Assigning evidence to actionability: An introduction to variant interpretation in precision cancer medicine.

Modern concepts in precision cancer medicine are based on increasingly complex genomic analyses and require standardized criteria for the functional evaluation and reporting of detected genomic alterations in order to assess their clinical relevance. In this article, we propose and address the necessary steps in systematic variant evaluation consisting of bioinformatic analysis, functional annotation and clinical interpretation, focusing on the latter two aspects. We discuss the role and clinical application of current variant classification systems and point out their scope and limitations. Finally, we highlight the significance of the molecular tumor board as a platform for clinical decision-making based on genomic analyses.

Germline testing for homologous recombination repair genes-opportunities and challenges.

Pathogenic variants in the BRCA1 and BRCA2 genes are well known causes of hereditary breast and ovarian cancer. Other genes involved in the homologous recombination pathway can also be associated with increased probability of cancer development, for example, breast and ovarian cancer, prostate and pancreatic cancer, colorectal cancer, and even childhood tumors like medulloblastoma. Traditionally, patients and families likely to harbor a genetic predisposition have been identified using personal and family history. Several checklists and risk prediction tools have proven to be useful in the clinic. Through the widespread application of next generation sequencing of tumor tissue, a growing number of individuals with genetic cancer predisposition is now identified molecularly, even in the absence of a suggestive family history. Any constitutional variant identified during molecular genetic testing has to be assessed for its relevance, both functionally and in the context of patient phenotype. Variant curation is time consuming, but has been increasingly standardized by introduction of several guidelines to allow reliable and reproducible classification of constitutional variants. Variant classification by expert panels using data mining tools, evidence-based decision trees and gene specific criteria represents the gold standard. Participation of geneticists in molecular tumor boards facilitates the curation of potential constitutional variants, germline validation and thus directing the patient to appropriate counselling and care pathways. Due to the high relevance of germline variants for treatment and surveillance of the index patient and predictive testing and surveillance of relatives, only pathogenic or likely pathogenic variants must be used for clinical decision-making.

Germ cell mosaicism for AUTS2 exon 6 deletion.

Haploinsufficiency of AUTS2 has been associated with neurodevelopmental disorders and dysmorphic features (MIM # 615834). More than 50 patients have been described, mostly carrying de novo deletions of one or more exons, including eight patients with exon 6 deletions. We report on two siblings, a girl and a boy aged 11 and 13 years, in whom the same pathogenic 85 kb deletion on 7q11.22 encompassing exon 6 of AUTS2 by SNP array analysis was identified. Both children had typical symptoms of AUTS2 syndrome such as intellectual impairment and behavioral problems, but with markedly different expression. SNP array analysis excluded the deletion in blood samples of both parents and a healthy brother. Conventional karyotyping of both parents and additional FISH analyses, marking the flanking regions of the deletion, did not show any structural rearrangements involving 7q11.22. A germ cell mosaicism was suggested as the most probable explanation for occurrence of the same deletion in these two siblings. To our knowledge this is the first report of germ cell mosaicism for AUTS2 syndrome. It additionally provides further evidence of intrafamilial phenotypic variability in AUTS2 syndrome and adds clinical information to the phenotypic spectrum of patients with AUTS2 exon 6 deletions.

Germline SDHB-inactivating mutation in gastric spindle cell sarcoma.

Gastrointestinal stromal tumors (GISTs) are the most frequent mesenchymal tumors of the gastrointestinal tract. Inactivating mutations or epigenetic deregulation of succinate dehydrogenase complex (SDH) genes are considered defining features of a subset of GIST occurring in the stomach. Based on comprehensive molecular profiling and biochemical analysis within a precision oncology program, we identified hallmarks of SDH deficiency (germline SDHB-inactivating mutation accompanied by somatic loss of heterozygosity, lack of SDHB expression, global DNA hypermethylation, and elevated succinate/fumarate ratio) in a 40-year-old woman with undifferentiated gastric spindle cell sarcoma that did not meet the diagnostic criteria for other mesenchymal tumors of the stomach, including GIST. These data reveal that the loss of SDH function can be involved in the pathogenesis of non-GIST sarcoma of the gastrointestinal tract.

