Role of psychological background in cancer susceptibility genetic testing distress: It is not only about a positive result.

Clinical and familial factors predict psychological distress after genetic testing for cancer susceptibility. However, the contribution of an individual's psychological background to such distress is unclear. This study aims to analyze the psychological impact of genetic testing and to identify the profile of individuals at higher risk. This is a longitudinal multicenter study of individuals undergoing genetic testing for cancer susceptibility. Demographic, clinical, genetic, familial, and psychological (personality types, cancer worry) characteristics were assessed by validated questionnaires the day of genetic testing. Distress, uncertainty, and positive experience perception (MICRA scale) were evaluated at the results disclosure visit, and 3 and 12 months afterwards. Multivariate analysis was performed. A total of 714 individuals were included. A high neuroticism score, high baseline cancer worry, and a positive genetic test result were independently associated with higher psychological impact (p-value < 0.05). The highest risk profile (10% of the cohort) included women with high level of neuroticism and a positive result. Uncertainty was mainly associated with a high level of neuroticism, regardless of the genetic test result. A holistic approach to personalized germline genetic counseling should include the assessment of personality dimensions.

Patients' and professionals' perspective of non-in-person visits in hereditary cancer: predictors and impact of the COVID-19 pandemic.

PURPOSE: To identify predictors of patient acceptance of non-in-person cancer genetic visits before and after the COVID-19 pandemic and assess the preferences of health-care professionals. METHODS: Prospective multicenter cohort study (N = 578, 1 February 2018-20 April 2019) and recontacted during the COVID-19 lockdown in April 2020. Health-care professionals participated in May 2020. Association of personality traits and clinical factors with acceptance was assessed with multivariate analysis. RESULTS: Before COVID-19, videoconference was more accepted than telephone-based visits (28% vs. 16% pretest, 30% vs. 19% post-test). Predictors for telephone visits were age (pretest, odds ratio [OR] 10-year increment = 0.79; post-test OR 10Y = 0.78); disclosure of panel testing (OR = 0.60), positive results (OR = 0.52), low conscientiousness group (OR = 2.87), and post-test level of uncertainty (OR = 0.93). Predictors for videoconference were age (pretest, OR 10Y = 0.73; post-test, OR 10Y = 0.75), educational level (pretest: OR = 1.61), low neuroticism (pretest, OR = 1.72), and post-test level of uncertainty (OR = 0.96). Patients' reported acceptance for non-in-person visits after COVID-19 increased to 92% for the pretest and 85% for the post-test. Health-care professionals only preferred non-in-person visits for disclosure of negative results (83%). CONCLUSION: These new delivery models need to recognize challenges associated with age and the psychological characteristics of the population and embrace health-care professionals' preferences.

Elucidating the molecular basis of MSH2-deficient tumors by combined germline and somatic analysis.

In a proportion of patients presenting mismatch repair (MMR)-deficient tumors, no germline MMR mutations are identified, the so-called Lynch-like syndrome (LLS). Recently, MMR-deficient tumors have been associated with germline mutations in POLE and MUTYH or double somatic MMR events. Our aim was to elucidate the molecular basis of MSH2-deficient LS-suspected cases using a comprehensive analysis of colorectal cancer (CRC)-associated genes at germline and somatic level. Fifty-eight probands harboring MSH2-deficient tumors were included. Germline mutational analysis of MSH2 (including EPCAM deletions) and MSH6 was performed. Pathogenicity of MSH2 variants was assessed by RNA analysis and multifactorial likelihood calculations. MSH2 cDNA and methylation of MSH2 and MSH6 promoters were studied. Matched blood and tumor DNA were analyzed using a customized next generation sequencing panel. Thirty-five individuals were carriers of pathogenic or probably pathogenic variants in MSH2 and EPCAM. Five patients harbored 4 different MSH2 variants of unknown significance (VUS) and one had 2 novel MSH6 promoter VUS. Pathogenicity assessment allowed the reclassification of the 4 MSH2 VUS and 6 probably pathogenic variants as pathogenic mutations, enabling a total of 40 LS diagnostics. Predicted pathogenic germline variants in BUB1, SETD2, FAN1 and MUTYH were identified in 5 cases. Three patients had double somatic hits in MSH2 or MSH6, and another 2 had somatic alterations in other MMR genes and/or proofreading polymerases. In conclusion, our comprehensive strategy combining germline and somatic mutational status of CRC-associated genes by means of a subexome panel allows the elucidation of up to 86% of MSH2-deficient suspected LS tumors.

Characteristics of Adrenocortical Carcinoma Associated With Lynch Syndrome.

