Brigitte Gubler

Epidermal growth factor receptor (EGFR) gene alterations and amplification are frequently reported in cases of glioblastoma (GBM). However, EGFR-activating mutations that confer proven sensitivity to tyrosine kinase inhibitors (TKIs) in lung cancer have not yet been reported in GBM. Using next-generation sequencing, array comparative genomic hybridization and droplet digital PCR, we identified the p.L861Q EGFR mutation in a case of GBM for the first time. The mutation was associated with gene amplification. L861Q may be a clinically valuable mutation because it is known to sensitize non-small-cell lung cancers to treatment with the second-generation EGFR TKI afatinib in particular. Furthermore, we used slice culture of the patient’s GBM explant to evaluate the tumour’s sensitivity to various EGFR-targeting drugs. Our results suggested that the tumour was not intrinsically sensitive to these drugs. Our results highlight (i) the value of comprehensive genomic analyses for identifying patient-specific, targetable alterations, and (ii) the need to combine genomic analyses with functional assays, such as tumour-derived slice cultures.

Chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) are hematological disorders that occur at different stages of B-cell development. It has been shown that CLL B-cells can differentiate into plasma cells in vitro and in vivo. CLL is the most frequent adult leukemia in the western world. It is a heterogeneous disease, characterized by clonal proliferation and the accumulation of mature CD5+ B lymphocytes (Zenz et al.). MM is a clonal plasma cell malignancy that accounts for more than 10% of all hematologic cancers (Kyle and Rajkumar, 2008). Although secondary cancers (particularly solid tumors (Hasskarl et al.;Morton et al.;Schollkopf et al., 2007)) can occur with CLL and MM, the concomitant occurrence of these two disorders in the same patient is rare (for a review of the few reported cases, see (Alley et al.)). The clonal relationship between these diseases has not always been clarified but is important in terms of understanding the pathogenesis and optimizing treatment. The clonal relationship between CLL and MM can be evaluated by (i) analyzing immunoglobulin heavy chain (IGH) and light chain (IGK, IGL) gene rearrangement, (ii) identifying and comparing somatic mutations, (iii) studying chromosomic aberrations. Nevertheless, immunoglobulin rearrangements must always be interpreted in the light of specific phenomena such as allelic exclusion, B-cell receptor (BCR) revision (VH and DH gene replacement), BCR editing and somatic hypermutations - events that were not considered in previous studies. These issues can be addressed by sequencing the rearranged immunoglobulin genes from sorted populations and interpreting the generated data. In the present study, we evaluated the putative clonal relationship between the two diseases by combining DNA copy number analysis with an assessment of Ig gene rearrangements (clonality assessment, V(D)J sequencing and somatic hypermutation analysis) in highly enriched CD19+ CD5+ (CLL) and CD38+ CD138+ (MM) cell populations. Array comparative genomic hybridization data suggested a possible phylogenic progression from CLL to MM. Moreover, V(D)J sequencing indicated that both CLL and MM cells used the same VH and JH genes but different DH genes. However, in-depth analysis and interpretation of Ig gene rearrangements ultimately suggested that the two diseases had distinct clonal origins.

Mutations in the activation segment of the v-raf murine sarcoma viral oncogene homolog B (BRAF) gene are present in approximately 50% of melanomas. The selective BRAF inhibitor vemurafenib has demonstrated significant clinical benefits in patients with melanomas harboring the most common mutations (V600E, V600K and V600R). However, the clinical activity of BRAF inhibitors in patients with rare mutations of codon 600 and the surrounding codons has not been documented. We used the BRAF inhibitor vemurafenib to treat a patient presenting a rare p.V600_K601delinsD-mutated melanoma. An objective response was evidenced by two months of progression-free survival. By cloning and sequencing BRAF exon 15, we confirmed that a dual mutation was present on a single allele and thus resulted in a BRAF V600DK601del mutant protein. We also performed an in silico crystal structure analysis of the mutated protein, in order to characterize the nature of the putative interaction between vemurafenib and the mutant protein. This clinical experience suggests that (i) patients with BRAF V600DK601del -mutation-positive melanoma can be treated successfully with the oral BRAF inhibitor vemurafenib and (ii) molecular screening in this context should encompass rare and complex mutations.

In B cells, B‐cell receptor (BCR) immunoglobulin revision is a common route for modifying unwanted antibody specificities via a mechanism called VH replacement. This in vivo process, mostly affecting heavy‐chain rearrangement, involves the replacement of all or part of a previously rearranged IGHV gene with another germline IGHV gene located upstream. Two different mechanisms of IGHV replacement have been reported: type 1, involving the recombination activating genes complex and requiring a framework region 3 internal recombination signal; and type 2, involving an unidentified mechanism different from that of type 1. In the case of light‐chain loci, BCR immunoglobulin editing ensures that a second V‐J rearrangement occurs. This helps to maintain tolerance, by generating a novel BCR with a new antigenic specificity. We report that human B cells can, surprisingly, undergo type 2 replacement associated with κ light‐chain rearrangements. The de novo IGKV‐IGKJ products result from the partial replacement of a previously rearranged IGKV gene by a new germline IGKV gene, in‐frame and without deletion or addition of nucleotides. There are wrcy/rgyw motifs at the ‘IGKV donor–IGKV recipient chimera junction’ as described for type 2 IGHV replacement, but activation‐induced cytidine deaminase (AID) expression was not detected. This unusual mechanism of homologous recombination seems to be a variant of gene conversion‐like recombination, which does not require AID. The recombination phenomenon described here provides new insight into immunoglobulin locus recombination and BCR immunoglobulin repertoire diversity.