Molecular Background of Colorectal Tumors From Patients With Lynch Syndrome Associated With Germline Variants in PMS2.

BACKGROUND & AIMS: Germline variants in mismatch repair genes MLH1, MSH2 (EPCAM), MSH6, or PMS2 cause Lynch syndrome. Patients with these variants have an increased risk of developing colorectal cancers (CRCs) that differ from sporadic CRCs in genetic and histologic features. It has been a challenge to study CRCs associated with PMS2 variants (PMS2-associated CRCs) because these develop less frequently and in older patients than CRCs with variants in other mismatch repair genes. METHODS: We analyzed 20 CRCs associated with germline variants in PMS2, 22 sporadic CRCs, 18 CRCs with germline variants in MSH2, and 24 CRCs from patients with germline variants in MLH1. Tumor tissue blocks were collected from Dutch pathology departments in 2017. After extraction of tumor DNA, we used a platform designed to detect approximately 3,000 somatic hotspot variants in 55 genes (including KRAS, APC, CTNNB1, and TP53). Somatic variant frequencies were compared using the Fisher exact test. RESULTS: None of the PMS2-associated CRCs contained any somatic variants in the catenin-ß1 gene (CTNNB1), which encodes ß-catenin, whereas 14 of 24 MLH1-associated CRCs (58%) contained variants in CTNNB1. Half the PMS2-associated CRCs contained KRAS variants, but only 20% of these were in hotspots that encoded G12D or G13D. These hotspot variants occurred more frequently in CRCs associated with variants in MLH1 (37.5%; P = .44) and MSH2 (71.4%; P = .035) than in those associated with variants in PMS2. CONCLUSIONS: In a genetic analysis of 84 colorectal tumors, we found tumors from patients with PMS2-associated Lynch syndrome to be distinct from colorectal tumors associated with defects in other mismatch repair genes. This might account for differences in development and less frequent occurrence.

Comprehensive Mutation Analysis of PMS2 in a Large Cohort of Probands Suspected of Lynch Syndrome or Constitutional Mismatch Repair Deficiency Syndrome.

Monoallelic PMS2 germline mutations cause 5%-15% of Lynch syndrome, a midlife cancer predisposition, whereas biallelic PMS2 mutations cause approximately 60% of constitutional mismatch repair deficiency (CMMRD), a rare childhood cancer syndrome. Recently improved DNA- and RNA-based strategies are applied to overcome problematic PMS2 mutation analysis due to the presence of pseudogenes and frequent gene conversion events. Here, we determined PMS2 mutation detection yield and mutation spectrum in a nationwide cohort of 396 probands. Furthermore, we studied concordance between tumor IHC/MSI (immunohistochemistry/microsatellite instability) profile and mutation carrier state. Overall, we found 52 different pathogenic PMS2 variants explaining 121 Lynch syndrome and nine CMMRD patients. In vitro mismatch repair assays suggested pathogenicity for three missense variants. Ninety-one PMS2 mutation carriers (70%) showed isolated loss of PMS2 in their tumors, for 31 (24%) no or inconclusive IHC was available, and eight carriers (6%) showed discordant IHC (presence of PMS2 or loss of both MLH1 and PMS2). Ten cases with isolated PMS2 loss (10%; 10/97) harbored MLH1 mutations. We confirmed that recently improved mutation analysis provides a high yield of PMS2 mutations in patients with isolated loss of PMS2 expression. Application of universal tumor prescreening methods will however miss some PMS2 germline mutation carriers.

Cancer risks by sex and variant type in PTEN hamartoma tumor syndrome.

