Deep targeted sequencing of TP53 in chronic lymphocytic leukemia: clinical impact at diagnosis and at time of treatment.

In chronic lymphocytic leukemia, TP53 mutations and deletion of chromosome 17p are well-characterized biomarkers associated with poor progression-free and overall survival following chemoimmunotherapy. Patients harboring low burden TP53 mutations with variant allele frequencies of 0.3-15% have been shown to have similar dismal outcome as those with high burden mutations. We here describe a highly sensitive deep targeted next-generation sequencing assay allowing for the detection of TP53 mutations as low as 0.2% variant allele frequency. Within a consecutive, single center cohort of 290 newly diagnosed patients with chronic lymphocytic leukemia, deletion of chromosome 17p was the only TP53 aberration significantly associated with shorter overall survival and treatment-free survival. We were unable to demonstrate any impact of TP53 mutations, whether high burden (variant allele frequency >10%) or low burden (variant allele frequency ≤10%), in the absence of deletion of chromosome 17p. In addition, the impact of high burden TP53 aberration (deletion of chromosome 17p and/or TP53 mutation with variant allele frequency >10%) was only evident for patients with IGHV unmutated status; no impact of TP53 aberrations on outcome was seen for patients with IGHV mutated status. In 61 patients at time of treatment, the prognostic impact of TP53 mutations over 1% variant allele frequency could be confirmed. This study furthers the identification of a clinical significant limit of detection for robust TP53 mutation analysis in chronic lymphocytic leukemia. Multicenter studies are needed for validation of ultra-sensitive TP53 mutation assays in order to define and implement a technical as well as a clinical lower limit of detection.

The BRCA2 c.68-7T > A variant is not pathogenic: A model for clinical calibration of spliceogenicity.

Although the spliceogenic nature of the BRCA2 c.68-7T > A variant has been demonstrated, its association with cancer risk remains controversial. In this study, we accurately quantified by real-time PCR and digital PCR (dPCR), the BRCA2 isoforms retaining or missing exon 3. In addition, the combined odds ratio for causality of the variant was estimated using genetic and clinical data, and its associated cancer risk was estimated by case-control analysis in 83,636 individuals. Co-occurrence in trans with pathogenic BRCA2 variants was assessed in 5,382 families. Exon 3 exclusion rate was 4.5-fold higher in variant carriers (13%) than controls (3%), indicating an exclusion rate for the c.68-7T > A allele of approximately 20%. The posterior probability of pathogenicity was 7.44 × 10-115 . There was neither evidence for increased risk of breast cancer (OR 1.03; 95% CI 0.86-1.24) nor for a deleterious effect of the variant when co-occurring with pathogenic variants. Our data provide for the first time robust evidence of the nonpathogenicity of the BRCA2 c.68-7T > A. Genetic and quantitative transcript analyses together inform the threshold for the ratio between functional and altered BRCA2 isoforms compatible with normal cell function. These findings might be exploited to assess the relevance for cancer risk of other BRCA2 spliceogenic variants.

Genomic profiling of newly diagnosed glioblastoma patients and its potential for clinical utility - a prospective, translational study.

Glioblastoma (GBM) is an incurable brain tumor for which new treatment strategies are urgently needed. Next-generation sequencing of GBM has most often been performed retrospectively and on archival tissue from both diagnostic and relapse surgeries with limited knowledge of clinical information, including treatment given. We sought to investigate the genomic composition prospectively in treatment-naïve patients, searched for possible targetable aberrations, and investigated for prognostic and/or predictive factors. A total of 108 newly diagnosed GBM patients were included. Clinical information, progression-free survival, and overall survival (OS) were noted. Tissues were analyzed by whole-exome sequencing, single nucleotide polymorphism (SNP) and transcriptome arrays, and RNA sequencing; assessed for mutations, fusions, tumor mutational burden (TMB), and chromosomal instability (CI); and classified into GBM subgroups. Each genomic report was discussed at a multidisciplinary tumor board meeting to evaluate for matching trials. From 111 consecutive patients, 97.3% accepted inclusion in this study. Eighty-six (77%) were treated with radiation therapy/temozolomide (TMZ) and adjuvant TMZ. One NTRK2 and three FGFR3-TACC3 fusions were identified. Copy number alterations in GRB2 and SMYD4 were significantly correlated with worse median OS together with known clinical variables like age, performance status, steroid dose, and O6-methyl-guanine-DNA-methyl-transferase status. Patients with CI-median or TMB-high had significantly worse median OS compared to CI-low/high or TMB-low/median. In conclusion, performing genomic profiling at diagnosis enables evaluation of genomic-driven therapy at the first progression. Furthermore, TMB-high or CI-median patients had worse median OS, which can support the possibility of offering experimental treatment already at the first line for this group.

Importance of Comprehensive Molecular Profiling for Clinical Outcome in Children With Recurrent Cancer.