CONTEXT: Lynch syndrome (LS) is the most common inherited colorectal and endometrial cancer syndrome, caused by germline mutations in DNA mismatch repair (MMR) genes. It is also characterized by an increased risk of other tumors with lower prevalence, such as adrenal cortical carcinoma (ACC), an endocrine tumor with an incidence of <2 cases/million individuals/year. Most ACC developed during childhood are associated with hereditary syndromes. In adults, this association is not as well established as in children. Previous studies showed a 3.2% prevalence of LS among patients with ACC. EVIDENCE ACQUISITION: The objective of this study is to determine the prevalence of ACC in a Spanish LS cohort and their molecular and histological characteristics. This retrospective study includes 634 patients from 220 LS families registered between 1999 and 2018. EVIDENCE SYNTHESIS: During the follow-up period, 3 patients were diagnosed with ACC (0.47%); all were carriers of a MSH2 germline mutation. The 3 ACC patients presented loss of expression of MSH2 and MSH6 proteins. One tumor analysis showed loss of heterozygosity of the MSH2 wildtype allele. Our findings support previous data that considered ACC as a LS spectrum tumor. CONCLUSION: MMR protein immunohistochemistry screening could be an efficient strategy to detect LS in patients with ACC.

Correction: Dueñas et al. Assessing Effectiveness of Colonic and Gynecological Risk Reducing Surgery in Lynch Syndrome Individuals. Cancers 2020, 12, 3419.

In the original article, there was a mistake in Figure 3 as published [...].

Paired Somatic-Germline Testing of 15 Polyposis and Colorectal Cancer-Predisposing Genes Highlights the Role of APC Mosaicism in de Novo Familial Adenomatous Polyposis.

Familial adenomatous polyposis (FAP) is an autosomal dominant syndrome responsible for 1% of colorectal cancers (CRCs). Up to 90% of classic FAPs are caused by inactivating mutations in APC, and mosaicism has been previously reported in 20% of de novo cases, usually linked to milder phenotypic manifestations. This study aimed to explore the prevalence of mosaicism in 11 unsolved cases of classic FAP and to evaluate the diagnostic yield of somatic testing. Paired samples of colorectal polyps, tumors, and/or mucosa were analyzed using a custom next-generation sequencing panel targeting 15 polyposis and CRC-predisposing genes. Whenever possible, the extension of mosaicism to blood or sperm was also examined. Of 11 patients with classic adenomatous polyposis, a mosaic pathogenic variant in APC was identified in 7 (64%). No other altered genes were identified. In two of seven patients (29%), mosaicism was found restricted to colonic tissues, whereas in five of seven patients (71%), it was extended to the blood. Germline affectation was confirmed in one patient. We report the first analysis at a somatic level of 15 genes associated with CRC susceptibility, which highlights the role of APC mosaicism in classic FAP etiology. The results further reinforce the importance of testing target tissues when blood test results are negative.

Improving Genetic Testing in Hereditary Cancer by RNA Analysis: Tools to Prioritize Splicing Studies and Challenges in Applying American College of Medical Genetics and Genomics Guidelines.

RNA analyses are a potent tool to identify spliceogenic effects of DNA variants, although they are time-consuming and cannot always be performed. We present splicing assays of 20 variants that represent a variety of mutation types in 10 hereditary cancer genes and attempt to incorporate these results into American College of Medical Genetics and Genomics (ACMG) classification guidelines. Sixteen single-nucleotide variants, 3 exon duplications, and 1 single-exon deletion were selected and prioritized by in silico algorithms. RNA was extracted from short-term lymphocyte cultures to perform RT-PCR and Sanger sequencing, and allele-specific expression was assessed whenever possible. Aberrant transcripts were detected in 14 variants (70%). Variant interpretation was difficult, especially comparing old classification standards to generic ACMG guidelines and a proposal was devised to weigh functional analyses at RNA level. According to the ACMG guidelines, only 12 variants were reclassified as pathogenic/likely pathogenic because the other two variants did not gather enough evidence. This study highlights the importance of RNA studies to improve variant classification. However, it also indicates the challenge of incorporating these results into generic ACMG guidelines and the need to refine these criteria gene specifically. Nevertheless, 60% of variants were reclassified, thus improving genetic counseling and surveillance for carriers of these variants.

ERCC3, a new ovarian cancer susceptibility gene?

