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.Acknowledgments: We thank the patients and their families who have made this research possible. We want to thank JacquesW. Lenders for his support. We further thank Alexander Krüger, Lydia Rossow and Franziska Stübner for technical support as well as Katharina Langton and Uwe Siemon for their assistance in patient administration.S

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

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

Abstract 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

Defective homologous recombination DNA repair as therapeutic target in advanced chordoma

Chordomas are rare bone tumors with limited therapeutic options. Here, the authors identify molecular alterations associated with defective homologous recombination DNA repair in advanced chordomas and report prolonged response in a patient treated with a PARP inhibitor, which later acquired resistance due to a newly gained PARP1 mutation

Diagnostic value of partial exome sequencing in developmental disorders.

Although intellectual disability is one of the major indications for genetic counselling, there are no homogenous diagnostic algorithms for molecular testing. While whole exome sequencing is increasingly applied, we questioned whether analyzing a partial exome, enriched for genes associated with Mendelian disorders, might be a valid alternative approach that yields similar detection rates but requires less sequencing capacities. Within this context 106 patients with different intellectual disability forms were analyzed for mutations in 4.813 genes after pre-exclusion of copy number variations by array-CGH. Subsequent variant interpretation was performed in accordance with the ACMG guidelines. By this, a molecular diagnosis was established in 34% of cases and candidate mutations were identified in additional 24% of patients. Detection rates of causative mutations were above 30%, regardless of further symptoms, except for patients with seizures (23%). We did not detect an advantage from partial exome sequencing for patients with severe intellectual disability (36%) as compared to those with mild intellectual disability (44%). Specific clinical diagnoses pre-existed for 20 patients. Of these, 5 could be confirmed and an additional 6 cases could be solved, but showed mutations in other genes than initially suspected. In conclusion partial exome sequencing solved >30% of intellectual disability cases, which is similar to published rates obtained by whole exome sequencing. The approach therefore proved to be a valid alternative to whole exome sequencing for molecular diagnostics in this cohort. The method proved equally suitable for both syndromic and non-syndromic intellectual disability forms of all severity grades

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

Comprehensive genomic characterization of gene therapy-induced T-cell acute lymphoblastic leukemia

Drug combinations that target critical pathways are a mainstay of cancer care. To improve current approaches to combination treatment of chronic lymphocytic leukemia (CLL) and gain insights into the underlying biology, we studied the effect of 352 drug combination pairs in multiple concentrations by analysing ex vivo drug response of 52 primary CLL samples, which were characterized by 'omics' profiling. Known synergistic interactions were confirmed for B-cell receptor (BCR) inhibitors with Bcl-2 inhibitors and with chemotherapeutic drugs, suggesting that this approach can identify clinically useful combinations. Moreover, we uncovered synergistic interactions between BCR inhibitors and afatinib, which we attribute to BCR activation by afatinib through BLK upstream of BTK and PI3K. Combinations of multiple inhibitors of BCR components (e.g., BTK, PI3K, SYK) had effects similar to the single agents. While PI3K and BTK inhibitors produced overall similar effects in combinations with other drugs, we uncovered a larger response heterogeneity of combinations including PI3K inhibitors, predominantly in CLL with mutated IGHV, which we attribute to the target's position within the BCR-signaling pathway. Taken together, our study shows that drug combination effects can be effectively queried in primary cancer cells, which could aid discovery, triage and clinical development of drug combinations

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-MIBG therapy approved only in the United States. We have, therefore, investigated the anti-tumor potential of novel molecular-targeted approaches in murine pheochromocytoma cell lines (MPC/MTT), immortalized mouse chromaffin Sdhb-/- cells, 3D-pheochromocytoma tumor models (MPC/MTT spheroids) and human pheochromocytoma primary cultures. We identified the specific PI3Kα inhibitor BYL719 and the mTORC1 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