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Abstract: Interphase fluorescent in situ hybridization on unstimulated peripheral blood mononuclear cells (I-FISH-PBMC) is used to detect chromosomal abnormalities such as 11q−, 13q−, 17p−, and trisomy 12 in chronic lymphocytic leukemia (CLL). A total of 56 samples from 49 patients with CLL were studied using commercially available probes for chromosome regions 11q22.3 (ATM), 13q14 (13S272), 17p13 (p53) and 12 centromere (D12Z3). We compared the results obtained with I-FISH-PBMC and those with I-FISH on TPA (tetradecanoylphorbol acetate; phorbol-12-myristate-13-acetate) stimulated peripheral blood cells (I-FISH-TPA) used for conventional cytogenetics, to evaluate the usefulness of I-FISH-TPA. The proportion of abnormal nuclei obtained with the I-FISH-TPA was higher than that found with I-FISH-PBMC (P < 0.001). Consequently, 15 cases with a negative or borderline result with I-FISH-PBMC became positive with I-FISH-TPA for 11q− (n = 2), 13q− (n = 9), and trisomy 12 (n = 4). In all but one of these, chromosomal abnormalities were confirmed by either metaphase FISH or conventional G-banding. Detection of cytogenetic aberrations thus increased from 51% with I-FISH-PBMC to 78% with I-FISH-TPA. Notably, all 15 of the cases that reached the diagnostic thresholds for 11q−, 13q−, and trisomy 12 had a slight lymphocytosis. An absolute lymphocyte count <8.7 × 109/L was found to be the critical threshold (P = 0.037) below which I-FISH-TPA rather than I-FISH-PBMC should be performed. Not only could I-FISH-TPA detect a higher proportion of abnormal interphase nuclei, but it could also identify abnormal CLL cases that might be overlooked with use of I-FISH-PBMC, especially those with low absolute lymphocyte counts. I-FISH-TPA is thus a reliable technique for clinical diagnostics in CLL. [Copyright &y& Elsevier]

A total of 127 adult de novo acute myelocytic leukemia (AML) patients were analyzed by comparative genomic hybridization (CGH) at diagnosis. Conventional cytogenetic analysis (CCA) showed a normal karyotype in 45 cases and an abnormal karyotype in 56 cases; in the remaining cases, CCA either failed to yield sufficient metaphase cells (19/26) or was not done (7/26). Abnormal CGH profiles were identified in 39 patients (30.7%). DNA copy number losses (61%) were high compared to gains (39%), whereas partial chromosome changes (76%) were more common than whole chromosomes changes (24%). Recurrent losses were detected on chromosomes 7, 5q (comprising bands 5q15 to 5q33), 7q (7q32∼q36), 16q (16q13∼q21), and 17p, and gains were detected on chromosomes 8, 22, and 3q (comprising bands 3q26.1∼q27). Furthermore, distinct amplifications were identified in chromosome regions 21q, 13q12∼q13, and 13q21.1. No cryptic recurrent chromosomal imbalances were identified by CGH in cases with normal karyotypes. The concordance between CGH results and CCA was 72.5%. In the remaining cases, CGH gave additional information compared to CCA (20%) and partially failed to identify the alterations previously detected by CCA (7.5%). The majority of discrepancies arose from the limitations of the CGH technique, such as insensitivity to detect unbalanced chromosomal changes when occurring in a low proportion of cells. CGH increased the detection of unbalanced chromosomal alterations and allowed precise defining of partial or uncharacterized cytogenetical abnormalities. Application of the CGH technique is thus a useful complementary diagnostic tool for CCA in de novo AML cases with abnormal karyotypes or with unsuccessful cytogenetics. [Copyright &y& Elsevier]

Expression analysis of apoptotic genes was performed for 15 patients with acute myelocytic leukemia (AML) at the time of diagnosis to identify genes and signaling pathways involved in the regulation of cell survival and apoptosis during leukemogenesis. cDNA array analysis revealed 34 genes whose expression was significantly different compared to others. Tumor suppressor genes TP53 and CDKN2A were downregulated and protooncogenes JUN and GRB10 were upregulated. Furthermore, several cellular signaling pathways acting either in cell cycle regulation or in apoptosis were altered. Deregulation was found in pathways that contribute to genomic stability (by downregulation of either TP53 or CSE1L and by upregulation of GADD45A) and regulate cell cycle progression (by downregulation of CDKN2A and upregulation of RBBP4, CDC37, and NEDD5). Alterations at the transcriptional level were identified, namely, upregulation of JUN and E2F5. Abnormalities were observed in the regulation of the caspases through upregulation of CASP8 and by altered expression of BCL2-related pathway. Extrinsic apoptotic signals mediated by IGFs were deregulated and the glutathione detoxification pathway was downregulated. These findings provide insight into the regulation of balance between apoptosis and cell proliferation signals, and suggest that these genes and pathways may have an important role in the pathogenesis of AML. [Copyright &y& Elsevier]