SNP-A may complement metaphase cytogenetics to improve the detection of chromosomal abnormalities in myeloid neoplasms.

BackgroundMultiple myeloma is a plasma cell neoplasm with acquired genetic abnormalities of clinical and prognostic importance. Multiple myeloma differs from other hematologic malignancies due to a high fraction of low proliferating malignant plasma cells and the paucity of plasma cells in bone marrow aspiration samples, making cytogenetic analysis a challenge. An abnormal karyotype is found in only one-third of patients with multiple myeloma and interphase fluorescence in situ hybridization is the most useful test for studying the chromosomal abnormalities present in almost 90% of cases. However, it is necessary to study the genetic abnormalities in plasma cells after their identification or selection by morphology, immunophenotyping or sorting. Other challenges are the selection of the most informative FISH panel and determining cut-off levels for FISH probes. This study reports the validation of interphase fluorescence in situ hybridization using CD138 positive cells, according to proposed guidelines published by the European Myeloma Network (EMN) in 2012.MethodBone marrow samples from patients with multiple myeloma were used to standardize a panel of five probes [1q amplification, 13q14 deletion, 17p deletion, t(4;14), and t(14;16)] in CD138+ cells purified by magnetic cell sorting.ResultsThis test was validated with a low turnaround time and good reproducibility. Five of six samples showed genetic abnormalities. Monosomy/deletion 13 plus t(4;14) were found in two cases.ConclusionThis technique together with magnetic cell sorting is effective and can be used in the routine laboratory practice. In addition, magnetic cell sorting provides a pure plasma cell population that allows other molecular and genomic studies.

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An 86-year-old male presented with three months of asthenia. He had a history of a vocal cord neoplasm eight years previously, which had been treated with radiotherapy. Physical examination showed only mucocutaneous pallor. Laboratory tests showed haemoglobin concentration 92 g/l, mean cell volume 101 fl, white blood cell count 1Á78 9 10 9 /l, neutrophils 0Á96 9 10 9 /l, lymphocytes 0Á44 9 10 9 /l and platelets 108 9 10 9 /l. Myeloblasts were present. Bone marrow aspiration showed marked hypocellularity with hypogranular and Pelgeroid neutrophils and 27Á7% blast cells, expressing CD34 and CD117. Bone marrow biopsy showed 10% cellularity with 20% CD34 + cells.The karyotype demonstrated a hypodiploid clone with numerous structural abnormalities, including 5q deletion, monosomy 7, triplication of part of 11q, a marker chromosome and a ring chromosome (left). Fluorescence in situ hybridization was compatible with deletion of EGR1, monosomy 7, RUNX1T1 trisomy, TP53 deletion and KMT2A (MLL) amplification in 50% of cells (right, dual-colour break-apart KMT2A probe). The patient was diagnosed with therapy-related acute myeloid leukaemia and showed a haematological and cytogenetic remission after one cycle of decitabine.KMT2A (located at 11q23) encodes a histone methyltransferase that has a role in epigenetic regulation of transcription, important for embryonic and haematopoietic development. Abnormalities of KMT2A, particularly rearrangements, are frequently seen in acute leukaemias. Amplification is a rare event and often associated with advanced age, a complex karyotype and a shorter overall survival. There is an association of KMT2A rearrangement with previous exposure to alkylating agents but whether there is a relationship of amplification to previous radiotherapy is unknown.

Resolução ?550 bandas é sugerida por programas de controle de qualidade em citogenética para estudo cariotípico de dismorfismos e deficiência intelectual. Entre os protocolos disponíveis estão o uso de agentes que inibem a condensação cromossômica por intercalação das bases de DNA, como actinomicinaD e brometo de etídio. A técnica de FISH pode ser utilizada para elucidação de anormalidades cariotípicas. Os objetivos deste trabalho foram: (a) padronização de protocolos para aumento do nível de resolução de bandas em cariótipo constitucional de sangue periférico e (b) avaliação por meio da técnica de FISH de alterações cromossômicas de origem desconhecida. Foram realizados cariótipos de 54 amostras, 11 com 10?L de actinomicinaD (1mg/mL), 26 com 2,5?L de brometo de etídio (10mg/mL) e 17 com 5,0?L de brometo de etídio (10mg/mL) e comparadas com culturas padrão (meio de cultura RPMI suplementado com 10% de soro fetal bovino e 1% de fitohemaglutinina incubadas a 37°C por 72h) quanto ao índice mitótico, qualidade das metáfases, nível de resolução de bandas e detecção de anormalidades. Após padronização foram estudados 5 casos com sondas de pintura cromossômica total [whole chromosome painting (wcp)] para os 24 cromossomos (Chromoprobe Multiprobe® OctochromeTM System, Cytocell, UK), envolvendo anormalidades citogenéticas não conclusivas (5q, 9p, 14q e pequenos cromossomos marcadores). A técnica de array-CGH CytoGenomics (Agilent, USA) foi aplicada em um caso (5q). Em relação ao índice mitótico, qualidade das metáfases e detecção de anormalidades [4 casos: +21, +21, XXY, fra(16)], não houve diferença entre os protocolos. As culturas com brometo de etídio 5,0?L apresentaram maior percentual de metáfases com ?550 bandas (53%) (p<0,05), [culturas padrão: 7%, actinomicinaD: 18% e brometo de etídio 2,5?L: 15%]. FISH identificou a origem dos materiais adicionais presentes nos cromossomos 5q, 9p e 14q, porém foi inconclusiva nos demais casos. A técnica de array-CGH foi elucidativa no caso selecionado. Preparações com 5?L de brometo de etídio resultaram em melhor resolução de bandas, mas comparada ao protocolo padrão não mostrou diferença nos achados citogenéticos. A técnica de FISH se mostrou ineficaz na análise de cromossomos marcadores muito pequenos. Técnicas citogenômicas, como array-CGH, poderão ser utilizadas como primeiro exame para identificar ganhos e perdas de material genômico, com utilização subsequente de cariótipo para estudo de risco de recorrência e aconselhamento genético.