The heterogeneous cancer phenotype of individuals with biallelic germline pathogenic variants in CHEK2.

PURPOSE: Females with biallelic CHEK2 germline pathogenic variants (gPVs) more often develop multiple breast cancers than individuals with monoallelic CHEK2 gPVs. This study is aimed at expanding the knowledge on the occurrence of other malignancies. METHODS: Exome sequencing of individuals who developed multiple primary malignancies identified 3 individuals with the CHEK2 (NM_007194.4) c.1100del p.(Thr367MetfsTer15) loss-of-function gPV in a biallelic state. We collected the phenotypes of an additional cohort of individuals with CHEK2 biallelic gPVs (n = 291). RESULTS: In total, 157 individuals (53.4%; 157/294 individuals) developed ≥1 (pre)malignancy. The most common (pre)malignancies next to breast cancer were colorectal- (n = 19), thyroid- (n = 19), and prostate (pre)malignancies (n = 12). Females with biallelic CHEK2 loss-of-function gPVs more frequently developed ≥2 (pre)malignancies and at an earlier age compared with females biallelic for the CHEK2 c.470T>C p.(Ile157Thr) missense variant. Furthermore, 26 males (31%; 26/84 males) with CHEK2 biallelic gPVs developed ≥1 (pre)malignancies of 15 origins. CONCLUSION: Our study suggests that CHEK2 biallelic gPVs likely increase the susceptibility to develop multiple malignancies in various tissues, both in females and males. However, it is possible that a substantial proportion of individuals with CHEK2 biallelic gPVs is missed as diagnostic testing for CHEK2 often is limited to individuals who developed breast cancer.

Mobile element insertions in rare diseases: a comparative benchmark and reanalysis of 60,000 exome samples.

Mobile element insertions (MEIs) are a known cause of genetic disease but have been underexplored due to technical limitations of genetic testing methods. Various bioinformatic tools have been developed to identify MEIs in Next Generation Sequencing data. However, most tools have been developed specifically for genome sequencing (GS) data rather than exome sequencing (ES) data, which remains more widely used for routine diagnostic testing. In this study, we benchmarked six MEI detection tools (ERVcaller, MELT, Mobster, SCRAMble, TEMP2 and xTea) on ES data and on GS data from publicly available genomic samples (HG002, NA12878). For all the tools we evaluated sensitivity and precision of different filtering strategies. Results show that there were substantial differences in tool performance between ES and GS data. MELT performed best with ES data and its combination with SCRAMble increased substantially the detection rate of MEIs. By applying both tools to 10,890 ES samples from Solve-RD and 52,624 samples from Radboudumc we were able to diagnose 10 patients who had remained undiagnosed by conventional ES analysis until now. Our study shows that MELT and SCRAMble can be used reliably to identify clinically relevant MEIs in ES data. This may lead to an additional diagnosis for 1 in 3000 to 4000 patients in routine clinical ES.

Combined loss of CDH1 and downstream regulatory sequences drive early-onset diffuse gastric cancer and increase penetrance of hereditary diffuse gastric cancer.

BACKGROUND: Germline CDH1 pathogenic or likely pathogenic variants cause hereditary diffuse gastric cancer (HDGC). Once a genetic cause is identified, stomachs' and breasts' surveillance and/or prophylactic surgery is offered to asymptomatic CDH1 carriers, which is life-saving. Herein, we characterized an inherited mechanism responsible for extremely early-onset gastric cancer and atypical HDGC high penetrance. METHODS: Whole-exome sequencing (WES) re-analysis was performed in an unsolved HDGC family. Accessible chromatin and CDH1 promoter interactors were evaluated in normal stomach by ATAC-seq and 4C-seq, and functional analysis was performed using CRISPR-Cas9, RNA-seq and pathway analysis. RESULTS: We identified a germline heterozygous 23 Kb CDH1-TANGO6 deletion in a family with eight diffuse gastric cancers, six before age 30. Atypical HDGC high penetrance and young cancer-onset argued towards a role for the deleted region downstream of CDH1, which we proved to present accessible chromatin, and CDH1 promoter interactors in normal stomach. CRISPR-Cas9 edited cells mimicking the CDH1-TANGO6 deletion display the strongest CDH1 mRNA downregulation, more impacted adhesion-associated, type-I interferon immune-associated and oncogenic signalling pathways, compared to wild-type or CDH1-deleted cells. This finding solved an 18-year family odyssey and engaged carrier family members in a cancer prevention pathway of care. CONCLUSION: In this work, we demonstrated that regulatory elements lying down-stream of CDH1 are part of a chromatin network that control CDH1 expression and influence cell transcriptome and associated signalling pathways, likely explaining high disease penetrance and very young cancer-onset. This study highlights the importance of incorporating scientific-technological updates and clinical guidelines in routine diagnosis, given their impact in timely genetic diagnosis and disease prevention.

