repair (MMR) genes (Lynch syndrome) predispose to colorectal (CRC) and endometrial (EC) cancer. However, mosaic variants in the MMR genes have been rarely described. We identified a likely de novo mosaic MSH6:c.1135_1139del p.Arg379* pathogenic variant in a patient diagnosed with suspected Lynch syndrome/Lynch-like syndrome. The patient developed MSH6-deficient EC and CRC at 54 and 58 years of age, respectively, without a detectable germline MMR pathogenic variant. Multigene panel sequencing of tumor and blood-derived DNA identified an MSH6 somatic mutation (MSH6:c.1135_1139del p.Arg379*) common to both the EC and CRC, raising suspicion of mosaicism. A droplet digital polymerase chain reaction (ddPCR) assay detected the MSH6 variant at 5.34% frequency in normal colonic tissue, 3.49% in saliva and 1.64% in blood DNA, demonstrating the presence of the MSH6 variant in all three germ layers. This study highlights the utility of tumor sequencing to guide sensitive ddPCR testing to detect low-level mosaicism in the MMR genes. Further investigation of the prevalence of MMR mosaicism is needed to inform routine diagnostic approaches and genetic counselling.

A tumor focused approach to resolving the etiology of DNA mismatch repair deficient tumors classified as suspected Lynch syndrome

Routine screening of tumors for DNA mismatch repair (MMR) deficiency (dMMR) in colorectal (CRC), endometrial (EC) and sebaceous skin (SST) tumors leads to a significant proportion of unresolved cases classified as suspected Lynch syndrome (SLS). SLS cases (n = 135) were recruited from Family Cancer Clinics across Australia and New Zealand. Targeted panel sequencing was performed on tumor (n = 137; 80×CRCs, 33×ECs and 24xSSTs) and matched blood-derived DNA to assess for microsatellite instability status, tumor mutation burden, COSMIC tumor mutational signatures and to identify germline and somatic MMR gene variants. MMR immunohistochemistry (IHC) and MLH1 promoter methylation were repeated. In total, 86.9% of the 137 SLS tumors could be resolved into established subtypes. For 22.6% of these resolved SLS cases, primary MLH1 epimutations (2.2%) as well as previously undetected germline MMR pathogenic variants (1.5%), tumor MLH1 methylation (13.1%) or false positive dMMR IHC (5.8%) results were identified. Double somatic MMR gene mutations were the major cause of dMMR identified across each tumor type (73.9% of resolved cases, 64.2% overall, 70% of CRC, 45.5% of ECs and 70.8% of SSTs). The unresolved SLS tumors (13.1%) comprised tumors with only a single somatic (7.3%) or no somatic (5.8%) MMR gene mutations. A tumor-focused testing approach reclassified 86.9% of SLS into Lynch syndrome, sporadic dMMR or MMR-proficient cases. These findings support the incorporation of tumor sequencing and alternate MLH1 methylation assays into clinical diagnostics to reduce the number of SLS patients and provide more appropriate surveillance and screening recommendations.

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.

Discordance between germline genetic findings and abnormal tumor immunohistochemistry staining of mismatch repair proteins in individuals with suspected Lynch syndrome.

Tumor immunohistochemical staining (IHC) of DNA mismatch repair (MMR) proteins is often used to guide germline genetic testing and variant classification for patients with suspected Lynch syndrome. This analysis examined the spectrum of germline findings in a cohort of individuals showing abnormal tumor IHC. We assessed individuals with reported abnormal IHC findings and referred for testing with a six-gene syndrome-specific panel (n=703). Pathogenic variants (PVs) and variants of uncertain significance (VUS) in MMR genes were designated expected/unexpected relative to IHC results. The PV positive rate was 23.2% (163/703; 95% confidence interval [CI], 20.1%-26.5%); 8.0% (13/163; 95% CI, 4.3%-13.3%) of PV carriers had a PV in an unexpected MMR gene. Overall, 121 individuals carried VUS in MMR genes expected to be mutated based on IHC results. Based on independent evidence, in 47.1% (57/121; 95% CI, 38.0%-56.4%) of these individuals the VUSs were later reclassified as benign and in 14.0% (17/121; 95% CI, 8.4%-21.5%) of these individuals the VUSs were reclassified as pathogenic. Among patients with abnormal IHC findings, IHC-guided single-gene genetic testing may miss 8% of individuals with Lynch syndrome. In addition, in patients with VUS identified in MMR genes predicted to be mutated by IHC, extreme caution must be taken when the IHC results are considered in variant classification.

