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

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Acute lymphoblastic leukemia (ALL) is a malignancy that can be subdivided into distinct entities based on clinical, immunophenotypic and genomic features, including mutations, structural variants (SVs), and copy number alterations (CNA). Chromosome banding analysis (CBA) and Fluorescent In-Situ Hybridization (FISH) together with Multiple Ligation-dependent Probe Amplification (MLPA), array and PCR-based methods form the backbone of routine diagnostics. This approach is labor-intensive, time-consuming and costly. New molecular technologies now exist that can detect SVs and CNAs in one test. Here we apply one such technology, optical genome mapping (OGM), to the diagnostic work-up of 41 ALL cases. Compared to our standard testing pathway, OGM identified all recurrent CNAs and SVs as well as additional recurrent SVs and the resulting fusion genes. Based on the genomic profile obtained by OGM, 32 patients could be assigned to one of the major cytogenetic risk groups compared to 23 with the standard approach. The latter identified 24/34 recurrent chromosomal abnormalities, while OGM identified 33/34, misinterpreting only 1 case with low hypodiploidy. The results of MLPA were concordant in 100% of cases.Overall, there was excellent concordance between the results. OGM increased the detection rate and cytogenetic resolution, and abrogated the need for cascade testing, resulting in reduced turnaround times. OGM also provided opportunities for better patient stratification and accurate treatment options. However, for comprehensive cytogenomic testing, OGM still needs to be complemented with CBA or SNP-array to detect ploidy changes and with BCR::ABL1 FISH to assign patients as soon as possible to targeted therapy.Katrina Rack and Jolien De Bie are co-first authors. Lucienne Michaux and Barbara Dewaele are co-senior authors.

Multiple cytogenetic subgroups have been described in adult Philadelphia chromosome (Ph)-negative B-cell precursor (BCP) acute lymphoblastic leukemia (ALL), often comprising small numbers of patients. In this study, we aimed to reassess the prognostic value of cytogenetic abnormalities in a large series of 617 adult patients with Ph-negative BCP-ALL (median age, 38 years), treated in the intensified Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL)-2003/2005 trials. Combined data from karyotype, DNA index, fluorescence in situ hybridization, and polymerase chain reaction screening for relevant abnormalities were centrally reviewed and were informative in 542 cases (88%), allowing classification in 10 exclusive primary cytogenetic subgroups and in secondary subgroups, including complex and monosomal karyotypes. Prognostic analyses focused on cumulative incidence of failure (including primary refractoriness and relapse), event-free survival, and overall survival. Only 2 subgroups, namely t(4;11)/ and 14q32/ translocations, displayed a significantly worse outcome in this context, still observed after adjustment for age and after censoring patients who received allogeneic stem cell transplantation (SCT) in first remission at SCT time. A worse outcome was also observed in patients with low hypodiploidy/near triploidy, but this was likely related to their higher age and worse tolerance to therapy. The other cytogenetic abnormalities, including complex and monosomal karyotypes, had no prognostic value in these intensive protocols designed for adult patients up to the age of 60 years.

We performed a multicentric study to assess the impact of two different culture procedures on the detection of chromosomal abnormalities in 217 consecutive unselected cases with chronic lymphocytic leukemia (CLL) referred for routine analysis either at the time of diagnosis (n = 172) or during disease evolution (n = 45). Parallel cultures of peripheral blood or bone marrow were set up with the addition of either the conventional B-cell mitogen 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or a combination of CpG oligonucleotide (CpG) and interleukin-2 (IL-2). Cytogenetic analyses were performed on both cultures. Clonal abnormalities were identified in 116 cases (53%). In 78 cases (36%), the aberrant clone was detected in both cultures. Among these, the percentages of aberrant metaphases were similar in both conditions in 17 cases, higher in the CpG/IL-2 culture in 43 cases, and higher in the TPA culture in 18 cases. Clonal aberrations were detected in only one culture, either in CpG/IL-2 or TPA in 33 (15%) and 5 (2%) cases, respectively. Taken together, abnormal karyotypes were observed in 51% with CpG/IL-2 and 38% with TPA (P < 0.0001). Application of FISH (n = 201) allowed the detection of abnormalities not visible by conventional cytogenetic analysis in 80 cases: del(13q) (n = 71), del(11q) (n = 5), +12 (n = 2), del(14q) (n = 1), and del(17p) (n = 1). In conclusion, our results confirm that CpG/IL-2 stimulation increases the detection rate of chromosomal abnormalities in CLL compared with TPA and that further improvement can be obtained by FISH. However, neither conventional cytogenetics nor FISH detected all aberrations, demonstrating the complementary nature of these techniques.

