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ZusammenfassungDas nationale Register „HerediCaRe“ für die Evaluation und Verbesserung der risiko-adjustierten Prävention bei erblichem Brust- und Eierstockkrebs ist eines von sechs vom BMBF geförderten „modellhaften Registern in der Versorgungsforschung“. In diesem Beitrag beschreiben und diskutieren wir die zur standardisierten Datenerfassung gewählte Dokumentations- und IT-Lösung auf der Basis der zuvor definierten speziellen funktionalen Anforderungen. Die Dokumentation gliedert sich in verschiedene patientenindividuell einzusetzende Module, die auf einem zuvor festgelegten Merkmalskatalog beruhen. Aufgrund spezieller funktionaler Anforderungen wurde eine eigene Datenerfassungsanwendung auf der Basis von ORACLE und ORACLE Forms entwickelt und implementiert. Die speziellen Anforderungen umfassten u. a. die Einbindung grafischer Stammbaumdarstellungen, den strukturierten Upload von Stammbaumdaten und molekulargenetischen Informationen, die automatisierte Altdatenübernahme aus dem Vorgängersystem, sowie die freie Programmierbarkeit von beliebig komplexen Datenbankabfragen zur zentralen Datenqualitätsprüfung. In die Anwendung ist eine Datenbank zur patienten-unabhängigen Verwaltung genetischer Risikovarianten nahtlos integriert und mit den patientenbezogenen Daten verknüpft. Die Vor- und Nachteile der gewählten IT-Lösung werden kritisch diskutiert. Insgesamt kommen wir zu der Schlussfolgerung, dass es angesichts der komplexen Dokumentation und der speziellen Funktionsanforderungen alternativ keine fertigen Softwareprodukte zu der von uns gewählten Eigenentwicklung existieren.

Variant-specific loss of heterozygosity (LOH) analyses may be useful to classify BRCA1/2 germline variants of unknown significance (VUS). The sensitivity and specificity of this approach, however, remains unknown. We performed comparative next-generation sequencing analyses of the BRCA1/2 genes using blood-derived and tumour-derived DNA of 488 patients with ovarian cancer enrolled in the observational AGO-TR1 trial (NCT02222883). Overall, 94 pathogenic, 90 benign and 24 VUS were identified in the germline. A significantly increased variant fraction (VF) of a germline variant in the tumour indicates loss of the wild-type allele; a decreased VF indicates loss of the variant allele. We demonstrate that significantly increased VFs predict pathogenicity with high sensitivity (0.84, 95% CI 0.77 to 0.91), poor specificity (0.63, 95% CI 0.53 to 0.73) and poor positive predictive value (PPV; 0.71, 95% CI 0.62 to 0.79). Significantly decreased VFs predict benignity with low sensitivity (0.26, 95% CI 0.17 to 0.35), high specificity (1.0, 95% CI 0.96 to 1.00) and PPV (1.0, 95% CI 0.85 to 1.00). Variant classification based on significantly increased VFs results in an unacceptable proportion of false-positive results. A significantly decreased VF in the tumour may be exploited as a reliable predictor for benignity, with no false-negative result observed. When applying the latter approach, VUS identified in four patients can now be considered benign. Trial registration number NCT02222883., publishedVersion

Purpose: Previously, we developed breast cancer BRCA1-like and BRCA2-like copy-number profile shrunken centroid classifiers predictive for mutation status and response to therapy, targeting homologous recombination deficiency (HRD). Therefore, we investigated BRCA1- and BRCA2-like classification in ovarian cancer, aiming to acquire classifiers with similar properties as those in breast cancer. Experimental Design: We analyzed DNA copy-number profiles of germline BRCA1- and BRCA2-mutant ovarian cancers and control tumors and observed that existing breast cancer classifiers did not sufficiently predict mutation status. Hence, we trained new shrunken centroid classifiers on this set and validated them in the independent The Cancer Genome Atlas dataset. Subsequently, we assessed BRCA1/2-like classification and obtained germline and tumor mutation and methylation status of cancer predisposition genes, among them several involved in HR repair, of 300 ovarian cancer samples derived from the consecutive cohort trial AGO-TR1 (NCT02222883). Results: The detection rate of the BRCA1-like classifier for BRCA1 mutations and promoter hypermethylation was 95.6%. The BRCA2-like classifier performed less accurately, likely due to a smaller training set. Furthermore, three quarters of the BRCA1/2-like tumors could be explained by (epi)genetic alterations in BRCA1/2, germline RAD51C mutations and alterations in other genes involved in HR. Around half of the non–BRCA-mutated ovarian cancer cases displayed a BRCA-like phenotype. Conclusions: The newly trained classifiers detected most BRCA-mutated and methylated cancers and all tumors harboring a RAD51C germline mutations. Beyond that, we found an additional substantial proportion of ovarian cancers to be BRCA-like.