Metabolome-guided genomics to identify pathogenic variants in isocitrate dehydrogenase, fumarate hydratase, and succinate dehydrogenase genes in pheochromocytoma and paraganglioma.

PURPOSE: Metabolic aberrations have been described in neoplasms with pathogenic variants (PV) in the Krebs cycle genes encoding succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH). In turn, accumulation of oncometabolites succinate, fumarate, and 2-hydroxyglutarate can be employed to identify tumors with those PV . Additionally, such metabolic readouts may aid in genetic variant interpretation and improve diagnostics. METHODS: Using liquid chromatography-mass spectrometry, 395 pheochromocytomas and paragangliomas (PPGLs) from 391 patients were screened for metabolites to indicate Krebs cycle aberrations. Multigene panel sequencing was applied to detect driver PV in cases with indicative metabolite profiles but undetermined genetic drivers. RESULTS: Aberrant Krebs cycle metabolomes identified rare cases of PPGLs with germline PV in FH and somatic PV in IDHx and SDHx, including the first case of a somatic IDH2 PV in PPGL. Metabolomics also reliably identified PPGLs with SDHx loss-of-function (LOF) PV. Therefore we utilized tumor metabolite profiles to further classify variants of unknown significance in SDHx, thereby enabling missense variants associated with SDHx LOF to be distinguished from benign variants. CONCLUSION: We propose incorporation of metabolome data into the diagnostics algorithm in PPGLs to guide genetic testing and variant interpretation and to help identify rare cases with PV in FH and IDHx.

Targeted capture-based NGS is superior to multiplex PCR-based NGS for hereditary BRCA1 and BRCA2 gene analysis in FFPE tumor samples.

BACKGROUND: With the introduction of Olaparib treatment for BRCA-deficient recurrent ovarian cancer, testing for somatic and/or germline mutations in BRCA1/2 genes in tumor tissues became essential for treatment decisions. In most cases only formalin-fixed paraffin-embedded (FFPE) samples, containing fragmented and chemically modified DNA of minor quality, are available. Thus, multiplex PCR-based sequencing is most commonly applied in routine molecular testing, which is predominantly focused on the identification of known hot spot mutations in oncogenes. METHODS: We compared the overall performance of an adjusted targeted capture-based enrichment protocol and a multiplex PCR-based approach for calling of pathogenic SNVs and InDels using DNA extracted from 13 FFPE tissue samples. We further applied both strategies to seven blood samples and five matched FFPE tumor tissues of patients with known germline exon-spanning deletions and gene-wide duplications in BRCA1/2 to evaluate CNV detection based solely on panel NGS data. Finally, we analyzed DNA from FFPE tissues of 11 index patients from families suspected of having hereditary breast and ovarian cancer, of whom no blood samples were available for testing, in order to identify underlying pathogenic germline BRCA1/2 mutations. RESULTS: The multiplex PCR-based protocol produced inhomogeneous coverage among targets of each sample and between samples as well as sporadic amplicon drop out, leading to insufficiently or non-covered nucleotides, which subsequently hindered variant detection. This protocol further led to detection of PCR-artifacts that could easily have been misinterpreted as pathogenic mutations. No such limitations were observed by application of an adjusted targeted capture-based protocol, which allowed for CNV calling with 86% sensitivity and 100% specificity. All pathogenic CNVs were confirmed in the five matched FFPE tumor samples from patients carrying known pathogenic germline mutations and we additionally identified somatic loss of the second allele in BRCA1/2. Furthermore we detected pathogenic BRCA1/2 variants in four the eleven FFPE samples from patients of whom no blood was available for analysis. CONCLUSIONS: We demonstrate that an adjusted targeted capture-based enrichment protocol is superior to commonly applied multiplex PCR-based protocols for reliable BRCA1/2 variant detection, including CNV-detection, using FFPE tumor samples.

Skewed X-inactivation in a family with DLG3-associated X-linked intellectual disability.