Microsatellite instability in noncolorectal and nonendometrial malignancies in patients with Lynch syndrome.

BACKGROUND: Individuals with Lynch syndrome are at increased hereditary risk of colorectal and endometrial carcinomas with microsatellite instability (MSI-H) and mismatch repair-deficiency (dMMR), which make these tumors vulnerable to therapy with immune checkpoint inhibitors. Our aim is to assess how often other tumor types in these individuals share these characteristics. METHODS: We retrieved the full tumor history of a historical clinic-based cohort of 1745 individuals with Lynch syndrome and calculated the standardized incidence ratio for all tumor types. MSI status, somatic second hit alterations, and immunohistochemistry-based MMR status were analyzed in 236 noncolorectal and nonendometrial malignant tumors. RESULTS: In individuals with Lynch syndrome MSI-H/dMMR occurred both in Lynch-spectrum and in non-Lynch-spectrum malignancies (85% vs 37%, P < .01). MSI-H/dMMR malignancies were found in nearly all non-Lynch-spectrum tumor types. Almost all breast carcinomas had medullary features, and most of them were MSI-H/dMMR. Breast carcinoma with medullary features were shown to be associated with Lynch syndrome (standardized incidence ratio = 38.8, 95% confidence interval = 16.7 to 76.5). CONCLUSIONS: In individuals with Lynch syndrome, MSI-H/dMMR occurs in more than one-half of the malignancies other than colorectal and endometrial carcinomas, including tumor types without increased incidence. The Lynch-spectrum tumors should be expanded to breast carcinomas with medullary features. All malignancies in patients with Lynch syndrome, independent of subtype, should be tested for MSI-H/dMMR in case therapy with immune checkpoint inhibitors is considered. Moreover, Lynch syndrome should be considered an underlying cause of all MSI-H/dMMR malignancies other than colorectal and endometrial carcinomas.

Germline mutations in WNK2 could be associated with serrated polyposis syndrome.

BACKGROUND: Patients with serrated polyposis syndrome (SPS) have multiple and/or large serrated colonic polyps and higher risk for colorectal cancer. SPS inherited genetic basis is mostly unknown. We aimed to identify new germline predisposition factors for SPS by functionally evaluating a candidate gene and replicating it in additional SPS cohorts. METHODS: After a previous whole-exome sequencing in 39 SPS patients from 16 families (discovery cohort), we sequenced specific genes in an independent validation cohort of 211 unrelated SPS cases. Additional external replication was also available in 297 SPS cases. The WNK2 gene was disrupted in HT-29 cells by gene editing, and WNK2 variants were transfected using a lentiviral delivery system. Cells were analysed by immunoblots, real-time PCR and functional assays monitoring the mitogen-activated protein kinase (MAPK) pathway, cell cycle progression, survival and adhesion. RESULTS: We identified 2 rare germline variants in the WNK2 gene in the discovery cohort, 3 additional variants in the validation cohort and 10 other variants in the external cohorts. Variants c.2105C>T (p.Pro702Leu), c.4820C>T (p.Ala1607Val) and c.6157G>A (p.Val2053Ile) were functionally characterised, displaying higher levels of phospho-PAK1/2, phospho-ERK1/2, CCND1, clonogenic capacity and MMP2. CONCLUSION: After whole-exome sequencing in SPS cases with familial aggregation and replication of results in additional cohorts, we identified rare germline variants in the WNK2 gene. Functional studies suggested germline WNK2 variants affect protein function in the context of the MAPK pathway, a molecular hallmark in this disease.

