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.

Cytogenetics is essential in myeloid neoplasms (MN) and pre-analytical variables are important for karyotyping. We assessed the relationship between pre-analytical variables (time from collection to sample processing, material type, sample cellularity, and diagnosis) and failures of karyotyping. Bone marrow (BM, n=352) and peripheral blood (PB, n=69) samples were analyzed from acute myeloid leukemia (n=113), myelodysplastic syndromes (n=73), myelodysplastic syndromes/myeloproliferative neoplasms (n=17), myeloproliferative neoplasms (n=137), and other with conclusive diagnosis (n=6), and reactive disorders/no conclusive diagnosis (n=75). The rate of unsuccessful karyotyping was 18.5% and was associated with the use of PB and a low number of nucleated cells (≤7×10 3 /µL) in the sample. High and low cellularity in BM and high and low cellularity in PB samples showed no metaphases in 3.9, 39.7, 41.9, and 84.6% of cases, respectively. Collecting a good BM sample is the key for the success of karyotyping in MN and avoids the use of expensive molecular techniques.

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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.