Mutations in DLG3 are a rare cause of non-syndromic X-linked intellectual disability (XLID) (MRX90, OMIM *300189). Only ten DLG3 mutations have been reported to date. The majority of female heterozygous mutation carriers was healthy and had random X-inactivation patterns. We report on an XLID family with a novel DLG3 mutation. The 12-year-old male index patient had moderate intellectual disability (ID) and dysmorphic features. The mutation was also present in four female relatives. A maternal aunt had moderate ID and significantly skewed X-inactivation favorably inactivating the normal DLG3 allele. The proband's healthy mother also had skewed X-inactivation but in the opposite direction (i.e., inactivation of the mutated allele). Two other female relatives had intermediate cognitive phenotypes and random X-inactivation. This family broadens the mutational and phenotypical spectrum of DLG3-associated XLID and demonstrates that heterozygous female mutation carriers can be as severely affected as males. Reports of additional families will be needed to elucidate the causes of unfavorable skewing in female XLID patients.

CATCH: A Prospective Precision Oncology Trial in Metastatic Breast Cancer.

PURPOSE: CATCH (Comprehensive Assessment of clinical feaTures and biomarkers to identify patients with advanced or metastatic breast Cancer for marker driven trials in Humans) is a prospective precision oncology program that uses genomics and transcriptomics to guide therapeutic decisions in the clinical management of metastatic breast cancer. Herein, we report our single-center experience and results on the basis of the first 200 enrolled patients of an ongoing trial. METHODS: From June 2017 to March 2019, 200 patients who had either primary metastatic or progressive disease, with any number of previous treatment lines and at least one metastatic site accessible to biopsy, were enrolled. DNA and RNA from tumor tissue and corresponding blood-derived nontumor DNA were profiled using whole-genome and transcriptome sequencing. Identified actionable alterations were brought into clinical context in a multidisciplinary molecular tumor board (MTB) with the aim of prioritizing personalized treatment recommendations. RESULTS: Among the first 200 enrolled patients, 128 (64%) were discussed in the MTB, of which 64 (50%) were subsequently treated according to MTB recommendation. Of 53 evaluable patients, 21 (40%) achieved either stable disease (n = 13, 25%) or partial response (n = 8, 15%). Furthermore, 16 (30%) of those patients showed improvement in progression-free survival of at least 30% while on MTB-recommended treatment compared with the progression-free survival of the previous treatment line. CONCLUSION: The initial phase of this study demonstrates that precision oncology on the basis of whole-genome and RNA sequencing is feasible when applied in the clinical management of patients with metastatic breast cancer and provides clinical benefit to a substantial proportion of patients.

Comprehensive Genomic and Transcriptomic Analysis for Guiding Therapeutic Decisions in Patients with Rare Cancers.

The clinical relevance of comprehensive molecular analysis in rare cancers is not established. We analyzed the molecular profiles and clinical outcomes of 1,310 patients (rare cancers, 75.5%) enrolled in a prospective observational study by the German Cancer Consortium that applies whole-genome/exome and RNA sequencing to inform the care of adults with incurable cancers. On the basis of 472 single and six composite biomarkers, a cross-institutional molecular tumor board provided evidence-based management recommendations, including diagnostic reevaluation, genetic counseling, and experimental treatment, in 88% of cases. Recommended therapies were administered in 362 of 1,138 patients (31.8%) and resulted in significantly improved overall response and disease control rates (23.9% and 55.3%) compared with previous therapies, translating into a progression-free survival ratio >1.3 in 35.7% of patients. These data demonstrate the benefit of molecular stratification in rare cancers and represent a resource that may promote clinical trial access and drug approvals in this underserved patient population. SIGNIFICANCE: Rare cancers are difficult to treat; in particular, molecular pathogenesis-oriented medical therapies are often lacking. This study shows that whole-genome/exome and RNA sequencing enables molecularly informed treatments that lead to clinical benefit in a substantial proportion of patients with advanced rare cancers and paves the way for future clinical trials.See related commentary by Eggermont et al., p. 2677.This article is highlighted in the In This Issue feature, p. 2659.

Acute Stanford type B aortic dissection-who benefits from genetic testing?