Dysregulation of MYC is the genetic hallmark of Burkitt lymphoma (BL) but it is encountered in other aggressive mature B-cell lymphomas. MYC dysregulation needs other cooperating events for BL development. We aimed to characterize these events and assess the differences between adult and paediatric BLs that may explain the different outcomes in these two populations. We analysed patterns of genetic aberrations in a series of 24 BLs: 11 adults and 13 children. We looked for genomic imbalances (copy number variations), copy-neutral loss of heterozygosity (CN-LOH) and mutations in TP53, CDKN2A, ID3 (exon 1), TCF3 (exon17) and CCND3 (exon 6). Young patients displayed more frequent 13q31.3q32.1 amplification, 7q32q36 gain and 5q23.3 CN-LOH, while 17p13 and 18q21.3 CN-LOH were only detected in adult BLs. ID3 mutations were present in all adult samples, but only in 42% of childhood cases. CCND3 and ID3 double-hit mutations, as well as 18q21 CN-LOH, seemed to be associated with poorer outcome. For the first time, we report different genetic anomalies between adult and paediatric BLs, suggesting age-related heterogeneity in Burkitt lymphomagenesis. This may explain the poorer prognosis of adult BLs. Additional studies are needed to confirm these results in the setting of clinical trials.

SummaryThe PRDM16 (1p36) gene is rearranged in acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) with t(1;3)(p36;q21), sharing characteristics with AML and MDS with MECOM (3q26.2) translocations. We used fluorescence in situ hybridization to study 39 haematological malignancies with translocations involving PRDM16 to assess the precise breakpoint on 1p36 and the identity of the partner locus. Reversetranscription polymerase chain reaction (PCR) was performed in selected cases in order to confirm the partner locus. PRDM16 expression studies were performed on bone marrow samples of patients, normal controls and CD34 + cells using TaqMan real-time quantitative PCR. PRDM16 was rearranged with the RPN1 (3q21) locus in 30 cases and with other loci in nine cases. The diagnosis was AML or MDS in most cases, except for two cases of lymphoid proliferation. We identified novel translocation partners of PRDM16, including the transcription factors ETV6 and IKZF1. Translocations involving PRDM16 lead to its overexpression irrespective of the consequence of the rearrangement (fusion gene or promoter swap). Survival data suggest that patients with AML/MDS and PRDM16 translocations have a poor prognosis despite a simple karyotype and a median age of 65 years. There seems to be an over-representation of lateonset therapy-related myeloid malignancies.

the secondary malignancies and cardiovascular events, may not directly relate to SCT itself.Long-term follow-up should also consider issues of quality of life (QOL). This initial analysis shows that chronic graft-versushost disease rates and the duration of required immune suppression were lower after RIC. Chronic graft-versus-host disease is a dominant factor determining QOL, suggesting better long-term QOL with RIC; however, formal QOL assessment is required to determine this question.The major limitation of this study is the nonrandomized comparison and the selection biases in allocating patients to one of the regimens. However, because patients were selected for RIC based on high risk for non-relapse mortality, and because OS was similar among these patients given RIC and eligible patients given MAC, it is possible that results in standard risk patients may be similar. Randomized studies, for patients in CR (preferentially CR1) with long-term follow-up and assessment of late complication and QOL, will be needed to determine the best approach in this setting. MAC and possibly the new reduced toxicity myeloablative regimens are still the best approach in patients with active disease.