Occurrence of variants of unknown clinical significance in genetic testing for hereditary breast and ovarian cancer syndrome and Lynch syndrome: a literature review and analytical observational retrospective cohort study

Background and purposeOver the last decade, the implementation of multigene panels for hereditary tumor syndrome has increased at our institution (Inselspital, University Hospital Berne, Switzerland). The aim of this study was to determine the prevalence of variants of unknown significance (VUS) in patients with suspected Lynch syndrome and suspected hereditary breast and ovarian cancer syndrome, the latter in connection with the trend toward ordering larger gene panels.ResultsRetrospectively collected data from 1057 patients at our institution showed at least one VUS in 126 different cases (11.9%). In patients undergoing genetic testing for BRCA1/2, the prevalence of VUS was 6%. When < 10 additional genes were tested in addition to BRCA1/2, the prevalence increased to 13.8%, and 31.8% for > 10 additional genes, respectively. The gene most frequently affected with a VUS was ATM. 6% of our patients who were tested for Lynch syndrome had a VUS result in either MLH1, MSH2 or MSH6.ConclusionsOur data demonstrate that panel testing statistically significantly increases VUS rates due to variants in non-BRCA genes. Good genetic counseling before and after obtaining results is therefore particularly important when conducting multigene panels to minimize patient uncertainty due to VUS results.

Hereditary Colorectal Cancer: State of the Art in Lynch Syndrome

Hereditary non-polyposis colorectal cancer is also known as Lynch syndrome. Lynch syndrome is associated with pathogenetic variants in one of the mismatch repair (MMR) genes. In addition to colorectal cancer, the inefficiency of the MMR system leads to a greater predisposition to cancer of the endometrium and other cancers of the abdominal sphere. Molecular diagnosis is performed to identify pathogenetic variants in MMR genes. However, for many patients with clinically suspected Lynch syndrome, it is not possible to identify a pathogenic variant in MMR genes. Molecular diagnosis is essential for referring patients to specific surveillance to prevent the development of tumors related to Lynch syndrome. This review summarizes the main aspects of Lynch syndrome and recent advances in the field and, in particular, emphasizes the factors that can lead to the loss of expression of MMR genes.

THE ROLE OF MICROSATELLITE INSTABILITY IN COLORECTAL CANCER:&#x0D; LITERATURE REVIEW

Relevance: According to Globocan 2020, colorectal cancer (CRC) incidence in Kazakhstan is lower than in all OECD countries except&#x0D; Mexico. However, the CRC incidence in Kazakhstan is steadily growing. In the structure of cancer incidence, CRC went up from 5th place in 2006&#x0D; to 3rd place in 2021. More than 80% of tumors associated with Lynch syndrome were found to have microsatellite instability (MSI). Thus, MSI is&#x0D; detected in 12% of sporadic colon tumors. At that, MSI in the tumor is caused by a decreased activity of DNA repair genes that can be due to both&#x0D; hereditary and somatic causes.&#x0D; The study aimed to systematize the current literature data to consider the need and adequacy of pre-scribing preventive chemotherapy, personalizing the treatment of patients, and predicting the course of the disease in CRC.&#x0D; Methods: A review was made of the published results of scientific and clinical studies for 2006-2021 from the PubMed, MedLine, and Cancer&#x0D; Observe database for the keywords “colon cancer,” “microsatellite resistance,” “adjuvant chemotherapy,” “PCR study,” “IGH-study.”&#x0D; Results: The value and adequacy of determining the IHC characteristics of the MSI status. The prognostic and predictive value of MSI in CRC&#x0D; has been proven. The optimal treatment options were determined depending on the status of MSI.&#x0D; Conclusion: Tumors with certain MSI status should be classified as a separate group of malignancies. Instability status determination is&#x0D; preferred in case of suspected Lynch syndrome, in patients with stage II CRC, as well as in clinical and histological features characteristic of MSI&#x0D; (proximal localization of the primary tumor, mucinous histotype, poorly differentiated tumors, lymphocytic infiltration of the tumor). Further determination of these tumors’ molecular characteristics will help stratify patients who may respond differently to chemotherapy. Also, the MSI status&#x0D; determination can be an importnant prognostic marker in patients whose tumors have a somatic mutation in the BRAF gene