BACKGROUND: Stanford type B aortic dissection is a rare, life-threatening complex phenotype associated with several modifiable and genetic risk factors. In the current study of a hospital-based, consecutive series of aortic dissection patients we propose a selection based on age and family history of aortic disease for genetic testing and detection of causative gene variants. METHODS: In this single center cohort study from 2013 to 2018 patients with acute Stanford type B aortic dissections were consecutively treated and analyzed by next generation sequencing based on selection criteria (age of disease onset ≤45 years and/or positive familial history for aortic disease) to detect genome-wide pathogenic variants in protein-coding sequences and to identify large copy number variants (CNV). Variants in a predefined panel of 30 genes associated with the familial thoracic aortic aneurysm and dissection (TAAD) syndrome were evaluated. RESULTS: From 105 patients nine matched selection criteria for genetic testing. Next-generation sequencing analysis revealed causal variants in FBN1 (fibrillin-1) in three patients: a pathogenic missense variant [c.6661T>C, p.(Cys2221Arg)] and two truncating variants [c.4786C>T, p.(Arg1596Ter)] and [c.6366C>CA, p.(Asp2123GlufsTer5)]. A fourth patient carried a large (>1,000,000 bp) CNV in the long arm of chromosome 10, deleting eleven genes, including the whole ACTA2 (actin alpha 2) gene. The latter two genetic findings have not been reported before. CONCLUSIONS: Selection of patients on the basis of young age and familial inheritance of aortic disease favors the identification of disease-causing genetic variants in a clinical cohort of patients with Stanford type B aortic dissection.

Correction: Diagnostic value of partial exome sequencing in developmental disorders.

[This corrects the article DOI: 10.1371/journal.pone.0201041.].

Targetable ERBB2 mutations identified in neurofibroma/schwannoma hybrid nerve sheath tumors.

BACKGROUNDNeurofibroma/schwannoma hybrid nerve sheath tumors (N/S HNSTs) are neoplasms associated with larger nerves that occur sporadically and in the context of schwannomatosis or neurofibromatosis type 2 or 1. Clinical management of N/S HNSTs is challenging, especially for large tumors, and established systemic treatments are lacking.METHODSWe used next-generation sequencing and array-based DNA methylation profiling to determine the clinically actionable genomic and epigenomic landscapes of N/S HNSTs.RESULTSWhole-exome sequencing within a precision oncology program identified an activating mutation (p.Asp769Tyr) in the catalytic domain of the ERBB2 receptor tyrosine kinase in a patient with schwannomatosis-associated N/S HNST, and targeted treatment with the small-molecule ERBB inhibitor lapatinib led to prolonged clinical benefit and a lasting radiographic and metabolic response. Analysis of a multicenter validation cohort revealed recurrent ERBB2 mutations (p.Leu755Ser, p.Asp769Tyr, p.Val777Leu) in N/S HNSTs occurring in patients who met diagnostic criteria for sporadic schwannomatosis (3 of 7 patients), but not in N/S HNSTs arising in the context of neurofibromatosis (6 patients) or outside a tumor syndrome (1 patient), and showed that ERBB2-mutant N/S HNSTs cluster in a distinct subgroup of peripheral nerve sheath tumors based on genome-wide DNA methylation patterns.CONCLUSIONThese findings uncover a key biological feature of N/S HNSTs that may have important diagnostic and therapeutic implications.FUNDINGThis work was supported by grant H021 from DKFZ-HIPO, the University Cancer Center Frankfurt, and the Frankfurt Research Funding Clinician Scientist Program.

The landscape of chromothripsis across adult cancer types.

Chromothripsis is a recently identified mutational phenomenon, by which a presumably single catastrophic event generates extensive genomic rearrangements of one or a few chromosome(s). Considered as an early event in tumour development, this form of genome instability plays a prominent role in tumour onset. Chromothripsis prevalence might have been underestimated when using low-resolution methods, and pan-cancer studies based on sequencing are rare. Here we analyse chromothripsis in 28 tumour types covering all major adult cancers (634 tumours, 316 whole-genome and 318 whole-exome sequences). We show that chromothripsis affects a substantial proportion of human cancers, with a prevalence of 49% across all cases. Chromothripsis generates entity-specific genomic alterations driving tumour development, including clinically relevant druggable fusions. Chromothripsis is linked with specific telomere patterns and univocal mutational signatures in distinct tumour entities. Longitudinal analysis of chromothriptic patterns in 24 matched tumour pairs reveals insights in the clonal evolution of tumours with chromothripsis.