IMPORTANCE: The GeparOcto randomized clinical trial compared the efficacy of 2 neoadjuvant breast cancer (BC) treatment regimens: sequential intense dose-dense epirubicin, paclitaxel, and cyclophosphamide (iddEPC) vs weekly paclitaxel and nonpegylated liposomal doxorubicin (PM) in patients with different biological BC subtypes. Patients with triple-negative BC (TNBC) randomized to the PM arm received additional carboplatin (PMCb). Overall, no difference in pathologic complete response (pCR) rates was observed between study arms. It remained elusive whether the germline variant status of BRCA1/2 and further BC predisposition genes are associated with treatment outcome. OBJECTIVE: To determine treatment outcome for BC according to germline variant status. DESIGN, SETTING, AND PARTICIPANTS: This retrospective biomarker study is a secondary analysis of the GeparOcto multicenter prospective randomized clinical trial conducted between December 2014 and June 2016. Genetic analyses assessing for variants in BRCA1/2 and 16 other BC predisposition genes in 914 of 945 women were performed at the Center for Familial Breast and Ovarian Cancer, Cologne, Germany, from August 2017 through December 2018. MAIN OUTCOMES AND MEASURES: Proportion of patients who achieved pCR (ypT0/is ypN0 definition) after neoadjuvant treatment according to germline variant status. RESULTS: In the study sample of 914 women with different BC subtypes with a mean (range) age at BC diagnosis of 48 (21-76) years, overall higher pCR rates were observed in patients with BRCA1/2 variants than in patients without (60.4% vs 46.7%; odds ratio [OR], 1.74; 95% CI, 1.13-2.68; P = .01); variants in non-BRCA1/2 BC predisposition genes were not associated with therapy response. Patients with TNBC with BRCA1/2 variants achieved highest pCR rates. In the TNBC subgroup, a positive BRCA1/2 variant status was associated with therapy response in both the PMCb arm (74.3% vs 47.0% without BRCA1/2 variant; OR, 3.26; 95% CI, 1.44-7.39; P = .005) and the iddEPC arm (64.7% vs 45.0%; OR, 2.24; 95% CI, 1.04-4.84; P = .04). A positive BRCA1/2 variant status was also associated with elevated pCR rates in patients with ERBB2-negative, hormone receptor–positive BC (31.8% vs 11.9%; OR, 3.44; 95% CI, 1.22-9.72; P = .02). CONCLUSIONS AND RELEVANCE: Effective chemotherapy for BRCA1/2-mutated TNBC is commonly suggested to be platinum based. With a pCR rate of 64.7%, iddEPC may also be effective in these patients, though further prospective studies are needed. The elevated pCR rate in BRCA1/2-mutated ERBB2-negative, hormone receptor–positive BC suggests that germline BRCA1/2 testing should be considered prior to treatment start. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02125344

The Li-Fraumeni cancer predisposition syndrome (LFS1) presents with a variety of tumor types and the TP53 gene is covered by most diagnostic cancer gene panels. We demonstrate that deleterious TP53 variants identified in blood-derived DNA of 523 patients with ovarian cancer (AGO-TR1 trial) were not causal for the patients' ovarian cancer in three out of six TP53-positive cases. In three out of six patients, deleterious TP53 mutations were identified with low variant fractions in blood-derived DNA but not in the tumor of the patient seeking advice. The analysis of the TP53 and PPM1D genes, both intimately involved in chemotherapy-induced and/or age-related clonal hematopoiesis (CH), in 523 patients and 1,053 age-matched female control individuals revealed that CH represents a frequent event following chemotherapy, affecting 26 of the 523 patients enrolled (5.0%). Considering that TP53 mutations may arise from chemotherapy-induced CH, our findings help to avoid false-positive genetic diagnoses of LFS1.

Background Germline mutations in the BRIP1 gene have been described as conferring a moderate risk for ovarian cancer (OC), while the role of BRIP1 in breast cancer (BC) pathogenesis remains controversial. Methods To assess the role of deleterious BRIP1 germline mutations in BC/OC predisposition, 6341 well-characterized index patients with BC, 706 index patients with OC, and 2189 geographically matched female controls were screened for loss-of-function (LoF) mutations and potentially damaging missense variants. All index patients met the inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for germline testing and tested negative for pathogenic BRCA1/2 variants. Results BRIP1 LoF mutations confer a high OC risk in familial index patients (odds ratio (OR) = 20.97, 95% confidence interval (CI) = 12.02–36.57, P

Scientific reports 9(1), 10227 (2019). doi:10.1038/s41598-019-46734-3, Published by Macmillan Publishers Limited, part of Springer Nature, [London]