THE ROLE OF MICROSATELLITE INSTABILITY IN COLORECTAL CANCER: A LITERATURE REVIEW

Relevance: According to Globocan 2020, colorectal cancer (CRC) incidence in Kazakhstan is lower than in all OECD countries except Mexico. However, the CRC incidence in Kazakhstan is steadily growing. In the structure of cancer incidence, CRC went up from 5th&#x0D; place in 2006 to 3rd place in 2021. More than 80% of tumors associated with Lynch syndrome were found to have microsatellite instability&#x0D; (MSI). Thus, MSI is detected in 12% of sporadic colon tumors., MSI in cancer is caused by a decreased activity of DNA repair genes due&#x0D; to hereditary and somatic causes.&#x0D; The study aimed to systematize the current literature data to consider the need and adequacy of prescribing preventive chemotherapy,&#x0D; personalizing the treatment of patients, and predicting the course of the disease in CRC.&#x0D; Methods: A review was made of the published results of scientific and clinical studies for 2006-2021 from the PubMed, MedLine,&#x0D; and Cancer Observe databases for the keywords “colon cancer,” “microsatellite resistance,” “adjuvant chemotherapy,” “PCR study,”&#x0D; “IGH-study.”&#x0D; Results: The value and adequacy of determining the IHC characteristics of the MSI status. The prognostic and predictive value of MSI&#x0D; in CRC has been proven. The optimal treatment options were selected depending on the status of MSI.&#x0D; Conclusion: Tumors with certain MSI status should be classified as a separate group of malignancies. Instability status determination is preferred in case of suspected Lynch syndrome in patients with stage II CRC, as well as in clinical and histological features&#x0D; characteristic of MSI (proximal localization of the primary tumor, mucinous histotype, poorly differentiated tumors, lymphocytic infiltration of the tumor). Further determination of these tumors’ molecular characteristics will help stratify patients who may respond&#x0D; differently to chemotherapy. Also, the MSI status determination can be an important prognostic marker in patients whose tumors have&#x0D; a somatic mutation in the BRAF gene.

A calibrated cell-based functional assay to aid classification of MLH1 DNA mismatch repair gene variants.

Functional assays provide important evidence for classifying the disease significance of germline variants in DNA mismatch repair genes. Numerous laboratories, including our own, have developed functional assays to study mismatch repair gene variants. However, previous assays are limited due to the model system employed, the manner of gene expression, or the environment in which function is assessed. Here, we developed a human cell-based approach for testing the function of variants of uncertain significance (VUS) in the MLH1 gene. Using clustered regularly interspaced short palindromic repeats gene editing, we knocked inMLH1 VUS into the endogenous MLH1 loci in human embryonic stem cells. We examined their impact on RNA and protein, including their ability to prevent microsatellite instability and instigate a DNA damage response. A statistical clustering analysis determined the range of functions associated with known pathogenic or benign variants, and linear regression was performed using existing odds in favor of pathogenicity scores for these control variants to calibrate our functional assay results. By converting the functional outputs into a single odds in favor of pathogenicity score, variant classification expert panels can use these results to readily reassess these VUS. Ultimately, this information will guide proper diagnosis and disease management for suspected Lynch syndrome patients.