BACKGROUND: Hereditary breast and ovarian cancer syndrome (HBOC) is an inherited disorder with an increased risk of breast cancer (BC) and ovarian cancers (OC). Mutations in BRCA1-BRCA2 explains less than a half of cases. In the last decade several genes with different penetrance have been associated with an increased risk of BC or OC. A recurrent heterozygous ERCC3 truncating mutation increases the risk for breast cancer in patients with Ashkenazi Jewish ancestry. Our study aimed to investigate the role of ERCC3 truncating variants in a cohort of patients with suspicion of HBOC. PATIENTS AND METHODS: ERCC3 screening by multigene-panel analysis in 1311 unrelated patients after our regional consensus for genetic testing in hereditary cancer was done. In addition, 453 Spanish cancer-free individuals and 51,343 GnomAD non-Finnish, non-cancer European individuals were used as control populations. RESULTS: We identified 13 patients with heterozygous ERCC3 truncating variants (0.99%). Five of them also carried a mutation in a high- /moderate-penetrance HBOC gene (BRCA1, BRCA2, CHEK2, and TP53) being Multilocus Inherited Neoplasia Alleles syndrome (MINAS) patients. The frequency in 453 Spanish controls was of 0.22%; similar to that observed in 51,343 non-Finnish European GnomAD population (0.24%). We found an almost statistically significant association of truncating ERCC3 variants with BC (odds ratio [OR] = 2.25, confidence interval [CI] = 0.6-5.93, P = 0.11), and we observed for the first time a significant association with OC (OR = 4.74, CI = 1-14.34, P = 0.028), that holds even after removing MINAS cases. CONCLUSIONS: To our knowledge, this is the largest HBOC series comprehensively analysed for ERCC3 mutations, and the first study identifying ERCC3 as a cancer risk for OC.

Assessing Effectiveness of Colonic and Gynecological Risk Reducing Surgery in Lynch Syndrome Individuals.

BACKGROUND: Colorectal (CRC) and endometrial cancer (EC) are the most common types of cancer in Lynch syndrome (LS). Risk reducing surgeries (RRS) might impact cancer incidence and mortality. Our objectives were to evaluate cumulative incidences of CRC, gynecological cancer and all-cause mortality after RRS in LS individuals. METHODS: Retrospective analysis of 976 LS carriers from a single-institution registry. Primary endpoints were cumulative incidence at 75 years of cancer (metachronous CRC in 425 individuals; EC and ovarian cancer (OC) in 531 individuals) and all-cause mortality cumulative incidence, comparing extended (ES) vs. segmental surgery (SS) in the CRC cohort and risk reducing gynecological surgery (RRGS) vs. surveillance in the gynecological cohort. RESULTS: Cumulative incidence at 75 years of metachronous CRC was 12.5% vs. 44.7% (p = 0.04) and all-cause mortality cumulative incidence was 38.6% vs. 55.3% (p = 0.31), for ES and SS, respectively. Cumulative, incidence at 75 years was 11.2% vs. 46.3% for EC (p = 0.001) and 0% vs. 12.7% for OC (p N/A) and all-cause mortality cumulative incidence was 0% vs. 52.7% (p N/A), for RRGS vs. surveillance, respectively. CONCLUSIONS: RRS in LS reduces the incidence of metachronous CRC and gynecological neoplasms, also indicating a reduction in all-cause mortality cumulative incidence in females undergoing RRGS.

Comprehensive Constitutional Genetic and Epigenetic Characterization of Lynch-Like Individuals.

The causal mechanism for cancer predisposition in Lynch-like syndrome (LLS) remains unknown. Our aim was to elucidate the constitutional basis of mismatch repair (MMR) deficiency in LLS patients throughout a comprehensive (epi)genetic analysis. One hundred and fifteen LLS patients harboring MMR-deficient tumors and no germline MMR mutations were included. Mutational analysis of 26 colorectal cancer (CRC)-associated genes was performed. Pathogenicity of MMR variants was assessed by splicing and multifactorial likelihood analyses. Genome-wide methylome analysis was performed by the Infinium Human Methylation 450K Bead Chip. The multigene panel analysis revealed the presence of two MMR gene truncating mutations not previously found. Of a total of 15 additional MMR variants identified, five -present in 6 unrelated individuals- were reclassified as pathogenic. In addition, 13 predicted deleterious variants in other CRC-predisposing genes were found in 12 probands. Methylome analysis detected one constitutional MLH1 epimutation, but no additional differentially methylated regions were identified in LLS compared to LS patients or cancer-free individuals. In conclusion, the use of an ad-hoc designed gene panel combined with pathogenicity assessment of variants allowed the identification of deleterious MMR mutations as well as new LLS candidate causal genes. Constitutional epimutations in non-LS-associated genes are not responsible for LLS.