Novel truncating PPM1D mutation in a patient with intellectual disability.

Truncating mutations in the last and penultimate exons of the PPM1D gene were recently described as a cause for mild to severe intellectual disability in fourteen patients. Feeding difficulties, periods of fever and vomiting as well as a high pain threshold were described as additional characteristic features and the disorder was subsequently termed "intellectual developmental disorder with gastrointestinal difficulties and high pain threshold (IDDGIP)" in the OMIM database (MIM # 617450). Here we report on an additional patient carrying a novel de novo truncating mutation NM_003620.3: c.1535del, p.(Asn512Ilefs*2) in the last exon of PPM1D. While the patient showed features overlapping with the reported phenotype, such as a short stature and small hands and feet, he also presented with additional features like cleft lip and palate and an aberrant right subclavian artery. Notably, the patient did not have any gastrointestinal difficulties or periods of fever, indicating variability of the phenotype of patients with PPM1D mutations.

Response to olaparib in a PALB2 germline mutated prostate cancer and genetic events associated with resistance.

Prostate cancers harboring DNA repair gene alterations are particularly sensitive to PARP inhibitor treatment. We report a case of an advanced prostate cancer patient profiled within the NCT-MASTER (Molecularly Aided Stratification for Tumor Eradication Research) precision oncology program using next-generation sequencing. Comprehensive genomic and transcriptomic analysis identified a pathogenic germline PALB2 variant as well as a mutational signature associated with disturbed homologous recombination together with structural genomic rearrangements. A molecular tumor board identified a potential benefit of targeted therapy and recommended PARP inhibition and platinum-based chemotherapy. Single-agent treatment with the PARP inhibitor olaparib as well as subsequent combination with platinum-based chemotherapy resulted in disease stabilization and substantial improvement of clinical symptoms. Upon progression, we performed whole-exome and RNA sequencing of a liver metastasis, which demonstrated up-regulation of several genes characteristic for the neuroendocrine prostate cancer phenotype as well as a novel translocation resulting in an in-frame, loss-of-function fusion of RB1. We suggest that multidimensional genomic characterization of prostate cancer patients undergoing PARP inhibitor therapy will be necessary to capture and understand predictive biomarkers of PARP inhibitor sensitivity and resistance.

Optimizing Genetic Workup in Pheochromocytoma and Paraganglioma by Integrating Diagnostic and Research Approaches.

Pheochromocytomas and paragangliomas (PPGL) are rare neuroendocrine tumors with a strong hereditary background and a large genetic heterogeneity. Identification of the underlying genetic cause is crucial for the management of patients and their families as it aids differentiation between hereditary and sporadic cases. To improve diagnostics and clinical management we tailored an enrichment based comprehensive multi-gene next generation sequencing panel applicable to both analyses of tumor tissue and blood samples. We applied this panel to tumor samples and compared its performance to our current routine diagnostic approach. Routine diagnostic sequencing of 11 PPGL susceptibility genes was applied to blood samples of 65 unselected PPGL patients at a single center in Dresden, Germany. Predisposing germline mutations were identified in 19 (29.2%) patients. Analyses of 28 PPGL tumor tissues using the dedicated PPGL panel revealed pathogenic or likely pathogenic variants in known PPGL susceptibility genes in 21 (75%) cases, including mutations in IDH2, ATRX and HRAS. These mutations suggest sporadic tumor development. Our results imply a diagnostic benefit from extended molecular tumor testing of PPGLs and consequent improvement of patient management. The approach is promising for determination of prognostic biomarkers that support therapeutic decision-making.

Synergistic Highly Potent Targeted Drug Combinations in Different Pheochromocytoma Models Including Human Tumor Cultures.

