Genomewide array-based comparative genomic hybridization analysis of acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is typically associated with the t(15;17) that generates the PML-RARA fusion protein. Animal models have shown that although the fusion protein is necessary, it is insufficient for the development of APL, implying that additional mechanisms are responsible for full-blown leukemia. The mutation of specific genes has been implicated in leukemogenesis; however, alterations in gene copy number have not been well investigated. Here, we applied the genomewide array-comparative genomic hybridization technique to 30 APL clinical samples and 2 APL cell lines. It was found that (1) approximately half the clinical samples (14 of 30 APL cases) had no detectable chromosomal imbalances; and (2) the remaining 16 cases, including the cell lines, exhibited recurrent chromosomal imbalances, such as loss of 1p36, 2p11, 16p, and 17p, and gain of 8p, 8q, and 13q. These results suggest that chromosomal imbalances are largely absent in APL, although some nonrandom chromosomal imbalances could be linked to the development of APL in a limited number of cases.     

New highly sensitive fluorescence in situ hybridization method to detect PML/RARA fusion in acute promyelocytic leukemia

We investigated a new fluorescence in situ hybridization (FISH) method to detect PML/RARA fusion and/or anomalies of the RARA gene (alias RARalpha) in interphase nuclei from patients with acute promyelocytic leukemia (APL). This method uses a commercially available product with two different colored fluorescent probes to detect both PML/RARA gene fusion products (double fusion signal or dual-color fluorescence in situ hybridization [D-FISH]). A total of 82 bone marrow specimens were studied, including 30 from normal bone marrow transplant donors, 33 from patients with untreated APL, 14 from patients with treated APL, and 5 from APL patients with known translocation variants or alternate translocations. The signal patterns and percentage of abnormal nuclei were determined in a blinded study on 500 interphase nuclei for each specimen. Based on 25 normal specimens, the normal cutoff was >0.6% and >1.6% for t(15;17) and t(17;var), respectively. The clinical sensitivity for this series of patients was 98% and the clinical specificity was 100%. The results suggest that the new D-FISH probe set can detect all t(15;17)(q22;q21) and all variant forms of this translocation associated with PML and RARA. In addition, this FISH method can detect all alternate translocations involving RARA and not PML. This FISH method can be used both for the accurate diagnosis of APL and to monitor low levels of disease in treated patients.     

Fluorescence in situ hybridization for identification of microorganisms in acute chorioamnionitis

The relevance of microorganisms in preterm birth is still under discussion. Using a diagnostic fluorescence in situ hybridization probe panel, we visualized Staphylococcus aureus and Streptococcus mitis group in two cases of acute chorioamnionitis. This technique provides spatial resolution and quantity of bacteria, clarifying the epidemiology and pathogenic pathways of acute chorioamnionitis.     

Fluorescence in situ hybridization characterization of different cryptic BCR-ABL rearrangements in chronic myeloid leukemia

Using fluorescence in situ hybridization probes, obtained from bacterial artificial chromosome (BAC) libraries that relate to sequences either side of the BCR and ABL genes, this study characterized four chronic myeloid leukemia cases with cryptic BCR-ABL rearrangements. Each case showed evidence of a different underlying mechanism: one case showed a microinsertion of BCR into ABL, another a microinsertion of ABL into BCR, and the third showed a complex rearrangement including deletion of adjacent flanking sequences, consistent with the reverse translocation model of cryptic rearrangement. The fourth case also showed evidence of a more complex rearrangement involving chromosome 1.     

Fluorescence in situ hybridization in diagnostic cytology

Fluorescence in situ hybridization (FISH) is a technique that uses fluorescently labeled DNA probes to detect chromosomal alterations in cells. FISH can detect various types of cytogenetic alterations including aneusomy (ie, abnormalities of chromosome copy number), duplication, amplification, deletion, and translocation. Because tumor cells generally contain chromosomal alterations, FISH is able to detect cells that have chromosomal abnormalities consistent with neoplasia in exfoliative and aspiration cytology specimens. This review will discuss the utility of FISH for the detection of bladder, lung, pancreatobiliary, and esophageal carcinoma in cytologic specimens.     

Fluorescence in situ hybridization patterns in newly diagnosed superficial bladder lesions and corresponding bladder washings

A multiprobe interphase fluorescence in situ hybridization (I-FISH) approach has become a useful ancillary tool in the follow-up protocol for patients with low-grade superficial bladder tumors. Nevertheless, reports contextually comparing I-FISH patterns in primary superficial tumor cells with those in concomitant washing cells at the time of initial tumor appearance are sparse. We comparatively evaluated I-FISH patterns of chromosomes 3, 7, 9, and 17 and of the CDKN2A and TP53 loci in newly diagnosed superficial bladder lesions and in corresponding bladder washings, to verify representatives of the latter type of sampling and to improve the efficacy of I-FISH follow-up. A total of 21 biopsies and 12 washings were examined. Samples obtained at the time of the tumor's first appearance showed the presence of cytogenetically abnormal clones in 80% of washings and 70% of biopsies. Five cases showed overlapping washing and biopsy I-FISH patterns; in three cases (and to a lesser extent in two others), consistent discrepancies between the two patterns was observed. The results indicate that knowledge of I-FISH patterns in both washing and biopsy cells on first tumor appearance may be of help in interpreting further follow-up I-FISH patterns, and that these should be considered in the context of the patient's entire clinical history.     