Segmental neurofibromatosis type 2: discriminating two hit from four hit in a patient presenting multiple schwannomas confined to one limb.

BACKGROUND: A clinical overlap exists between mosaic Neurofibromatosis Type 2 and sporadic Schwannomatosis conditions. In these cases a molecular analysis of tumors is recommended for a proper genetic diagnostics. This analysis is challenged by the fact that schwannomas in both conditions bear a somatic double inactivation of the NF2 gene. However, SMARCB1-associated schwannomas follow a four-hit, three-step model, in which both alleles of SMARCB1 and NF2 genes are inactivated in the tumor, with one of the steps being always the loss of a big part of chromosome 22 involving both loci. CASE PRESENTATION: Here we report a 36-year-old woman who only presented multiple subcutaneous schwannomas on her right leg. To help discriminate between both possible diagnoses, an exhaustive molecular genetic and genomic analysis was performed on two schwannomas of the patient, consisting in cDNA and DNA sequencing, MLPA, microsatellite multiplex PCR and SNP-array analyses. The loss of a big part of chromosome 22 (22q12.1q13.33) was identified in both tumors. However, this loss involved the NF2 but not the SMARCB1 locus. SNP-array analysis revealed the presence of the same deletion breakpoint in both schwannomas, indicating that this alteration was actually the first NF2 inactivating hit. In addition, a distinct NF2 point mutation in each tumor was identified, representing independent second hits. In accordance with these results, no deletions or point mutations in the SMARCB1 gene were identified. None of the mutations were present in the blood. Two of the patient's children inherited chromosome 22 deleted in schwannomas of the mother, but in its wild type form. CONCLUSIONS: These results conclusively confirm the segmental mosaic NF2 nature of the clinical phenotype presented.

Prevalence of germline MUTYH mutations among Lynch-like syndrome patients.

BACKGROUND AND AIMS: Individuals with tumours showing mismatch repair (MMR) deficiency not linked to germline mutations or somatic methylation of MMR genes have been recently referred as having 'Lynch-like syndrome' (LLS). The genetic basis of these LLS cases is unknown. MUTYH-associated polyposis patients show some phenotypic similarities to Lynch syndrome patients. The aim of this study was to investigate the prevalence of germline MUTYH mutations in a large series of LLS patients. METHODS: Two hundred and twenty-five probands fulfilling LLS criteria were included in this study. Screening of MUTYH recurrent mutations, whole coding sequencing and a large rearrangement analysis were undertaken. Age, sex, clinical, pathological and molecular characteristics of tumours including KRAS mutations were assessed. RESULTS: We found a prevalence of 3.1% of MAP syndrome in the whole series of LLS (7/225) and 3.9% when only cases fulfilling clinical criteria were considered (7/178). Patients with MUTYH biallelic mutations had more adenomas than monoallelic (P=0.02) and wildtype patients (P<0.0001). Six out of nine analysed tumours from six biallelic MUTYH carriers harboured KRAS-p.G12C mutation. This mutation was found to be associated with biallelic MUTYH germline mutation when compared with reported series of unselected colorectal cancer cohorts (P<0.0001). CONCLUSIONS: A proportion of unexplained LLS cases is caused by biallelic MUTYH mutations. The obtained results further justify the inclusion of MUTYH in the diagnostic strategy for Lynch syndrome-suspected patients.

In vitro antisense therapeutics for a deep intronic mutation causing Neurofibromatosis type 2.

Neurofibromatosis type 2 (NF2) is an autosomal-dominant disorder affecting about 1:33 000 newborns, mainly characterized by the development of tumors of the nervous system and ocular abnormalities. Around 85% of germline NF2 mutations are point mutations. Among them, ∼25% affect splicing and are associated with a variable disease severity. In the context of our NF2 Multidisciplinary Clinics, we have identified a patient fulfilling clinical criteria for the disease and exhibiting a severe phenotype. The patient carries a deep intronic mutation (g. 74409T>A, NG_009057.1) that produces the insertion of a cryptic exon of 167pb in the mature mRNA between exons 13 and 14, resulting in a truncated merlin protein (p.Pro482Profs*39). A mutation-specific antisense phosphorodiamidate morpholino oligomer was designed and used in vitro to effectively restore normal NF2 splicing in patient-derived primary fibroblasts. In addition, merlin protein levels were greatly recovered after morpholino treatment, decreasing patient's fibroblasts in vitro proliferation capacity and restoring cytoeskeleton organization. To our knowledge, this is the first NF2 case caused by a deep intronic mutation in which an in vitro antisense therapeutic approximation has been tested. These results open the possibility of using this approach in vivo for this type of mutation causing NF2.