BACKGROUND: PTEN Hamartoma Tumor Syndrome (PHTS) is a rare syndrome with a broad phenotypic spectrum, including increased risks of breast (BC, 67%-78% at age 60 years), endometrial (EC, 19%-28%), and thyroid cancer (TC, 6%-38%). Current risks are likely overestimated due to ascertainment bias. We aimed to provide more accurate and personalized cancer risks. METHODS: This was a European, adult PHTS cohort study with data from medical files, registries, and/or questionnaires. Cancer risks and hazard ratios were assessed with Kaplan-Meier and Cox regression analyses, and standardized incidence ratios were calculated. Bias correction consisted of excluding cancer index cases and incident case analyses. RESULTS: A total of 455 patients were included, including 50.5% index cases, 372 with prospective follow-up (median 6 years, interquartile range = 3-10 years), and 159 of 281 females and 39 of 174 males with cancer. By age 60 years, PHTS-related cancer risk was higher in females (68.4% to 86.3%) than males (16.4% to 20.8%). Female BC risks ranged from 54.3% (95% confidence interval [CI] = 43.0% to 66.4%) to 75.8% (95% CI = 60.7% to 88.4%), with two- to threefold increased risks for PTEN truncating and approximately twofold for phosphatase domain variants. EC risks ranged from 6.4% (95% CI = 2.1% to 18.6%) to 22.1% (95% CI = 11.6% to 39.6%) and TC risks from 8.9% (95% CI = 5.1% to 15.3%) to 20.5% (95% CI = 11.3% to 35.4%). Colorectal cancer, renal cancer, and melanoma risks were each less than 10.0%. CONCLUSIONS: Females have a different BC risk depending on their PTEN germline variant. PHTS patients are predominantly at risk of BC (females), EC, and TC. This should be the main focus of surveillance. These lower, more unbiased and personalized risks provide guidance for optimized cancer risk management.

Radiologic Characteristics of Spinal Hemangioblastomas in von Hippel Lindau Disease as Guidance in Clinical Interventions.

OBJECTIVE: Hemangioblastomas in the central nervous system are the most common manifestation of von Hippel-Lindau (VHL) disease. Because the growth rate of hemangioblastomas is unpredictable, regular follow-up is mandatory, focusing on clinical symptoms and imaging of the central nervous system. However, clinical symptoms may be subtle and nonspecific, and data about the relationship between the radiologic findings and clinical symptoms are sparse. This study aims to evaluate if and how findings of magnetic resonance imaging (MRI) regarding spinal hemangioblastomas are associated with symptoms of VHL disease, with special attention to peritumoral edema and spinal cysts. METHODS: Serial spinal MRI scans of 43 genetically or clinically established VHL patients with at least 2 years of follow-up were reevaluated to examine the volume, growth rate, and location of spinal hemangioblastomas and the presence, size, and growth rate of peritumoral edema and cysts. Findings were compared with clinical symptoms using the Fisher exact test. RESULTS: We observed a total of 77 spinal hemangioblastomas in 28 patients. Eight of the 28 patients showed peritumoral edema and spinal cysts, and 1 patient showed peritumoral edema without cyst formation; 6 of these 9 patients showed clinical symptoms. Both peritumoral edema and spinal cysts were associated with clinical symptoms (P = 0.023 and P = 0.011, respectively). CONCLUSIONS: The presence of peritumoral edema and/or spinal cysts shown on MRI in VHL patients with spinal hemangioblastomas is associated with symptoms in more than half of the patients and may alert the clinician to intensify clinical and radiologic surveillance.

Clinical value of a screening tool for tumor predisposition syndromes in childhood cancer patients (TuPS): a prospective, observational, multi-center study.

Recognizing a tumor predisposition syndrome (TPS) in a child with cancer is of clinical relevance. Earlier we developed a screening tool to increase diagnostic accuracy and clinical efficiency of identifying TPSs in children with cancer. Here we report on the value of this tool in clinical practice. TuPS is a prospective, observational, multi-center study including children newly diagnosed with cancer from 2016 to 2019 in the Netherlands. Children in whom a TPS had been diagnosed before the cancer diagnosis were excluded. The screening tool consists of a checklist, 2D and 3D photographic series and digital assessment of these by a clinical geneticist. If a TPS was suspected, the patient was assessed positive and referred for routine genetic consultation. Primary aim was to assess the clinical value of this new screening tool. Of the 363 included patients, 57% (208/363) were assessed positive. In 15% of patients (32/208), the 2D photographic series with (n = 12) or without (n = 20) 3D photographs were decisive in the positive assessment. In 2% (4/208) of positive assessed patients, a TPS was diagnosed, and in an additional 2% (4/208) a germline variant of uncertain significance was found. Thirty-five negatively assessed patients were evaluated through routine genetic consultation as controls, in none a TPS was detected. Using the screening tool, 57% of the patients were assessed as suspected for having a TPS. No false negative results were identified in the negative control group in the clinical care setting. The observed prevalence of TPS was lower than expected, due to selection bias in the cohort.

Germline hypermethylation of MLH1 and EPCAM deletions are a frequent cause of Lynch syndrome.