579 Background: The phase II trial WSG-ADAPT TN randomized triple-negative breast cancer (TNBC) patients to receive 12 weeks of neoadjuvant nab-paclitaxel (nab-pac) combined with carboplatin (carbo) vs gemcitabine (gem) and showed a substantial improvement of pathological complete response (pCR: ypT0/is, ypN0) with carbo (45.9% vs 28.7%). pCR had a strong favorable impact on iDFS after 3-year follow-up. Distribution of tumor mutations in BC-associated genes and impact of BRCA mutation status on pCR and outcome are analyzed here. Methods: NGS-based mutational analysis of BRCA1/2 and 18 further (potentially) BC-associated genes was performed on DNA derived from pretreatment FFPE samples (gem: n = 158, carbo: n = 108) using a customized gene panel. Variants with a variant fraction of ≥5% were included and classified according to IARC and ENIGMA guidelines. Results: In 42 of the 266 analyzed samples, at least one deleterious BRCA1/2-variant was found (15.8%; BRCA1 n = 37, BRCA2 n = 3, BRCA1+ BRCA2 n = 2) one of which displayed an additional STK11-mutation. In the BRCA1/2-negative cohort, a mutation in one of 14 further analyzed (potential) BC-risk genes was found in 19 samples (7.1%; BARD1 n = 3, CHEK2 n = 2, CDH1 n = 2, FANCM n = 3, PALB2 n = 5, RAD50 n = 1, RAD51C n = 1, RAD51D n = 1, XRCC2 n = 1; no deleterious mutations were found in ATM, BRIP1, MRE11A, NBN). At least one deleterious variant in TP53, PIK3CA, PTEN or MAP3K1 was seen in 89.1% (n = 237; TP53 n = 233, PIK3CA n = 22 PTEN n = 15, MAP3K1 n = 1). In 22 samples (8.3%) no deleterious mutation was identified in the analyzed genes. Overall, patients with tumor BRCA mutation (carbo n = 14, gem n = 28) had 45.2% vs 34.4% pCR (OR = 1.58, 95%-CI: 0.81-3.07, p =.18) without a mutation. pCR in the small group with mutation receiving carbo (n = 14) was 64.3% vs. 34.5% in all others (OR = 3.41, 95%-CI: 1.11-10.50; p =.03); direct comparison to BRCA-positive patients receiving gem (n = 28, 35.7%, OR = 3.2, 95%-CI: 0.85-12.36, p = 0.079) did not reach statistical significance. The results suggest that the strong favorable impact of pCR on iDFS is preserved even among BRCA-positive patients (n = 42, p =.07), as well as in the BRCA-negative subgroup (p

Neuroblastoma is a malignancy of the developing sympathetic nervous system that is often lethal when relapse occurs. We here used whole-exome sequencing, mRNA expression profiling, array CGH and DNA methylation analysis to characterize 16 paired samples at diagnosis and relapse from individuals with neuroblastoma. The mutational burden significantly increased in relapsing tumors, accompanied by altered mutational signatures and reduced subclonal heterogeneity. Global allele frequencies at relapse indicated clonal mutation selection during disease progression. Promoter methylation patterns were consistent over disease course and were patient specific. Recurrent alterations at relapse included mutations in the putative CHD5 neuroblastoma tumor suppressor, chromosome 9p losses, DOCK8 mutations, inactivating mutations in PTPN14 and a relapse-specific activity pattern for the PTPN14 target YAP. Recurrent new mutations in HRAS, KRAS and genes mediating cell-cell interaction in 13 of 16 relapse tumors indicate disturbances in signaling pathways mediating mesenchymal transition. Our data shed light on genetic alteration frequency, identity and evolution in neuroblastoma.

The prevalence of germ line mutations in non-BRCA1/2 genes associated with hereditary breast cancer (BC) is low, and the role of some of these genes in BC predisposition and pathogenesis is conflicting. In this study, 5589 consecutive BC index patients negative for pathogenic BRCA1/2 mutations and 2189 female controls were screened for germ line mutations in eight cancer predisposition genes (ATM, CDH1, CHEK2, NBN, PALB2, RAD51C, RAD51D, and TP53). All patients met the inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for germ line testing. The highest mutation prevalence was observed in the CHEK2 gene (2.5%), followed by ATM (1.5%) and PALB2 (1.2%). The mutation prevalence in each of the remaining genes was 0.3% or lower. Using Exome Aggregation Consortium control data, we confirm significant associations of heterozygous germ line mutations with BC for ATM (OR: 3.63, 95% CI: 2.67-4.94), CDH1 (OR: 17.04, 95% CI: 3.54-82), CHEK2 (OR: 2.93, 95% CI: 2.29-3.75), PALB2 (OR: 9.53, 95% CI: 6.25-14.51), and TP53 (OR: 7.30, 95% CI: 1.22-43.68). NBN germ line mutations were not significantly associated with BC risk (OR: 1.39, 95% CI: 0.73-2.64). Due to their low mutation prevalence, the RAD51C and RAD51D genes require further investigation. Compared with control datasets, predicted damaging rare missense variants were significantly more prevalent in CHEK2 and TP53 in BC index patients. Compared with the overall sample, only TP53 mutation carriers show a significantly younger age at first BC diagnosis. We demonstrate a significant association of deleterious variants in the CHEK2, PALB2, and TP53 genes with bilateral BC. Both, ATM and CHEK2, were negatively associated with triple-negative breast cancer (TNBC) and estrogen receptor (ER)-negative tumor phenotypes. A particularly high CHEK2 mutation prevalence (5.2%) was observed in patients with human epidermal growth factor receptor 2 (HER2)-positive tumors.