Immunological and Genomic Analysis Reveals Clinically Relevant Distinctions between Angiosarcoma Subgroups.

Angiosarcomas (AS) are extremely rare and aggressive vascular malignancies subdivided in de novo primary AS (pAS) and secondary AS (sAS). We hypothesize that the combination of immunological and genomic profiles significantly differs between primary and secondary AS, with potential impact on treatment strategies and a role for immunotherapy. Tumor-infiltrating lymphocytes were analyzed using multiplex immunohistochemistry from 79 pAS and 178 sAS. Median cell density was significantly higher in sAS for CD3+ T-cells (p < 0.001), CD8+ cytotoxic T-cells (p = 0.033), CD4+ T-helper cells (p < 0.001) and FoxP3+ T-regulatory cells (p < 0.001). CD20+ B-cell density was comparable (p = 0.417). Comprehensive genomic profiling was performed in 25 pAS and 25 sAS. A (likely) pathogenic mutation was detected in 80% of pAS vs. 88% of sAS (p = 0.702). Amplifications were found in 15% of pAS vs. 84% of sAS (p < 0.001). DNA damage response (DDR) pathway mutations (p = 0.021) and MYC amplifications (p < 0.001) were predominantly seen in sAS. In conclusion we observed a clear and clinical relevant distinction in immune infiltration and genomic profiles between pAS and sAS. The T-cell infiltrated tumor microenvironment and frequent DDR gene mutations, especially in sAS, warrant clinical trials with immunotherapy.

Solving the genetic aetiology of hereditary gastrointestinal tumour syndromes- a collaborative multicentre endeavour within the project Solve-RD.

BACKGROUND: Patients and families with suspected, but genetically unexplained (unsolved) genetic tumour risk syndromes lack appropriate treatment and prevention, leading to preventable morbidity and mortality. To tackle this problem, patients from the European Reference Network on Genetic Tumour Risk Syndromes (ERN GENTURIS) are analysed in the European Commission's research project "Solving the unsolved rare diseases" (Solve-RD). The aim is to uncover known and novel cancer predisposing genes by reanalysing available whole-exome sequencing (WES) data of large cohorts in a combined manner, and applying a multidimensional omics approach. APPROACH: Around 500 genetically unsolved cases with suspected hereditary gastrointestinal tumour syndromes (polyposis, early-onset/familial colorectal cancer and gastric cancer) from multiple European centres are aimed to be included. Currently, clinical and germline WES data from 294 cases have been analysed. In addition, an extensive molecular profiling of gastrointestinal tumours from these patients is planned and deep learning techniques will be applied. The ambitious, multidisciplinary project is accompanied by a number of methodical, technical, and logistic challenges, which require the development and implementation of new analysis tools, the standardisation of bioinformatics pipelines, and strategies to exchange data and knowledge. RESULTS: and Outlook. The first re-analysis of 229 known and proposed cancer predisposition genes allowed solving 2-3% of previously unsolved GENTURIS cases. The integration of expert knowledge and new technologies will help to identify the genetic basis of additional unsolved cases within the ongoing project. The ERN GENTURIS approach might serve as a model for other genomic initiatives.

Clonal Hematopoiesis Is Associated With Low CD4 Nadir and Increased Residual HIV Transcriptional Activity in Virally Suppressed Individuals With HIV.

Clonal hematopoiesis, a common age-related phenomenon marked by expansion of cells with clonal hematopoiesis driver mutations, has been associated with all-cause mortality, cancer, and cardiovascular disease. People with HIV (PWH) are at risk for non-AIDS-related comorbidities such as atherosclerotic cardiovascular disease and cancer. In a cross-sectional cohort study, we compared clonal hematopoiesis prevalence in PWH on stable antiretroviral therapy with prevalence in a cohort of overweight individuals and a cohort of age- and sex-matched population controls. The prevalence of clonal hematopoiesis adjusted for age was increased and clone size was larger in PWH compared to population controls. Clonal hematopoiesis is associated with low CD4 nadir, increased residual HIV-1 transcriptional activity, and coagulation factors in PWH. Future studies on the effect of clonal hematopoiesis on the HIV reservoir and non-AIDS-related comorbidities are warranted.