Purpose: Pediatric cancers are often difficult to classify and can be complex to treat. To ensure precise diagnostics and identify relevant treatment targets, we implemented comprehensive molecular profiling of consecutive pediatric patients with cancer relapse. We evaluated the clinical impact of extensive molecular profiling by assessing the frequency of identified biological onco-drivers, altered diagnosis, and/or identification of new relevant targeted therapies. Patients and Methods: Forty-six tumor samples (44 fresh-frozen; two formalin-fixed paraffin embedded), two bone marrow aspirates, three cerebrospinal fluid samples, and one archived DNA were obtained from 48 children (0-17 years; median 9.5) with relapsed or refractory cancer, where the disease was rapidly progressing in spite of their current treatment or they had exhausted all treatment options. The samples were analyzed by whole-exome sequencing (WES), RNA sequencing (RNAseq), transcriptome arrays, and SNP arrays. Final reports were available within 3-4 weeks after patient inclusion and included mutation status, a description of copy number alterations, differentially expressed genes, and gene fusions, as well as suggestions for targeted treatment. Results: Of the 48 patients, 33 had actionable findings. The most efficient method for the identification of actionable findings was WES (39%), followed by SNP array (37%). Of note, gene fusions were identified by RNAseq in 21% of the samples. Eleven findings led to clinical intervention, i.e., oncogenetic counseling, targeted treatment, and treatment based on changed diagnosis. Four patients received compassionate use targeted therapy. Six patients experienced direct benefits in the form of stable disease or response. Conclusion: The application of comprehensive genetic diagnostics in children with recurrent cancers allowed for discovery and implementation of effective targeted therapies and hereby improvement of outcome in some patients.

Circulating tumor DNA as a marker of treatment response in BRAF V600E mutated non-melanoma solid tumors.

PURPOSE: We evaluated longitudinal tracking of BRAF V600E in circulating cell-free DNA (cfDNA) as a marker of treatment response to BRAF inhibitor (BRAFi) combination therapies in non-melanoma solid tumors included in the Copenhagen Prospective Personalized Oncology (CoPPO) program. EXPERIMENTAL DESIGN: Patients with BRAF V600E-mutated tumors were treated with combination therapies including BRAFi. Quantification of mutant cfDNA from plasma was determined and correlated to clinical outcomes. Exome sequencing was performed to identify possible resistance mutations. RESULTS: Twenty-three patients had BRAF-mutated tumors out of 455 patients included in CoPPO and 17 started BRAFi combination (EGFRi/MEKi) therapy. Tumor responses were achieved in 8 out of 16 evaluable patients and the median overall- and progression-free survival (OS and PFS) was 15 and 4.8 months, respectively. Longitudinal measurements of BRAF V600E-mutant cfDNA indicated disease progression prior to radiological evaluation and a reduction in the mutant fraction of more than 50% after 4 and 12 weeks of therapy was associated with a significantly longer PFS (p=0.003 and p=0.029) and OS (p=0.029 and p=0.017). Furthermore, the baseline mutant fraction and total level of cfDNA positively correlated with tumor burden (p=0.026 and p=0.024). Finally, analysis of cfDNA at progression revealed novel mutations potentially affecting the MAPK pathway. CONCLUSION: BRAFi combination therapies showed a response rate of 50% in BRAF V600E-mutated non-melanoma tumors. The fraction of BRAF-mutant cfDNA represent a sensitive indicator for clinical outcomes with plasma collected at week 4 and 12 as crucial time points for monitoring response and disease progression.

Next-Generation Sequencing-Based Detection of Germline Copy Number Variations in BRCA1/BRCA2: Validation of a One-Step Diagnostic Workflow.

Genetic testing of BRCA1/2 includes screening for single nucleotide variants and small insertions/deletions and for larger copy number variations (CNVs), primarily by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). With the advent of next-generation sequencing (NGS), it has become feasible to provide CNV information and sequence data using a single platform. We report the use of NGS gene panel sequencing on the Illumina MiSeq platform and JSI SeqPilot SeqNext software to call germline CNVs in BRCA1 and BRCA2. For validation 18 different BRCA1/BRCA2 CNVs previously identified by MLPA in 48 Danish breast and/or ovarian cancer families were analyzed. Moreover, 120 patient samples previously determined as negative for BRCA1/BRCA2 CNVs by MLPA were included in the analysis. Comparison of the NGS data with the data from MLPA revealed that the sensitivity was 100%, whereas the specificity was 95%. Taken together, this study validates a one-step bioinformatics work-flow to call germline BRCA1/2 CNVs using data obtained by NGS of a breast cancer gene panel. The work-flow represents a robust and easy-to-use method for full BRCA1/2 screening, which can be easily implemented in routine diagnostic testing and adapted to genes other than BRCA1/2.