It was shown that Lynch syndrome can be caused by germline hypermethylation of the MLH1 and MSH2 promoters. Furthermore, it has been demonstrated very recently that germline deletions of the 3' region of EPCAM cause transcriptional read-through which results in silencing of MSH2 by hypermethylation. We wanted to determine the prevalence of germline MLH1 promoter hypermethylation and of germline and somatic MSH2 promoter hypermethylation in a large group of Lynch syndrome-suspected patients. From a group of 331 Lynch Syndrome-suspected patients we selected cases, who had no germline MLH1, MSH2, or MSH6 mutation and whose tumors showed loss of MLH1 or MSH2, or, if staining was unavailable, had a tumor with microsatellite instability. Methylation assays were performed to test these patients for germline MLH1 and/or MSH2 promoter hypermethylation. Two patients with germline MLH1 promoter hypermethylation and no patients with germline MSH2 promoter hypermethylation were identified. In the subgroup screened for germline MSH2 promoter hypermethylation, we identified 3 patients with somatic MSH2 promoter hypermethylation in their tumors, which was caused by a germline EPCAM deletion. In the group of 331 Lynch Syndrome-suspected patients, the frequencies of germline MLH1 promoter hypermethylation and somatic MSH2 promoter hypermethylation caused by germline EPCAM deletions are 0.6 and 0.9%, respectively. These mutations, therefore, seem to be rather infrequent. However, the contribution of germline MLH1 hypermethylation and EPCAM deletions to the genetically proven Lynch syndrome cases in this cohort is very high. Previously 27 pathogenic mutations were identified; the newly identified mutations now represent 16% of all mutations.

The incidence of consecutive manifestations in Von Hippel-Lindau disease.

Von Hippel-Lindau (VHL) disease is an autosomal dominant rare tumor syndrome characterized by high penetrance. VHL mutation carriers develop numerous manifestations in multiple organs during life. The natural course of development of new and growth of existing VHL-related manifestations is still unclear. In this study we aimed to gain insight into the development of subsequent manifestations in VHL disease. We retrospectively scored each new VHL-related manifestation as detected by standard follow-up (retina, central nervous system, kidneys and pancreas, excluding adrenal and endolymfatic sac manifestations) in 75 VHL mutation carriers. The Kaplan-Meier method was used to plot the cumulative proportions of all consecutive manifestations in each organ against age. The cumulative average number of manifestations in all organs during life was calculated by summating these cumulative proportions. Poisson model parameters were used to calculate average time to the detection of consecutive VHL manifestations in each organ. Consecutive VHL-related kidney and retina manifestations during life occur linearly according to Poisson distribution model. The total number of VHL manifestations rises linearly, with an average of seven VHL-related lesions at age 60 years. The incidence of consecutive VHL-related manifestations is constant during life in VHL mutation carriers. Our data is consistent with the notion that somatic inactivation of the remaining allele (Knudson's "two-hit" hypothesis) is the determining factor in developing new VHL-related manifestations.

The Apparent Genetic Anticipation in PMS2-Associated Lynch Syndrome Families Is Explained by Birth-cohort Effect.

BACKGROUND: PMS2-associated Lynch syndrome is characterized by a relatively low colorectal cancer penetrance compared with other Lynch syndromes. However, age at colorectal cancer diagnosis varies widely, and a strong genetic anticipation effect has been suggested for PMS2 families. In this study, we examined proposed genetic anticipation in a sample of 152 European PMS2 families. METHODS: The 152 families (637 family members) that were eligible for analysis were mainly clinically ascertained via clinical genetics centers. We used weighted Cox-type random effects model, adjusted by birth cohort and sex, to estimate the generational effect on the age of onset of colorectal cancer. Probands and young birth cohorts were excluded from the analyses. Weights represented mutation probabilities based on kinship coefficients, thus avoiding testing bias. RESULTS: Family data across three generations, including 123 colorectal cancers, were analyzed. When compared with the first generation, the crude HR for anticipation was 2.242 [95% confidence interval (CI), 1.162-4.328] for the second generation and 2.644 (95% CI, 1.082-6.464) for the third generation. However, after correction for birth cohort and sex, the effect vanished [HR = 1.302 (95% CI, 0.648-2.619) and HR = 1.074 (95% CI, 0.406-2.842) for second and third generations, respectively]. CONCLUSIONS: Our study did not confirm previous reports of genetic anticipation in PMS2-associated Lynch syndrome. Birth-cohort effect seems the most likely explanation for observed younger colorectal cancer diagnosis in subsequent generations, particularly because there is currently no commonly accepted biological mechanism that could explain genetic anticipation in Lynch syndrome. IMPACT: This new model for studying genetic anticipation provides a standard for rigorous analysis of families with dominantly inherited cancer predisposition.

