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BackgroundLi–Fraumeni syndrome (LFS) is a rare autosomal dominant cancer predisposition syndrome. Most families fulfilling the classical diagnostic criteria harbour TP53 germline mutations. However, TP53 germline mutations may also occur in less obvious phenotypes. As a result, different criteria are in use to decide which patients qualify for TP53 mutation analysis, including the LFS, Li–Fraumeni-like (LFL) and Chompret criteria. We investigated which criteria for TP53 mutation analysis resulted in the highest mutation detection rate and sensitivity in Dutch families. We describe the tumour spectrum in TP53-positive families and calculated tumour type specific relative risks.MethodA total of 180 Dutch families referred for TP53 mutation analysis were evaluated. Tumour phenotypes were verified by pathology reports or clinical records.ResultsA TP53 germline mutation was identified in 24 families. When the Chompret criteria were used 22/24 mutations were detected (sensitivity 92%, mutation detection rate 21%). In LFS and LFL families 18/24 mutations were found (sensitivity 75%). The two mutations detected outside the ‘Chompret group’ were found in a child with rhabdomyosarcoma and a young woman with breast cancer. In the mutation carriers, in addition to the classical LFS tumour types, colon and pancreatic cancer were also found significantly more often than in the general population.ConclusionWe suggest TP53 mutation testing for all families fulfilling the Chompret criteria. In addition, TP53 mutation testing can be considered in the event of childhood sarcoma and breast cancer before 30 years. In addition to the risk for established LFS tumour types, TP53-positive individuals may also have an elevated risk for pancreatic and colon cancer.

Background We previously showed that BRCA-like profiles can be used to preselect individuals with the highest risk of carrying BRCA mutations but could also indicate which patients would benefit from double-strand break inducing chemotherapy. A simple, robust, and reliable assay for clinical use that utilizes limited amounts of formalin-fixed, paraffin-embedded tumor tissue to assess BRCAness status in both ER-positive and ER-negative breast cancer (BC) is currently lacking. Methods A digital multiplex ligation-dependent probe amplification (digitalMLPA) assay was designed to detect copy number alterations required for the classification of BRCA1-like and BRCA2-like BC. The BRCA1-like classifier was trained on 71 tumors, enriched for triple-negative BC; the BRCA2-like classifier was trained on 55 tumors, enriched for luminal-type BC. A shrunken centroid-based classifier was developed and applied on an independent validation cohort. A total of 114 cases of a randomized controlled trial were analyzed, and the association of the classifier result with intensified platinum-based chemotherapy response was assessed. Results The digitalMLPA BRCA1-like classifier correctly classified 91% of the BRCA1-like samples and 82% of the BRCA2-like samples. Patients with a BRCA-like tumor derived significant benefit of high-dose chemotherapy (adjusted hazard ratio (HR) 0.12, 95% CI 0.04–0.44) which was not observed in non-BRCA-like patients (HR 0.9, 95% CI 0.37–2.18) ( p  = 0.01). Analysis stratified for ER status showed borderline significance. Conclusions The digitalMLPA is a reliable method to detect a BRCA1- and BRCA2-like pattern on clinical samples and predicts platinum-based chemotherapy benefit in both triple-negative and luminal-type BC.

Background While new defects in BRCA1 are still being found, it is unclear whether current breast cancer diagnostics misses many BRCA1 -associated cases. A reliable test that is able to indicate the involvement of BRCA1 deficiency in cancer genesis could support decision making in genetic counselling and clinical management. To find BRCA1 -specific markers and explore the effectiveness of the current diagnostic strategy, we designed a classification method, validated it and examined whether we could find BRCA1 -like breast tumours in a group of patients initially diagnosed as non- BRCA1/2 mutation carriers. Methods A classifier was built based on array-CGH profiles of 18 BRCA1 -related and 32 control breast tumours, and validated on independent sets of 16 BRCA1 -related and 16 control breast carcinomas. Subsequently, we applied the classifier to 48 breast tumours of patients from Hereditary Breast and Ovarian Cancer (HBOC) families in whom no germ line BRCA1/BRCA2 mutations were identified. Results The classifier showed an accuracy of 91% when applied to the validation sets. In 48 non- BRCA1/2 patients, only two breast tumours presented a BRCA1-like CGH profile. Additional evidence for BRCA1 dysfunction was found in one of these tumours. Conclusion We here describe the specific chromosomal aberrations in BRCA1 -related breast carcinomas. We developed a predictive genetic test for BRCA1 -association and show that BRCA1 -related tumours can still be identified in HBOC families after routine DNA diagnostics.

