Purpose: Gene copy number alteration (CNA) is common in malignant melanoma and is associated with tumor development and progression. The concordance between molecular cytogenetic techniques used to determine CNA has not been evaluated on a large set of loci in malignant melanoma. Experimental Design: A panel of 16 locus-specific fluorescence in situ hybridization (FISH) probes located on eight chromosomes was used to identify CNA in touch preparations of frozen tissue samples from 19 patients with metastatic melanoma (SWOG-9431). A subset (n = 11) was analyzed using bacterial artificial chromosome (BAC) array comparative genomic hybridization (aCGH) of DNA isolated directly from touch-preparation slides. Results: By FISH, most samples showed loss near or at WISP3/6p21, CCND3/6q22, and CDKN2A/9p21 (>75% of samples tested). More than one third of CDKN2A/9p21 losses were biallelic. Gains of NEDD9/6p24, MET/7q31, and MYC/8q24 were common (57%, 47%, and 41%, respectively) and CNA events involving 9p21/7p12.3 and MET were frequently coincident, suggesting gain of the whole chromosome 7. Changes were confirmed by aCGH, which also uncovered many discreet regions of change, larger than a single BAC. Overlapping segments observed in >45% of samples included many of the loci analyzed in the FISH study, in addition to other WNT pathway members, and genes associated withTP53 pathways and DNA damage response, repair, and stability. Conclusions: This study outlines a set of CNAs at the gene and regional level, using FISH and aCGH, which may provide a benchmark for future studies and may be important in selection of individual therapy for patients with metastatic malignant melanoma.Skin cancer occurs in more than 1,200,000 Americans annually (1). Most cases are highly curable squamous and basal cell cancers, but f62,500 new cases of melanoma will be diagnosed this year in the United States. The incidence of melanoma has steadily been increasing, and melanoma causes more than 8,400 deaths per year in this country, accounting for the vast majority of skin cancer deaths (1). Early detection and surgery are key to improved survival. Once metastatic melanoma is diagnosed, median survival duration of only 6 to 9 months is expected.Primary melanoma lesions have largely been characterized by Breslow's depth, Clark's level, ulceration, microscopic involvement of regional lymph nodes, as well as mitotic index, lymphocyte infiltration, and signs of regression. Metastatic melanoma is staged based on location of lesions, such as skin, lymph nodes, lung, liver, and other organ sites, and finally, serum lactate dehydrogenase. With advances in genetic technology, new tools have been developed that may facilitate the collection of a set of potentially robust prognostic genetic indicators (2, 3) to augment the anatomic classifications.Cytogenetic, molecular, and biological studies indicate that multiple genetic alterations are involved in the development and progression of malignant melanoma, with several genes and chromosomal sites del...

This study demonstrates that copy number alterations can be robustly and reproducibly detected by array comparative genomic hybridization in DNA isolated from challenging tumor types and sources, including archival materials, low DNA yield, and heterogeneous tissues. Furthermore, bacterial artificial chromosome array comparative genomic hybridization offers the advantage over the Agilent oligonucleotide platform of presenting fewer outliers, which could affect data interpretation.

SummaryChromosome rearrangements involving band 3q26.2 are associated with myeloid malignancies, aberrant expression of the human ecotropic virus integration site‐1 (EVI1) gene, an unfavourable prognosis and an aggressive clinical course. The 3q26.2 rearrangements are characteristically heterogeneous and typically difficult to detect in poor quality metaphases. To develop a dual‐colour fluorescence in situ hybridisation (FISH) assay for the detection of 3q26.2/EVI1 aberrations, a series of 10 BAC clones corresponding to the EVI1 gene region were systematically evaluated and narrowed down to two probe sets; one probe set encompassed the EVI1 gene extending centromeric, while the second probe set covered the EVI1 gene and extends telomeric. Both probe sets were evaluated on 35 patient samples with cytogenetically defined 3q26.2 rearrangements collected at various treatment time points, the inv(3)(q21q26.2) Kasumi‐4 cell line, and 10 known negative samples. The two‐probe set strategy identified all samples, despite the vast breakpoint heterogeneity observed. In samples from acute myeloid leukaemia and myelodysplastic syndrome cases, the majority of inversion breakpoints were 3′ to EVI1 whereas 3q26.2 translocation breakpoints frequently mapped 5′ to EVI1. However, two 3q26.2 translocation samples had breakpoints 3′ to EVI1. Most inv(3q) chronic myeloid leukaemia samples showed breakpoints within the EVI1 gene. This study demonstrated that, despite the extensive breakpoint heterogeneity observed with 3q26.2 aberrations, this FISH strategy is effective for the detection of 3q26.2 abnormalities in myeloid malignancies.

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Bonhoure et al. 8 reported that sustained SPHK1 overexpresion can render HL60 cells chemoresistant by decreasing the cellular ceramide level and that a novel SPHK1 inhibitor, F-12509a, could recover chemosensitivity. Considering the molecular target of chemotherapy, enzyme inhibitors are more practical than agents for enzyme activation (NSMase2 in our case). Inhibitor of SPHK1 is almost at the stage of clinical investigation. In the present study, we could analyze only one time point of patients (mostly at their first diagnosis before chemotherapy) and could not measure enzyme activities due to the paucity of samples. The measurement of SPHK1 gene expression and/or SPHK enzyme activity of each patient might be necessary to assess the efficacy of enzyme inhibitors in future clinical settings. Sequential analysis of the same patients will also add further information.The localization and activation of enzymes are also important factors to determine the final cellular ceramide/S1P rheostat. SPHK1 was reportedly activated by phosphorylation by agonists and translocated to membranes. 2 Therefore, further analysis of enzyme activation is necessary to conclude firmly that sphingolipid metabolizing enzymes such as SPHK1 are a novel and promising molecular target for acute leukemia chemotherapy.Taken together, this is, to our knowledge, the first report of a gene expression profile of major sphingolipid metabolizing enzymes of AL and MDS using quantitative RT-PCR, It documents the increase of SPHK1 gene expression in AL and RAEB-t and the decrease of NSMase2 gene expression in AL and RAEB, suggesting that sphingolipid metabolizing enzymes such as SPHK1 could be a novel target for the chemotherapy of AL. AcknowledgementsWe express sincere thanks to Professor S Nakamura and other members