SNP association study in PMS2-associated Lynch syndrome.

Lynch syndrome (LS) patients are at high risk of developing colorectal cancer (CRC). Phenotypic variability might in part be explained by common susceptibility loci identified in Genome Wide Association Studies (GWAS). Previous studies focused mostly on MLH1, MSH2 and MSH6 carriers, with conflicting results. We aimed to determine the role of GWAS SNPs in PMS2 mutation carriers. A cohort study was performed in 507 PMS2 carriers (124 CRC cases), genotyped for 24 GWAS SNPs, including SNPs at 11q23.1 and 8q23.3. Hazard ratios (HRs) were calculated using a weighted Cox regression analysis to correct for ascertainment bias. Discrimination was assessed with a concordance statistic in a bootstrap cross-validation procedure. Individual SNPs only had non-significant associations with CRC occurrence with HRs lower than 2, although male carriers of allele A at rs1321311 (6p21.31) may have increased risk of CRC (HR = 2.1, 95% CI 1.2-3.0). A polygenic risk score (PRS) based on 24 HRs had an HR of 2.6 (95% CI 1.5-4.6) for the highest compared to the lowest quartile, but had no discriminative ability (c statistic 0.52). Previously suggested SNPs do not modify CRC risk in PMS2 carriers. Future large studies are needed for improved risk stratification among Lynch syndrome patients.

High Yield of Pathogenic Germline Mutations Causative or Likely Causative of the Cancer Phenotype in Selected Children with Cancer.

Purpose: In many children with cancer and characteristics suggestive of a genetic predisposition syndrome, the genetic cause is still unknown. We studied the yield of pathogenic mutations by applying whole-exome sequencing on a selected cohort of children with cancer.Experimental Design: To identify mutations in known and novel cancer-predisposing genes, we performed trio-based whole-exome sequencing on germline DNA of 40 selected children and their parents. These children were diagnosed with cancer and had at least one of the following features: (1) intellectual disability and/or congenital anomalies, (2) multiple malignancies, (3) family history of cancer, or (4) an adult type of cancer. We first analyzed the sequence data for germline mutations in 146 known cancer-predisposing genes. If no causative mutation was found, the analysis was extended to the whole exome.Results: Four patients carried causative mutations in a known cancer-predisposing gene: TP53 and DICER1 (n = 3). In another 4 patients, exome sequencing revealed mutations causing syndromes that might have contributed to the malignancy (EP300-based Rubinstein-Taybi syndrome, ARID1A-based Coffin-Siris syndrome, ACTB-based Baraitser-Winter syndrome, and EZH2-based Weaver syndrome). In addition, we identified two genes, KDM3B and TYK2, which are possibly involved in genetic cancer predisposition.Conclusions: In our selected cohort of patients, pathogenic germline mutations causative or likely causative of the cancer phenotype were found in 8 patients, and two possible novel cancer-predisposing genes were identified. Therewith, our study shows the added value of sequencing beyond a cancer gene panel in selected patients, to recognize childhood cancer predisposition. Clin Cancer Res; 24(7); 1594-603. ©2018 AACR.

Cancer Risks for PMS2-Associated Lynch Syndrome.

