Home page: www.blodet.dkThe European Myeloma Network has organized two workshops on fluorescence in situ hybridization in multiple myeloma. The first aimed to identify specific indications and consensus technical approaches of current practice. A second workshop followed a quality control exercise in which 21 laboratories analyzed diagnostic cases of purified plasma cells for recurrent abnormalities. The summary report was discussed at the EHA Myeloma Scientific Working Group Meeting 2010. During the quality control exercise, there was acceptable agreement on more than 1,000 tests. The conclusions from the exercise were that the primary clinical applications for FISH analysis were for newly diagnosed cases of MM or frank relapse cases. A range of technical recommendations included: 1) material should be part of the first draw of the aspirate; 2) samples should be sent at suitable times to allow for the lengthy processing procedure; 3) most importantly, PCs must be purified or specifically identified; 4) positive cut-off levels should be relatively conservative: 10% for fusion or breakapart probes, 20% for numerical abnormalities; 5) informative probes should be combined to best effect; 6) in specialist laboratories, a single experienced analyst is considered adequate; 7) at least 100 PC should be scored; 8) essential abnormalities to test for are t(4;14), t(14;16) and 17p13 deletions; 9) suitable commercial probes should be available for clinically relevant abnormalities; 10) the clinical report should be expressed clearly and must state the percentage of PC involved and the method used for identification; 11) a retrospective European based FISH data bank linked to clinical data should be generated; and 12) prospective analysis should be centralized for upcoming trials based on the recommendations made. The European Myeloma Network aims to build on these recommendations to establish standards for a common European data base to define subgroups with prognostic significance.Key words: myeloma, cytogenetic, interphase FISH, recommendation.Citation: Ross FM, Avet-Loiseau H, Ameye G, Gutiérrez NC, Liebisch P, O´Connor S, Dalva K, Fabris S, Testi AM, Jarosova M, Hodkinson C, Collin A, Kerndrup G, Kuglik P, Ladon D, Bernasconi P, Maes B, Zemanova Z, Michalova K, Michau L, Neben K, Hermansen NEU, Rack K, Rocci A, Protheroe R, Chiecchio L, Poirel HA, Sonneveld P, Nyegaard M, and myeloma and related disorders. Haematologica 2012;97(8):1272-1277. doi:10.3324/haematol.2011 This is an open-access paper. ABSTRACT© F e r r a t a S t o r t i F o u n d a t i o n

Ewing sarcoma family of tumors share recurrent translocations that fuse EWS from 22q12 to ®ve dierent members of transcription factors namely FLI-1, ERG, ETV1, E1AF and FEV. Dierent classes of DNA binding proteins, ATF1, WT1 and CHOP are fused to EWS generating distinct tumor phenotypes: clear cell sarcoma, desmoplastic small round cell tumor, and myxoid liposarcoma, respectively. We have cloned a novel gene located at 22q12 fused to EWS by a submicroscopic inversion of 22q in a small round cell sarcoma showing a translocation (t(1;22)(p36.1;q12). The gene, designated ZSG (Zinc ®nger Sarcoma Gene), is a putative Cys 2 -His 2 zinc ®nger protein which contains a POZ transcriptional repressor-like domain at the Nterminus. The rearrangement involves intron 8 of EWS and exon 1 of ZSG creating a chimeric sequence containing the transactivation domain of EWS fused to zinc ®nger domain of ZSG. This product lacks the transcriptional repressor domain at the N-terminus of ZSG. A rearrangement of the second ZSG allele was also found in tumor cells. This is the ®rst example of an intra-chromosomal rearrangement of chromosome 22, undetectable by cytogenetics, activating EWS in soft tissue sarcoma. Oncogene (2000) 19, 3799 ± 3804.Keywords: sarcoma; EWS; fusion; POZ/BTB domain; zinc ®ngerWe present here the ®rst report of an intrachromosomal rearrangement of chromosome 22 activating EWS by fusion with a novel zinc ®nger gene in a soft tissue sarcoma.The tumor occurred in a male boy aged 16 years, presenting with a pulmonary metastasis 2 years after a primary tumor of the chest wall. Histologically both primary and metastatic tumors were highly suggesting for pPNET, possibly Askin-Rosai type owing to the primary site. Immunophenotyping performed on primary and metastatic tumor showed reactivity for synaptophysin (AO10, Dako) and NSE (MIG-N3, Sambio) and the same small clusters and scattered cells decorated with desmin (D33, Dako) and lowweight cytocheratins (CAM 5.2, Becton Dickinson). A negative staining was observed with neuro®laments (RPN1105, Amersham) and MIC2 antigen (013, Signet). The latter ®nding, being MIC2 a hallmark of pPNET, prompted the diagnosis of a small round cell tumor with multidirectional dierentiation rather than of pPNET.Cytogenetic and¯uorescence in situ hybridization (FISH) analyses of the small round cell sarcoma under study revealed a translocation t(1;22)(p36.1;q12) (Figure 1a ± c). No structural involvement of chromosome 11, indicative of the translocation t(11;22) described in 90% of Ewing's tumors (Turc-Carel et al., 1983), (Sorensen et al., 1994) was observed.To rule out a molecular fusion of EWS with already known partners, we performed RT ± PCR on tumor RNA to assess the presence of EWS/FLI-1 (May et al., and Gerald, 1994;Gerald et al., 1995) fusion products. We could not detect PCR products with any primer set (data not shown).We tested the tumor DNA for the presence of EWS gene alterations by Southern blot analyses using as probe a partial EWS cDNA clone (Delattre et al., 1992). An abnormal r...