Heterogeneity in the psychosocial and behavioral responses associated with a diagnosis of suspected Lynch syndrome in women with endometrial cancer

BackgroundA suspected Lynch syndrome (SLS) diagnosis is made when a tumor exhibits DNA mismatch repair deficiency but cannot be definitively assigned to an inherited or non-inherited etiology. This diagnosis poses challenges for healthcare professionals, patients, and their families in managing future cancer risks and clinical care.MethodsThis qualitative study aimed to explore the psychosocial and behavioral responses of endometrial cancer (EC) patients receiving a SLS diagnosis (EC-SLS). Semi-structured telephone interviews were conducted with 15 EC-SLS women, transcribed, and thematically analyzed.ResultsMost who interpreted their result as negative for Lynch syndrome (LS) believed they were at population-level risk of cancer and felt happy and relieved. Many participants who interpreted their result as inconclusive/not definitive for LS were confused about their cancer risk and experienced negative emotions of anger and frustration. Despite variation in colorectal cancer screening recommendations reported by participants, most adhered to the advice given. Almost all participants communicated their genetic test result to immediate family members; however, communication of family cancer risk management advice was more limited due to most participants reporting not receiving family screening advice. A family history of cancer and a professional healthcare background influenced participants’ engagement in regular cancer screening.ConclusionThese findings highlight variability in the psychosocial and behavioral responses associated with EC-SLS, providing insight into how healthcare professionals can optimally manage and support such individuals.

A case of strongly suspected Lynch syndrome with colorectal neuroendocrine carcinoma

BackgroundNeuroendocrine cell carcinomas (NEC) of the colon and rectum are uncommon, representing ~ 0.1% of all colorectal carcinomas. They are associated with a much worse prognosis compared to adenocarcinoma of the colon and rectum, as death occurs in approximately half of all patients within 1 year. Lynch syndrome (LS) is the most common cause of inherited colorectal cancer, accounting for 2–4% of newly diagnosed colorectal cancer cases. This case is extremely rare which was strongly suspected LS as the background, and NEC as the histological type of colorectal cancer.Case presentationThe patient was a 44-year-old man presenting with vomiting as the main complaint. He had undergone ileocecal resection for cecal cancer at age 29. The diagnosis was obstructive descending colorectal cancer, and colonoscopy revealed tumors in the rectum and sigmoid colon in addition. Due to multiple occurrences of colorectal cancer and its prevalence in the patient’s family, LS was suspected. The operation which was a subtotal proctocolectomy was performed. Pathological analysis revealed complete curative resection and the descending colon cancer of the obstructed portion was at the most advanced pathological Stage IIIC in UICC TNM classification, and the tissue type was a NEC. The Ki-67 index was 70%. The results of the microsatellite instability (MSI) test showed high-frequency MSI. The BRAF V600E variant was negative. The immunoexpression of MLH1 was positive, MSH2 was negative, PMS2 was positive, and MSH6 was negative.ConclusionsExtended surgery is recommended for incipient colorectal cancer in LS cases in order to reliably reduce the risk of developing metachronous colorectal cancer. The survival outcome of surgery alone on digestive tract NECs, even locoregional lesions that are completely resection, is extremely poor. It is currently unclear if digestive tract NECs develop more readily in patients with LS. The accumulation of additional cases is necessary.

Splicing analyses for variants in MMR genes: best practice recommendations from the European Mismatch Repair Working Group

Over 20% of the DNA mismatch repair (MMR) germline variants in suspected Lynch syndrome patients are classified as variants of uncertain significance (VUS). Well-established functional assays are pivotal for assessing the biological impact of these variants and provide relevant evidence for clinical classification. In our collaborative European Mismatch Repair Working Group (EMMR-WG) we compared three different experimental approaches for evaluating the effect of seven variants on mRNA splicing in MMR genes: (i) RT-PCR of full-length transcripts (FLT), (ii) RT-PCR of targeted transcript sections (TTS), both from patient biological samples and (iii) minigene splicing assays. An overall good concordance was observed between splicing patterns in TTS, FLT and minigene analyses for all variants. The FLT analysis depicted a higher number of different isoforms and mitigated PCR-bias towards shorter isoforms. TTS analyses may miss aberrant isoforms and minigene assays may under/overestimate the severity of certain splicing defects. The interpretation of the experimental findings must be cautious to adequately discriminate abnormal events from physiological complex alternative splicing patterns. A consensus strategy for investigating the impact of MMR variants on splicing was defined. First, RNA should be obtained from patient’s cell cultures (such as fresh lymphocyte cultures) incubated with/without a nonsense-mediated decay inhibitor. Second, FLT RT-PCR analysis is recommended to oversee all generated isoforms. Third, TTS analysis and minigene assays are useful independent approaches for verifying and clarifying FLT results. The use of several methodologies is likely to increase the strength of the experimental evidence which contributes to improve variant interpretation.