Germline mutations in BRCA1/2 increase the lifetime risk for breast and ovarian cancer dramatically. Identification of such mutations is important for optimal treatment decisions and pre-symptomatic mutation screening in family members. Although current DNA diagnostics is able to identify many different mutations, it remains unclear, how many BRCA2 -associated breast cancer cases remain unidentified as such. In addition, mutation scanning detects many unclassified variants (UV) for which the clinical relevance is uncertain. Therefore, our aim was to develop a test to identify BRCA2 -association in breast tumors based on the genomic signature. A BRCA2-classifier was built using array-CGH profiles of 28 BRCA2 -mutated and 28 sporadic breast tumors. The classifier was validated on an independent group of 19 BRCA2 -mutated and 19 sporadic breast tumors. Subsequently, we tested 89 breast tumors from suspected hereditary breast (and ovarian) cancer (HBOC) families, in which either no BRCA1/2 mutation or an UV had been found by routine diagnostics. The classifier showed a sensitivity of 89% and specificity of 84% on the validation set of known BRCA2 -mutation carriers and sporadic tumor cases. Of the 89 HBOC cases, 17 presented a BRCA2-like profile. In three of these cases additional indications for BRCA2 -deficiency were found. Chromosomal aberrations that were specific for BRCA2 -mutated tumors included loss on chromosome arm 13q and 14q, and gain on 17q. Since we could separate BRCA1-like, BRCA2-like, and sporadic-like tumors, using our current BRCA2- and previous BRCA1-classifier, this method of breast tumor classification could be applied as additional test for current diagnostics to help clinicians in decision making and classifying sequence variants of unknown significance.

Two decades after the genomics revolution, oncology is rapidly transforming into a genome-driven discipline, yet routine cancer diagnostics is still mainly microscopy based, except for tumor type-specific predictive molecular tests. Pathology laboratories struggle to quickly validate and adopt biomarkers identified by genomics studies of new targeted therapies. Consequently, clinical implementation of newly approved biomarkers suffers substantial delays, leading to unequal patient access to these therapies. Whole-genome sequencing (WGS) can successfully address these challenges by providing a stable molecular diagnostic platform that allows detection of a multitude of genomic alterations in a single cost-efficient assay and facilitating rapid implementation, as well as by the development of new genomic biomarkers. Recently, the Whole-genome sequencing Implementation in standard Diagnostics for Every cancer patient (WIDE) study demonstrated that WGS is a feasible and clinically valid technique in routine clinical practice with a turnaround time of 11 workdays. As a result, WGS was successfully implemented at the Netherlands Cancer Institute as part of routine diagnostics in January 2021. The success of implementing WGS has relied on adhering to a comprehensive protocol including recording patient information, sample collection, shipment and storage logistics, sequencing data interpretation and reporting, integration into clinical decision-making and data usage. This protocol describes the use of fresh-frozen samples that are necessary for WGS but can be challenging to implement in pathology laboratories accustomed to using formalin-fixed paraffin-embedded samples. In addition, the protocol outlines key considerations to guide uptake of WGS in routine clinical care in hospitals worldwide. Key points Whole-genome sequencing analysis detects DNA changes in the whole cancer genome, allowing identification of new, clinically relevant biomarkers, and opening the doors to a new era of tumor diagnostic and personalized cancer medicine. The protocol provides a detailed workflow covering patient selection, sample handling, and interpretation and reporting of the results, facilitating implementation of whole-genome sequencing analysis in routine oncology clinical practice. This protocol presents a comprehensive workflow for the implementation of whole-genome sequencing in routine tumor diagnostics, exemplified by the pipeline used in the Netherlands Cancer Institute.

When hereditary breast and ovarian cancer (HBOC) due to a BRCA1/BRCA2 germline pathogenic variant is diagnosed, the proband will be asked to inform other at-risk family members. In the Netherlands, a guideline was introduced in 2012 which provided detailed recommendations regarding this proband-mediated procedure. We now evaluated the uptake of predictive BRCA1/BRCA2 testing in 40 consecutive HBOC families diagnosed in our centre in 2014. We performed a retrospective observational study of all 40 families in which a pathogenic BRCA1/BRCA2 germline variant was identified during 2014. We scored the uptake of predictive and confirmatory testing by the end of 2018 and explored factors associated with the level of uptake. Two families were excluded. In the remaining 38 families, among 239 family members ≥18 years at 50% risk of being a mutation carrier or at 25% risk if the family member at 50% risk was deceased, 102 (43%) were tested. Among 108 females 25–75 years of age at 50% risk, 59 (55%) underwent predictive or confirmatory testing, and among 43 males at 50% risk with daughters ≥18 years, 22 (51%) were tested. Factors which complicated cascade screening included family members living abroad, probands not wanting to share information and limited pedigree information. In conclusion, the standard proband-mediated procedure of informing relatives seems to be far from optimal. We suggest a tailored approach for each family, including the option of a direct approach to at-risk family members by the geneticist. In addition, we suggest detailed monitoring and follow-up of families.