Fluorescence in situ hybridization analysis of complex translocations in two newly diagnosed Philadelphia chromosome-positive chronic myelogenous leukemia patients.

Two different complex translocations from newly diagnosed cases of Philadelphia chromosome-positive chronic myelogenous leukemia (CML) were characterized by G-banding and fluorescence in situ hybridization (FISH) analysis. In one case, a unique balanced t(9;22;9;11) (q34;q11;p22;q23) was identified by G-banding, and confirmed by FISH using MBCR/ABL and painting probes. In the second case, an apparently balanced t(19;22) was identified by G-banding analysis. FISH using MBCR/ABL probe detected the fusion gene on the derivative chromosome 22, indicating the involvement of chromosome 9. Further FISH analysis with selected painting probes showed that the t(19;22) was a result of a complex translocation involving chromosomes 9, 19, 21, and 22.     

荧光原位杂交技术检测慢性粒细胞白血病微小残留病

目的 研究应用荧光原位杂交技术(FISH)检测慢性粒细胞白血病微小残留病，及用FISH技术对缓解期慢性粒细胞白血病(CML)患者外周血进行检测，评价其体内微小残留病的意义。方法 应用CG和I-FI舛对30例CML(13例给予化疗、17例移植后)患者初发和／或缓解期的骨髓及外周血标本进行分析，分别检测Ph染色体和BCR/ABL融合基因的存在。结果 对13例临床给予化疗的患者初发期的外周血和骨髓进行CG分析，Ph检出率分别为15％(2／13)、100％(13／13)。同时进行I-FISH分析，均可检出BCR/ABL融合基因。对初发期骨髓的CG、I-FISH分析结果进行统计学分析，两组无显著差异；对外周血的CG、I-FISH分析结果进行统计学分析，两组有显著差异。对其缓解期骨髓CG、I-FISH分析结果进行统计学分析，两组有显著差异；对缓解期外周血CG、I-FISH结果进行统计学分析，两组有显著差异；对缓解期外周血I-FISH和骨髓I-FISH结果进行统计学分析，两组呈显著相关。对17例移植后患者CG、I-FISH结果进行统计学分析，两组有显著差异。结论 FISH技术检测慢性粒细胞白血病微小残留病敏感性大大高于常规细胞遗传学分析；采集缓解期外周血进行荧光原位杂交分析，可作为一种方便易行的手段评价微小残留病。     

Cytogenetic studies of acute promyelocytic leukemia

Translocation t(15;17) is reported in bone marrow cells from six of seven patients with active acute promyelocytic leukemia (APL). One patient who showed t(15;17) at final relapse did not show it in directly prepared or cultured cells taken from a previous relapse. Bone marrow samples from two patients showed only cells with a normal karyotype in the direct preparation, whereas more than 60% of cells cultured for 24 hr showed t(15;17). R-Banding, G-banding, and an attempt at high-resolution banding indicated the break points t(15;17)(q24;21) for one of our patients.     

Fluorescence in situ hybridization: A brief review

Fluorescence in situ hybridization (FISH) is used for many purposes, including analysis of chromosomal damage, gene mapping, clinical diagnostics, molecular toxicology and cross-species chromosome homology. FISH allows an investigator to identify the presence and location of a region of cellular DNA or RNA within morphologically preserved chromosome preparations, fixed cells or tissue sections. This report describes in situ hybridization, and discusses the past, present and future applications of this method for genetic analysis and molecular toxicology. © 1996 Wiley-Liss, Inc.     

CD79 alpha expression in acute myeloid leukemia. High frequency of expression in acute promyelocytic leukemia.

CD79 alpha is a subunit of an intracytoplasmic protein reported to be specific for B lymphocytes, including immature B lineage cells. To evaluate expression of the CD79 alpha antigen in acute myeloid leukemia (AML), we studied forty-eight cases of AML by paraffin section immunohistochemistry. The cases included four MO, nine M1, nine M2, ten M3, ten M4, and six M5 AMLs using criteria of the French-American-British cooperative group. Eleven cases demonstrated cytoplasmic staining for the CD79 alpha antigen, including one M1, nine M3, and one M5 AML. These CD79 alpha-positive cases represented 5% of all non-promyelocytic AMLs and 90% of all acute promyelocytic leukemias studied. All acute promyelocytic leukemias had the characteristic t(15;17)(q24;q21), including two cases of the microgranular variant (M3v). No other B-lineage-associated antigens were found in the CD79 alpha-positive cases, with the exception of a subpopulation of CD19-positive leukemic cells in one patient. The two non-promyelocytic leukemias that expressed CD79 alpha had no evidence of t(15;17) and did not express any additional B-lineage-associated antigens that might suggest a mixed lineage proliferation. This study demonstrates that CD79 alpha expression in acute leukemia is not restricted to B-lineage acute lymphoblastic leukemias and that CD79 alpha expression is frequently associated with t(15;17) acute myeloid leukemia.