There are no officially approved therapies for metastatic pheochromocytomas apart from ultratrace 131I-metaiodbenzylguanidine therapy, which is approved only in the United States. We have, therefore, investigated the antitumor potential of molecular-targeted approaches in murine pheochromocytoma cell lines [monocyte chemoattractant protein (MPC)/monocyte chemoattractant protein/3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)], immortalized mouse chromaffin Sdhb-/- cells, three-dimensional pheochromocytoma tumor models (MPC/MTT spheroids), and human pheochromocytoma primary cultures. We identified the specific phosphatidylinositol-3-kinase α inhibitor BYL719 and the mammalian target of rapamycin inhibitor everolimus as the most effective combination in all models. Single treatment with clinically relevant doses of BYL719 and everolimus significantly decreased MPC/MTT and Sdhb-/- cell viability. A targeted combination of both inhibitors synergistically reduced MPC and Sdhb-/- cell viability and showed an additive effect on MTT cells. In MPC/MTT spheroids, treatment with clinically relevant doses of BYL719 alone or in combination with everolimus was highly effective, leading to a significant shrinkage or even a complete collapse of the spheroids. We confirmed the synergism of clinically relevant doses of BYL719 plus everolimus in human pheochromocytoma primary cultures of individual patient tumors with BYL719 attenuating everolimus-induced AKT activation. We have thus established a method to assess molecular-targeted therapies in human pheochromocytoma cultures and identified a highly effective combination therapy. Our data pave the way to customized combination therapy to target individual patient tumors.

Defective homologous recombination DNA repair as therapeutic target in advanced chordoma.

Chordomas are rare bone tumors with few therapeutic options. Here we show, using whole-exome and genome sequencing within a precision oncology program, that advanced chordomas (n = 11) may be characterized by genomic patterns indicative of defective homologous recombination (HR) DNA repair and alterations affecting HR-related genes, including, for example, deletions and pathogenic germline variants of BRCA2, NBN, and CHEK2. A mutational signature associated with HR deficiency was significantly enriched in 72.7% of samples and co-occurred with genomic instability. The poly(ADP-ribose) polymerase (PARP) inhibitor olaparib, which is preferentially toxic to HR-incompetent cells, led to prolonged clinical benefit in a patient with refractory chordoma, and whole-genome analysis at progression revealed a PARP1 p.T910A mutation predicted to disrupt the autoinhibitory PARP1 helical domain. These findings uncover a therapeutic opportunity in chordoma that warrants further exploration, and provide insight into the mechanisms underlying PARP inhibitor resistance.

Next-generation panel sequencing identifies NF1 germline mutations in three patients with pheochromocytoma but no clinical diagnosis of neurofibromatosis type 1.

OBJECTIVE: Our objective was to improve molecular diagnostics in patients with hereditary pheochromocytoma and paraganglioma (PPGL) by using next-generation sequencing (NGS) multi-gene panel analysis. Derived from this study, we here present three cases that were diagnosed with NF1 germline mutations but did not have a prior clinical diagnosis of neurofibromatosis type 1 (NF1). DESIGN: We performed genetic analysis of known tumor predisposition genes, including NF1, using a multi-gene NGS enrichment-based panel applied to a total of 1029 PPGL patients. We did not exclude genes known to cause clinically defined syndromes such as NF1 based on missing phenotypic expression as is commonly practiced. METHODS: Genetic analysis was performed using NGS (TruSight Cancer Panel/customized panel by Illumina) for analyzing patients' blood and tumor samples. Validation was carried out by Sanger sequencing. RESULTS: Within our cohort, three patients, who were identified to carry pathogenic NF1 germline mutations, attracted attention, since none of the patients had a clinical suspicion of NF1 and one of them was initially suspected to have MEN2A syndrome due to co-occurrence of a medullary thyroid carcinoma. In these cases, one splice site, one stop and one frameshift mutation in NF1 were identified. CONCLUSIONS: Since phenotypical presentation of NF1 is highly variable, we suggest analysis of the NF1 gene also in PPGL patients who do not meet diagnostic NF1 criteria. Co-occurrence of medullary thyroid carcinoma and PPGL was found to be a clinical decoy in NF1 diagnostics. These observations underline the value of multi-gene panel NGS for PPGL patients.