An inherited single nucleotide variant (SNV) in the 5′UTR of the BRCA1 gene c.-107A > T was identified to be related to BRCA1 promoter hypermethylation and a hereditary breast and ovarian cancer phenotype in two UK families. We investigated whether this BRCA1 variant was also present in a Dutch cohort of breast and ovarian cancer patients with tumor BRCA1 promoter hypermethylation. We selected all breast and ovarian cancer cases that tested positive for tumor BRCA1 promoter hypermethylation at the Netherlands Cancer Institute and Sanger sequenced the specific mutation in the tumor DNA. In total, we identified 193 tumors with BRCA1 promoter hypermethylation in 178 unique patients. The wild-type allele was identified in 100% (193/193) of sequenced tumor samples. In a large cohort of 178 patients, none had tumors harboring the previously identified c.-107A > T SNV in BRCA1. We therefore can conclude that the germline SNV is not pervasive in patients with tumor BRCA1 promoter hypermethylation.

Since the 1980s the genetic cause of many hereditary tumor syndromes has been elucidated. As a consequence, carriers of a deleterious mutation in these genes may opt for prenatal diagnoses (PND). We studied the uptake of prenatal diagnosis for five hereditary cancer syndromes in the Netherlands. Uptake for retinoblastoma (Rb) was compared with uptake for Von Hippel–Lindau disease (VHL), Li–Fraumeni syndrome (LFS), familial adenomatous polyposis (FAP), and hereditary breast ovarian cancer (HBOC). A questionnaire was completed by all nine DNA-diagnostic laboratories assessing the number of independent mutation-positive families identified from the start of diagnostic testing until May 2013, and the number of PNDs performed for these syndromes within these families. Of 187 families with a known Rb-gene mutation, 22 had performed PND (11.8%), this was significantly higher than uptake for FAP (1.6%) and HBOC (<0.2%). For VHL (6.5%) and LFS (4.9%) the difference was not statistically significant. PND for Rb started 3 years after introduction of diagnostic DNA testing and remained stable over the years. For the other cancer syndromes PND started 10–15 years after the introduction and uptake for PND showed an increase after 2009. We conclude that uptake of PND for Rb was significantly higher than for FAP and HBOC, but not different from VHL and LFS. Early onset, high penetrance, lack of preventive surgery and perceived burden of disease may explain these differences.

Extensive expression profiling studies have shown that sporadic breast cancer is composed of five clinically relevant molecular subtypes. However, although BRCA1-related tumours are known to be predominantly basal-like, there are few published data on other classes of familial breast tumours. We analysed a cohort of 75 BRCA1, BRCA2 and non-BRCA1/2 breast tumours by gene expression profiling and found that 74% BRCA1 tumours were basal-like, 73% of BRCA2 tumours were luminal A or B, and 52% non-BRCA1/2 tumours were luminal A. Thirty-four tumours were also analysed by single nucleotide polymorphism-comparative genomic hybridization (SNP-CGH) arrays. Copy number data could predict whether a tumour was basal-like or luminal with high accuracy, but could not predict its mutation class. Basal-like BRCA1 and basal-like non-BRCA1 tumours were very similar, and contained the highest number of chromosome aberrations. We identified regions of frequent gain containing potential driver genes in the basal (8q and 12p) and luminal A tumours (1q and 17q). Regions of homozygous loss associated with decreased expression of potential tumour suppressor genes were also detected, including in basal tumours (5q and 9p), and basal and luminal tumours (10q). This study highlights the heterogeneity of familial tumours and the clinical consequences for treatment and prognosis.

PurposeCHEK2 c.1100delC is associated with an increased breast cancer risk in women. While this variant is prevalent in the Netherlands (1% in the general population), knowledge of aetiology and prognosis of breast cancer and other tumours in CHEK2 c.1100delC carriers is lacking. The nationwide HEreditary Breast and Ovarian cancer study the Netherlands (Hebon) cohort aims to answer study questions in families with an increased risk of breast cancer and ovarian cancer. While initially focusing on BRCA1/2-variant families, Hebon gradually expanded to include pathogenic variants in other genes associated with breast and/or ovarian cancer over time. This provides an excellent setting to establish a cohort to ultimately study the impact of CHEK2 c.1100delC on cancer risk prediction and surveillance, breast cancer treatment and prognosis.ParticipantsWe invited all heterozygous and homozygous CHEK2 c.1100delC indexes and tested female relatives. 1802 women were included, of whom 1374 were heterozygotes and 938 were breast cancer cases. Pedigrees were collected from all clinical genetic departments. Furthermore, participants completed a detailed questionnaire on hormonal and lifestyle factors, family history, cancer diagnosis and treatment.Findings to dateMean age at study inclusion was 53 years. Linkage with the Netherlands Cancer Registry showed a younger age at diagnosis in homozygotes (mean age 41.7 years) and heterozygotes (47.9 years) than non-carriers (51.2 years). Furthermore, carriers were more often diagnosed with grade 2, oestrogen receptor-positive breast cancer and more often developed contralateral breast cancer than non-carriers. Most women consumed alcohol regularly and about half never smoked.Future plansFurther data linkages with the Netherlands Cancer Registry will allow prospective follow-up and breast cancer risk assessment in unaffected women at the time of genetic testing, risk of contralateral breast cancer and survival in patients with breast cancer. Also, linkage with the nationwide network and registry of histopathology and cytopathology in The Netherlands (PALGA) allows us to retrieve tumour samples to study tumourigenesis.