The biotrophic fungus Ustilago maydis causes smut disease on maize (Zea mays), which is characterized by immense plant tumours. To establish disease and reprogram organ primordia to tumours, U. maydis deploys effector proteins in an organ-specific manner. However, the cellular contribution to leaf tumours remains unknown. We investigated leaf tumour formation at the tissue- and cell type-specific levels. Cytology and metabolite analysis were deployed to understand the cellular basis for tumourigenesis. Laser-capture microdissection was performed to gain a cell type-specific transcriptome of U. maydis during tumour formation. In vivo visualization of plant DNA synthesis identified bundle sheath cells as the origin of hyperplasic tumour cells, while mesophyll cells become hypertrophic tumour cells. Cell type-specific transcriptome profiling of U. maydis revealed tailored expression of fungal effector genes. Moreover, U. maydis See1 was identified as the first cell type-specific fungal effector, being required for induction of cell cycle reactivation in bundle sheath cells. Identification of distinct cellular mechanisms in two different leaf cell types and of See1 as an effector for induction of proliferation of bundle sheath cells are major steps in understanding U. maydis-induced tumour formation. Moreover, the cell type-specific U. maydis transcriptome data are a valuable resource to the scientific community.

Importance Germline mutations in established moderately or highly penetrant risk genes for breast cancer (BC) and/or ovarian cancer (OC), including BRCA1 and BRCA2 , explain fewer than half of all familial BC and/or OC cases. Based on the genotyping of 2 loss-of-function (LoF) variants c.5101C>T (p.GIn1701Ter [rs147021911]) and c.5791C>T (p.Arg1931Ter [rs144567652]), the FANCM gene has been suggested as a novel BC predisposition gene, while the analysis of the entire coding region of the FANCM gene in familial index cases and geographically matched controls is pending. Objectives To assess the mutational spectrum within the FANCM gene, and to determine a potential association of LoF germline mutations within the FANCM gene with BC and/or OC risk. Design, Setting, and Participants For the purpose of identification and characterization of novel BC and/or OC predisposition genes, a total of 2047 well-characterized familial BC index cases, 628 OC cases, and 2187 geographically matched controls were screened for LoF mutations within the FANCM gene by next-generation sequencing. All patients previously tested negative for pathogenic BRCA1 and BRCA2 mutations. All data collection occurred between June 1, 2013, and April 30, 2016. Data analysis was performed from May 1, 2016, to July 1, 2016. Main Outcomes and Measures FANCM LoF mutation frequencies in patients with BC and/or OC were compared with the FANCM LoF mutation frequencies in geographically matched controls by univariate logistic regression. Positive associations were stratified by age at onset and cancer family history. Results In this case-control study, 2047 well-characterized familial female BC index cases, 628 OC cases, and 2187 geographically matched controls were screened for truncating FANCM alterations. Heterozygous LoF mutations within the FANCM gene were significantly associated with familial BC risk, with an overall odds ratio (OR) of 2.05 (95% CI, 0.94-4.54; P = .049) and a mutation frequency of 1.03% in index cases. In familial patients whose BC onset was before age 51 years, an elevated OR of 2.44 (95% CI, 1.08-5.59; P = .02) was observed. A more pronounced association was identified for patients with a triple-negative BC tumor phenotype (OR, 3.75; 95% CI, 1.00-12.85; P = .02). No significant association was detected for unselected OC cases (OR, 1.74; 95% CI, 0.57-5.08; P = .27). Conclusions and Relevance Based on the significant associations of heterozygous LoF mutations with early-onset or triple-negative BC, FANCM should be included in diagnostic gene panel testing for individual risk assessment. Larger studies are required to determine age-dependent disease risks for BC and to assess a potential role of FANCM mutations in OC pathogenesis.