Activating ERBB2/HER2 mutations indicate susceptibility to pan-HER inhibitors in Lynch and Lynch-like colorectal cancer.

OBJECTIVE: Microsatellite instability (MSI) is detected in approximately 15% of all colorectal cancers (CRC) and virtually in all cases with Lynch syndrome. The MSI phenotype is caused by dysfunctional mismatch repair (MMR) and leads to accumulation of DNA replication errors. Sporadic MSI CRC often harbours BRAF(V600E); however, no consistent data exist regarding targeted treatment approaches in BRAF(wt) MSI CRC. DESIGN: Mutations and quantitative MSI were analysed by deep sequencing in 196 formalin fixed paraffin embedded (FFPE) specimens comprising Lynch and Lynch-like CRCs from the German Hereditary Nonpolyposis Colorectal Cancer registry. Functional relevance of recurrent ERBB2/HER2 mutations was investigated in CRC cell lines using reversible and irreversible HER-targeting inhibitors, EGFR-directed antibody cetuximab, HER2-directed antibody trastuzumab and siRNA-mediated ERBB2/HER2 knockdown. RESULTS: Quantification of nucleotide loss in non-coding mononucleotide repeats distinguished microsatellite status with very high accuracy (area under curve=0.9998) and demonstrated progressive losses with deeper invasion of MMR-deficient colorectal neoplasms (p=0.008). Characterisation of BRAF(wt) MSI CRC revealed hot-spot mutations in well-known oncogenic drivers, including KRAS (38.7%), PIK3CA (36.5%), and ERBB2 (15.0%). L755S and V842I substitutions in ERBB2 were highly recurrent. Functional analyses in ERBB2-mutated MSI CRC cell lines revealed a differential response to HER-targeting compounds and superiority of irreversible pan-HER inhibitors. CONCLUSIONS: We developed a high-throughput deep sequencing approach for concomitant MSI and mutational analyses in FFPE specimens. We provided novel insights into clinically relevant alterations in MSI CRC and a rationale for targeting ERBB2/HER2 mutations in Lynch and Lynch-like CRC.

Comprehensive Analysis of Disease-Related Genes in Chronic Lymphocytic Leukemia by Multiplex PCR-Based Next Generation Sequencing.

BACKGROUND: High resolution molecular studies have demonstrated that the clonal acquisition of gene mutations is an important mechanism that may promote rapid disease progression and drug resistance in chronic lymphocytic leukemia (CLL). Therefore, the early and sensitive detection of such mutations is an important prerequisite for future predictive CLL diagnostics in the clinical setting. MATERIAL & METHODS: Here, we describe a novel, target-specific next generation sequencing (NGS) approach, which combines multiplex PCR-based target enrichment and library generation with ultra-deep high-throughput parallel sequencing using a MiSeq platform. We designed a CLL specific target panel, covering hotspots or complete coding regions of 15 genes known to be recurrently mutated and/or related to B-cell receptor signaling. RESULTS: High-throughput sequencing was performed using as little as 40 ng of peripheral blood B-cell DNA from 136 CLL patients and a dilution series of two ATM- or TP53-mutated cell lines, the latter of which demonstrated a limit of mutation detection below 5%. Using a stringent functional assessment algorithm, 102 mutations in 8 genes were identified in CLL patients, including hotspot regions of TP53, SF3B1, NOTCH1, ATM, XPO1, MYD88, DDX3X and the B-cell receptor signaling regulator PTPN6. The presence of mutations was significantly associated with an advanced disease status und molecular markers of an inferior prognosis, such as an unmutated IGHV mutation status or positivity for ZAP70 by flow cytometry. CONCLUSION: In summary, targeted sequencing using an amplicon based library technology allows a resource-efficient and sensitive mutation analysis for diagnostic or exploratory purposes and facilitates molecular subtyping of patient sets with adverse prognosis.

Deep ion sequencing of amplicon adapter ligated libraries: a novel tool in molecular diagnostics of formalin fixed and paraffin embedded tissues.

Due to the advanced progress in personalised therapy concepts for non-small cell lung cancer (NSCLC), we applied the ion semiconductor sequencing (ISS) approach to molecular diagnosis of NSCLC, analysing a set of therapy relevant gene loci. DNA from macrodissected tumour samples of formalin fixed biopsies was used for PCR amplification of EGFR exons 18, 19, 21 and KRAS exon 1. A total of 128 PCR products were analysed by conventional termination sequencing as well as by ISS. Sensitivity of ISS was additionally determined using 100-10 000 copies of reference mutants. All somatic mutations detected by direct Sanger sequencing were also identified by ISS. No additional mutants were detected. Running samples with limited copies of mutated alleles revealed high sensitivity, detecting less than 10% (2500 copies) mutants in a human wild type background. In conclusion, multiplexed mutation analyses by ISS is an efficient technology that can easily be linked to existing PCR approaches in molecular pathology.

Psychological treatments for fibromyalgia: a meta-analysis.

The aims of the present analysis were to investigate the short- and long-term efficacies and treatment moderators of psychological interventions for fibromyalgia. A literature search using PubMed, PsychINFO, the Cochrane Library, and manual searches identified 23 eligible studies including 30 psychological treatment conditions and 1396 patients. Meta-analytic integration resulted in a significant but small effect size for short-term pain reduction (Hedges's g=0.37, 95% confidence interval (CI): 0.27-0.48) and a small-to-medium effect size for long-term pain reduction over an average follow-up phase of 7.4 months (Hedges's g=0.47, 95% CI: 0.3-0.65) for any psychological intervention. Psychological treatments also proved effective in reducing sleep problems (Hedges's g=0.46, 95% CI: 0.28-0.64), depression (Hedges's g=0.33, 95% CI: 0.20-0.45), functional status (Hedges's g=0.42, 95% CI: 0.25-0.58), and catastrophizing (Hedges's g=0.33, 95% CI: 0.17-0.49). These effects remained stable at follow-up. Moderator analyses revealed cognitive-behavioral treatment to be significantly better than other psychological treatments in short-term pain reduction (Hedges's g=0.60, 95% CI: 0.46-0.76). Higher treatment dose was associated with better outcome. Publication-bias analyses demonstrated that the effect sizes were robust. The results suggest that the effects of psychological treatments for fibromyalgia are relatively small but robust and comparable to those reported for other pain and drug treatments used for this disorder. Cognitive-behavioral therapy was associated with the greatest effect sizes.