PURPOSE: Lynch syndrome due to pathogenic variants in the DNA mismatch repair genes MLH1, MSH2, and MSH6 is predominantly associated with colorectal and endometrial cancer, although extracolonic cancers have been described within the Lynch tumor spectrum. However, the age-specific cumulative risk (penetrance) of these cancers is still poorly defined for PMS2-associated Lynch syndrome. Using a large data set from a worldwide collaboration, our aim was to determine accurate penetrance measures of cancers for carriers of heterozygous pathogenic PMS2 variants. METHODS: A modified segregation analysis was conducted that incorporated both genotyped and nongenotyped relatives, with conditioning for ascertainment to estimates corrected for bias. Hazard ratios (HRs) and corresponding 95% CIs were estimated for each cancer site for mutation carriers compared with the general population, followed by estimation of penetrance. RESULTS: In total, 284 families consisting of 4,878 first- and second-degree family members were included in the analysis. PMS2 mutation carriers were at increased risk for colorectal cancer (cumulative risk to age 80 years of 13% [95% CI, 7.9% to 22%] for males and 12% [95% CI, 6.7% to 21%] for females) and endometrial cancer (13% [95% CI, 7.0%-24%]), compared with the general population (6.6%, 4.7%, and 2.4%, respectively). There was no clear evidence of an increased risk of ovarian, gastric, hepatobiliary, bladder, renal, brain, breast, prostate, or small bowel cancer. CONCLUSION: Heterozygous PMS2 mutation carriers were at small increased risk for colorectal and endometrial cancer but not for any other Lynch syndrome-associated cancer. This finding justifies that PMS2-specific screening protocols could be restricted to colonoscopies. The role of risk-reducing hysterectomy and bilateral salpingo-oophorectomy for PMS2 mutation carriers needs further discussion.

Childhood tumours with a high probability of being part of a tumour predisposition syndrome; reason for referral for genetic consultation.

INTRODUCTION: Recognising a tumour predisposition syndrome (TPS) in childhood cancer patients is of major clinical relevance. The presence of a TPS may be suggested by the type of tumour in the child. We present an overview of 23 childhood tumours that in themselves should be a reason to refer a child for genetic consultation. METHODS: We performed a PubMed search to review the incidence of TPSs in children for 85 tumour types listed in the International Classification of Childhood Cancer third edition (ICCC-3). The results were discussed during a national consensus meeting with representative clinical geneticists from all six academic paediatric oncology centres in The Netherlands. A TPS incidence of 5% or more was considered a high probability and therefore in itself a reason for referral to a clinical geneticist. RESULTS: The literature search resulted in data on the incidence of a TPS in 26 tumours. For 23/26 tumour types, a TPS incidence of 5% or higher was reported. In addition, during the consensus meeting the experts agreed that children with any carcinoma should always be referred for clinical genetic consultation as well, as it may point to a TPS. CONCLUSION: We present an overview of 23 paediatric tumours with a high probability of a TPS; this will facilitate paediatric oncologists to decide which patients should be referred for genetic consultation merely based on type of tumour.

Cancer risk and genotype-phenotype correlations in PTEN hamartoma tumor syndrome.

Patients with germline PTEN mutations are at high risk of developing benign and malignant tumours. We aimed to evaluate the cumulative risk of several types of cancer and of dysplastic cerebellar gangliocytoma (Lhermitte-Duclos disease, LDD). In addition, genotype-phenotype correlations in PTEN hamartoma tumour syndrome (PHTS) were assessed. Data on patients with PTEN mutations were collected from clinical genetic centres in Western Europe, Australia, and the USA. The cumulative risk of developing cancers of the breast, thyroid, endometrium, skin, kidneys, colorectum, and lungs, and also LDD was calculated by Kaplan-Meier methods. Associations between mutations and cancer were assessed by Chi square means. A total of 180 germline PTEN mutation carriers, 81 males (45%), from nine countries were included. The cumulative risk of developing any cancer and/or LDD at age 60 was 56% for males and 87% for females (p = 0.001). Females had significant higher risks of developing breast cancer, thyroid cancer, and LDD than males. The only genotype-phenotype correlation identified was a lower frequency of thyroid cancer in patients with missense mutations (p = 0.014). In conclusion, PHTS patients, particularly females, have a substantial risk of developing one or more tumours from a broad tumour spectrum. Major genotype-phenotype associations could not be identified.

Split hand/foot malformation due to chromosome 7q aberrations(SHFM1): additional support for functional haploinsufficiency as the causative mechanism.

We report on three patients with split hand/foot malformation type 1 (SHFM1). We detected a deletion in two patients and an inversion in the third, all involving chromosome 7q21q22. We performed conventional chromosomal analysis, array comparative genomic hybridization and fluorescence in situ hybridization. Both deletions included the known genes associated with SHFM1 (DLX5, DLX6 and DSS1), whereas in the third patient one of the inversion break points was located just centromeric to these genes. These observations confirm that haploinsufficiency due to either a simultaneous deletion of these genes or combined downregulation of gene expression due to a disruption in the region between these genes and a control element could be the cause of the syndrome. We review previously reported studies that support this hypothetical mechanism.