A 37-year-old man was referred to our department with pancytopenia. His hemoglobin level was 5.6 g/dL, white blood cell count was 2.41 × 10³/µL, and platelet count was 32.0 × 10³/µL. A blood smear examination revealed significantly large numbers of schistocytes and marked anisopoikilocytosis. His bone marrow contained 37.0% blasts with the morphological and cytochemical characteristics of proerythroblasts, and showed trilineage dysplasia. Flow cytometric and electron microscopic analyses provided persuasive evidence for the immature erythroid features of blasts. Metaphases obtained from the bone marrow carried complex cytogenetic abnormalities, including del(5q) and −7, both of which were detected in approximately 70% of bone marrow cell nuclei by fluorescence in situ hybridization. The patient was treated with induction chemotherapy consisting of cytarabine and idarubicin, leading to a complete hematological response and the normalization of erythrocyte morphology. Schistocytosis as well as anisopoikilocytosis in the blood may reflect defective erythrocyte development in the bone marrow. Despite the erythroid features of the blasts, the presence of del(5q) and −7, and the trilineage dysplasia indicated that the leukemia of this case was acute myeloid leukemia with myelodysplasia-related changes, according to the current WHO classification.

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 Amplification of the MLL gene has been recognized as a recurrent cytogenetic abnormality that occurs in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). The first case was a 74-year-old man who had a 9-year-long treatment history for hepatocellular carcinoma and concurrent laryngeal cancer. He presented with pancytopenia and his bone marrow picture corresponded to refractory anemia with excess of blasts-2. He died of leukemia progression 80 days after presentation. G-banded karyotype revealed del(7)(q31), −13, two types of ring chromosomes, and one double minute (dmin). The second case was a 68-year-old man who had been treated for bladder, laryngeal, and esophageal cancers for the preceding 12 years. He developed AML with myelodysplasia-related changes and died shortly. Clonally occurring abnormalities were add(2), −5, der(7), der(11), −14, add(15), and a ring chromosome which appeared to be derived from chromosome 11 [r(11)]. Fluorescence in situ hybridization (FISH) using the Vysis LSI MLL dual-color, breakapart rearrangement probe revealed MLL amplification at one of the rings and the dmin in case 1, while in case 2, the gene was duplicated at the atypical banded region of der(11) and amplified at the r(11). del(7q) in case 1 and del(5q) and del(7q) in case 2 were confirmed by FISH, using the respective probes. The clinical features of the two cases corresponded well with those of MLL-amplification-associated AML/MDS. It is likely that the long-lasting cytotoxic treatment for the multiple cancers was responsible for the development of both diseases.

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Purpose: Ten years ago, we introduced fluorescence in situ hybridization (FISH) into clinical practice as a companion diagnostic test for HER2 gene amplification in breast cancer. We summarize here our FISH data during the last 3 years, and discuss the points at issue of this diagnostic test.
Patients and methods: Of a total of 521 cases diagnosed with breast cancer and investigated for HER2 expression by immunohistochemistry (IHC) between 2011 and 2013, 91 cases with IHC score of 2+ were subjected to the FISH test. Using the PathVysion HER-2 DNA Probe Kit, we counted the number of HER2 and CEP17 signals in 20 or more cancer cells, and a ratio of HER2/CEP17 ≥2 was defined as positive for HER2 amplification, while a ratio <2 was defined as negative.
Results and discussion: Fourteen (15%) cases were FISH positive and 77 (85%) cases were FISH negative for HER2 amplification. In the FISH-positive group, the mean HER2 signal number per nucleus was 6.5 (range, 2~30) and that of CEP17 was 2.0 (range, 1~5), while in the FISH-negative group, the mean HER2 signal number was 3.2 (range, 1~14) and that of CEP17 was 2.8 (range 1~9). Increased HER2 signal numbers in the negative group were mainly attributable to polysomy of chromosome 17. Although the assessment of test results in the majority of cases was undemanding, there were equivocal cases in which the HER2/CEP17 ratio was close to 2.0. In FISH specimens prepared from formalin-fixed paraffin-embedded tissues, nuclear truncation by sectioning leading to loss of DNA material can occur, and non-overlapping intact nuclei for scoring and areas of invasive tumor may not necessarily be identified. Thus, a considerable amount of practice with FISH assay is necessary to correctly define the presence of a FISH abnormality.

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 A 75-year-old woman with acute myeloid leukemia (AML), who carried the inv(2)(p23q13)/RANBP2- ALK fusion gene and monosomy 7 and exhibited nuclear membrane expression of ALK protein, showed florid relapse of AML. Fluorescence in situ hybridization (FISH) of a bone marrow smear slide using an ALK break-apart probe revealed that over 80% of the cells had ALK rearrangement. We obtained her written consent and treated her with crizotinib, an inhibitor of ALK tyrosine kinase. Leukemia cells in the blood rapidly disappeared and ALK rearrangement-positive cells identified by FISH in the bone marrow decreased below the cut-off level. The response at 3 months of crizotinib treatment was comparable to the definition of complete remission (CR) of AML. On day 135 of treatment, however, her AML relapsed. G-banded karyotyping revealed del(1)(p13p22) in addition to inv(2) and −7 after crizotinib. The ALK gene carried the heterozygous mutation c.3806G>C within exon 25, causing p.G1269A amino acid substitution within the kinase domain of ALK. As both del(1)(p13p22) at the cytogenetic level and G1269A at the nucleotide level newly appeared after crizotinib treatment, it is likely that they were secondarily acquired alterations involved in crizotinib resistance. Although the initial response to crizotinib was marked, crizotinib alone was not capable of producing a long-term response. We should have performed consolidating chemotherapy using conventional cytotoxic drugs immediately after the achievement of CR by crizotinib.

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