Cancer Risk C (CR-C), a functional genomics test is a sensitive and rapid test for germline mismatch repair deficiency

PurposeHeritable pathogenic variants in the DNA mismatch repair (MMR) pathway cause Lynch syndrome, a condition that significantly increases risk of colorectal and other cancers. At least half of individuals tested using gene panel sequencing have a variant of uncertain significance or no variant identified leading to no diagnosis. To fill this diagnostic gap, we developed Cancer Risk C (CR-C), a flow variant assay test. MethodsIn response to treatment with an alkylating agent, individual assays of the nuclear translocation of MLH1, MSH2, BARD1, PMS2, and BRCA2 proteins and the nuclear phosphorylation of the ATM and ATR proteins distinguished pathogenic/likely pathogenic (P/LP) from benign/likely benign variants in MMR genes. ResultsA risk classification score based on MLH1, MSH2, and ATR assays was 100% sensitive and 98% specific. Causality of MMR P/LP variants was shown through gene editing and rescue. In individuals with suspected Lynch syndrome but no P/LP, CR-C identified most (73%) as having germline MMR defects. Direct comparison of CR-C on matched blood samples and lymphoblastoid cell lines yielded comparable results (r2 > 0.9). ConclusionFor identifying germline MMR defects, CR-C provides augmentation to traditional panel sequencing through greater accuracy, shorter turnaround time (48 hours), and performance on blood with minimal sample handling.

Identification of Germline Mutations in Upper Tract Urothelial Carcinoma With Suspected Lynch Syndrome

ObjectiveWhole-exon sequencing (WES) is a commercially available tool for hereditary disease testing. However, little is known about hereditary upper-tract urothelial carcinoma (UTUC) in the Chinese population. This study aims to investigate the prevalence of Lynch syndrome (LS) in UTUC patients with high-risk features and identify the germline mutations of genetic predisposition gene mutations in those patients.MethodsIn total, 354 consecutive UTUC patients undergoing surgery were universally recruited, of whom 108 patients under 60 years old or with a personal/family history of cancer underwent universal immunohistochemistry staining to detect the expression of mismatch repair (MMR) proteins (MLH1, MSH2, MSH6 and PMS2). Patients with deficient or weak MMR protein staining or meeting the Amsterdam II criterion were defined as suspected LS patients, who further experienced microsatellite instability (MSI) (BAT25, BAT26, BAT40, D2S123, D5S346, D17S250) detection and performed WES analysis to explore germline pathogenic/likely pathogenic (P/LP) alterations.ResultsOf 108 patients, 90 (83.3%) cases were included due to younger than 60 years, and 18 cases due to personal/family history. IHC staining identified 21 patients with deficient MMR protein staining and 15 cases with weak MMR protein staining. Three cases met the Amsterdam II criterion but with proficient MMR protein staining. Finally, WES analysis was performed in 38 suspected LS patients and P/LP germline mutations were identified in 22 individuals. Genetic testing confirmed 5 LS cases, including 3 cases with novel mutations. MSI-harboring tumor was discovered in 4 LS cases, one of whom had weak MMR protein staining. Germline P/LP variants in DNA damage repair genes were found in 11 cases. In addition, we found that 11 patients had high- or moderate- penetrance P/LP mutations other than MMR genes. The common P/LP variants in high- or moderate-penetrance genes were 4 in ATM, 3 in MSH6 and KIT, and 2 in APC, NF1 and DICER.ConclusionsWe identified approximately 11% of UTUC cases as suspected LS and at least 1.4% patients with confirmed LS-associated UTUC. In addition, broader germline genetic testing could be considered to screen for cancer severity in hereditary UTUC patients.