PLX4032/vemurafenib is a first-in-class small-molecule BRAF(V600E) inhibitor with clinical activity in patients with BRAF mutant melanoma. Nevertheless, drug resistance develops in treated patients, and strategies to overcome primary and acquired resistance are required. To explore the molecular mechanisms involved in primary resistance to PLX4032, we investigated its effects on cell proliferation and signaling in a panel of 27 genetically characterized patient-derived melanoma cell lines. Cell sensitivity to PLX4032 was dependent on BRAF(V600E) and independent from other gene alterations that commonly occur in melanoma such as PTEN loss, BRAF, and MITF gene amplification. Two cell lines lacking sensitivity to PLX4032 and harboring a different set of genetic alterations were studied as models of primary resistance. Treatment with the MEK inhibitor UO126 but not with PLX4032 inhibited cell growth and ERK activation. Resistance to PLX4032 was maintained after CRAF down-regulation by siRNA indicating alternative activation of MEK-ERK signaling. Genetic characterization by multiplex ligation-dependent probe amplification and analysis of phosphotyrosine signaling by MALDI-TOF mass spectrometry analysis revealed the activation of MET and SRC signaling, associated with the amplification of MET and of CTNNB1 and CCND1 genes, respectively. The combination of PLX4032 with drugs or siRNA targeting MET was effective in inhibiting cell growth and reducing cell invasion and migration in melanoma cells with MET amplification; similar effects were observed after targeting SRC in the other cell line, indicating a role for MET and SRC signaling in primary resistance to PLX4032. Our results support the development of classification of melanoma in molecular subtypes for more effective therapies.

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Most mesenchymal neoplasms of the gastrointestinal tract belong to the category of gastrointestinal stromal tumors (GISTs) and are characterized by the immunohistochemical expression of KIT receptor. In cases without detectable KIT receptor expression several differential diagnoses have to be taken into consideration. Here, we report a case of a 41-year-old man with a tumor of the small bowel composed of large epithelioid tumor cells arranged in solid and alveolar sheets including scattered osteoclast-like multinucleated giant cells. Immunohistochemically, the tumor cells expressed strongly S-100 protein, vimentin, and to a lesser extent, bcl-2. HMB-45, melan-A, KIT receptor, desmin, smooth-muscle actin, and CD-34 were not detectable. Ki-67 index was 20%. The diagnosis was established by 2 different FISH strategies demostrating the presence of a t(12;22)(q13;q12) translocation, the diagnostic hallmark of clear cell sarcoma of soft parts. Our results provide further evidence for the existence of a new tumor entity designated gastrointestinal clear cell sarcoma with osteoclast-like giant cells. The diagnosis of this entity should be considered in the presence of S-100-positive tumors of the gastrointestinal tract containing multinucleated giant cells and can be established by FISH analysis.