Mutational signature analysis in non-small cell lung cancer patients with a high tumor mutational burden.

BACKGROUND: Lung cancer is the leading cause of cancer death worldwide. With the growing number of targeted therapies and the introduction of immuno-oncology (IO), personalized medicine has become standard of care in patients with metastatic disease. The development of predictive and prognostic biomarkers is of great importance. Mutational signatures harbor potential clinical value as predictors of therapy response in cancer. Here we set out to investigate particular mutational processes by assessing mutational signatures and associations with clinical features, tumor mutational burden (TMB) and targetable mutations. METHODS: In this retrospective study, we studied tumor DNA from patients with non-small cell lung cancer (NSCLC) irrespective of stage. The samples were sequenced using a 2 megabase (Mb) gene panel. On each sample TMB was determined and defined as the total number of single nucleotide mutations per Mb (mut/Mb) including non-synonymous mutations. Mutational signature profiling was performed on tumor samples in which at least 30 somatic single base substitutions (SBS) were detected. RESULTS: In total 195 samples were sequenced. Median total TMB was 10.3 mut/Mb (range 0-109.3). Mutational signatures were evaluated in 76 tumor samples (39%; median TMB 15.2 mut/Mb). SBS signature 4 (SBS4), associated with tobacco smoking, was prominently present in 25 of 76 samples (33%). SBS2 and/or SBS13, both associated with activity of the AID/APOBEC family of cytidine deaminases, were observed in 11 of 76 samples (14%). SBS4 was significantly more present in early stages (I and II) versus advanced stages (III and IV; P = .005). CONCLUSION: In a large proportion of NSCLC patients tissue panel sequencing with a 2 Mb panel can be used to determine the mutational signatures. In general, mutational signature SBS4 was more often found in early versus advanced stages of NSCLC. Further studies are needed to determine the clinical utility of mutational signature analyses.

The role of haematological parameters in predicting the response to radical chemoradiotherapy in patients with anal squamous cell cancer.

BACKGROUND: Historically, the treatment of choice for anal cancer had been abdominoperineal resection (APR). Radical radiotherapy with concurrent 5-fluorouracil plus mitomycin C chemotherapy was later established as standard therapy, although with a failure rate of 20-30%. The aim of this study was to evaluate the outcomes after radical chemoradiotherapy (CRT), prognostic and predictive factors and patterns of failure. PATIENTS AND METHODS: This study included 47 patients treated with radical CRT for patohistologicaly confirmed anal squamous cell carcinoma. Analysed haematological parameters included: neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and haemoglobin level. The final logistic regression model included treatment break period. Tumour response was assessed at 24 weeks from CRT completion. Follow-up was performed every 3 months during the first two years, and every 6 months thereafter. RESULTS: A complete clinical response (CR) was detected in 30 patients (63.8%). Patients who did not achieve a 6-months CR and those who had a CR after 6 months but then relapsed were referred to surgical treatment. With combined CRT and surgical salvage treatment the CR rate was 80.9%. Patients with CR after 6 months had significantly longer disease-free survival (DFS), progression-free survival (PFS), and overall survival (OS). A significant effect on the 6-month response was confirmed for PLR (p = 0.03). CONCLUSIONS: Important prognostic factors associated with CR were baseline haemoglobin level and period of treatment interruptions. Potential haematological prognostic factors could be PLR and NLR, which can be routinely determined by low-cost and minimally invasive methods.

Challenges of Neoantigen Targeting in Lynch Syndrome and Constitutional Mismatch Repair Deficiency Syndrome.

Lynch syndrome (LS) and constitutional mismatch repair deficiency (CMMRD) are hereditary disorders characterised by a highly increased risk of cancer development. This is due to germline aberrations in the mismatch repair (MMR) genes, which results in a high mutational load in tumours of these patients, including insertions and deletions in genes bearing microsatellites. This generates microsatellite instability and cause reading frameshifts in coding regions that could lead to the generation of neoantigens and opens up avenues for neoantigen targeting immune therapies prophylactically and therapeutically. However, major obstacles need to be overcome, such as the heterogeneity in tumour formation within and between LS and CMMRD patients, which results in considerable variability in the genes targeted by mutations, hence challenging the choice of suitable neoantigens. The machine-learning methods such as NetMHC and MHCflurry that predict neoantigen- human leukocyte antigen (HLA) binding affinity provide little information on other aspects of neoantigen presentation. Immune escape mechanisms that allow MMR-deficient cells to evade surveillance combined with the resistance to immune checkpoint therapy make the neoantigen targeting regimen challenging. Studies to delineate shared neoantigen profiles across patient cohorts, precise HLA binding algorithms, additional therapies to counter immune evasion and evaluation of biomarkers that predict the response of these patients to immune checkpoint therapy are warranted.

A mosaic PIK3CA variant in a young adult with diffuse gastric cancer: case report.

Hereditary diffuse gastric cancer (HDGC) is associated with germline deleterious variants in CDH1 and CTNNA1. The majority of HDGC-suspected patients are still genetically unresolved, raising the need for identification of novel HDGC predisposing genes. Under the collaborative environment of the SOLVE-RD consortium, re-analysis of whole-exome sequencing data from unresolved gastric cancer cases (n = 83) identified a mosaic missense variant in PIK3CA in a 25-year-old female with diffuse gastric cancer (DGC) without familial history for cancer. The variant, c.3140A>G p.(His1047Arg), a known cancer-related somatic hotspot, was present at a low variant allele frequency (18%) in leukocyte-derived DNA. Somatic variants in PIK3CA are usually associated with overgrowth, a phenotype that was not observed in this patient. This report highlights mosaicism as a potential, and understudied, mechanism in the etiology of DGC.

Solve-RD: systematic pan-European data sharing and collaborative analysis to solve rare diseases.

For the first time in Europe hundreds of rare disease (RD) experts team up to actively share and jointly analyse existing patient's data. Solve-RD is a Horizon 2020-supported EU flagship project bringing together >300 clinicians, scientists, and patient representatives of 51 sites from 15 countries. Solve-RD is built upon a core group of four European Reference Networks (ERNs; ERN-ITHACA, ERN-RND, ERN-Euro NMD, ERN-GENTURIS) which annually see more than 270,000 RD patients with respective pathologies. The main ambition is to solve unsolved rare diseases for which a molecular cause is not yet known. This is achieved through an innovative clinical research environment that introduces novel ways to organise expertise and data. Two major approaches are being pursued (i) massive data re-analysis of >19,000 unsolved rare disease patients and (ii) novel combined -omics approaches. The minimum requirement to be eligible for the analysis activities is an inconclusive exome that can be shared with controlled access. The first preliminary data re-analysis has already diagnosed 255 cases form 8393 exomes/genome datasets. This unprecedented degree of collaboration focused on sharing of data and expertise shall identify many new disease genes and enable diagnosis of many so far undiagnosed patients from all over Europe.

Solving patients with rare diseases through programmatic reanalysis of genome-phenome data.

Reanalysis of inconclusive exome/genome sequencing data increases the diagnosis yield of patients with rare diseases. However, the cost and efforts required for reanalysis prevent its routine implementation in research and clinical environments. The Solve-RD project aims to reveal the molecular causes underlying undiagnosed rare diseases. One of the goals is to implement innovative approaches to reanalyse the exomes and genomes from thousands of well-studied undiagnosed cases. The raw genomic data is submitted to Solve-RD through the RD-Connect Genome-Phenome Analysis Platform (GPAP) together with standardised phenotypic and pedigree data. We have developed a programmatic workflow to reanalyse genome-phenome data. It uses the RD-Connect GPAP's Application Programming Interface (API) and relies on the big-data technologies upon which the system is built. We have applied the workflow to prioritise rare known pathogenic variants from 4411 undiagnosed cases. The queries returned an average of 1.45 variants per case, which first were evaluated in bulk by a panel of disease experts and afterwards specifically by the submitter of each case. A total of 120 index cases (21.2% of prioritised cases, 2.7% of all exome/genome-negative samples) have already been solved, with others being under investigation. The implementation of solutions as the one described here provide the technical framework to enable periodic case-level data re-evaluation in clinical settings, as recommended by the American College of Medical Genetics.