Sim-Leng Tien

Effect of Fixation Period on HER2/neu Gene Amplification Detected by Fluorescence In Situ Hybridization in Invasive Breast Carcinoma

This study investigated if formalin fixation duration affects HER2/neu gene amplification detection by fluorescence in situ hybridization (FISH) in breast cancer. Tumor tissues from 35 cases were divided into three groups and subjected to two formalin fixation protocols per group (12 hr, 27 hr in the first; 2 hr, 17.5 hr in the second; 28.5 hr, 541 hr in the third) before FISH analysis. There was no significant difference in FISH signal detection between the two different fixation protocols in the first two groups. In the third, no signal was detected in 4/6 cases fixed for an extended duration.

Evidence for deletion of 9q as a two-step process in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is characterized by the Philadelphia translocation t(9;22)(q34;q11) resulting in the BCR/ABL fusion gene. Submicroscopic deletion of the derivative chromosome 9 occurs in a subset of these patients and is associated with poor prognosis. In the current study, we present two unusual cases of CML selected from a series of 54 consecutive cases. A detailed study using classical cytogenetics and fluorescence in situ hybridization (FISH) analysis was done using dual color extra signal FISH and whole chromosome paint in order to elucidate the mechanism of 9q deletion. One case had two clones on interphase FISH, one with and one without chromosome 9q deletion. The other case had two clones on both cytogenetic and FISH analyses, one with and one without a marker chromosome carrying chromosome 9q sequences. In this latter case, the clone with deletion of the derivative chromosome 9 comprised 21.1% at diagnosis, increasing to 36.8% after 11 months, suggesting a growth advantage. We report here evidence that deletions on 9q in CML may occur through breakage and rearrangement of chromosomes resulting in derivative chromosomes and either a marker chromosome or fragment/episome, followed by loss of chromosome material from the cell.

Highly Complex Chromosomal Rearrangement of Chromosome 9 in a Case of Chronic Myeloid Leukemia

A 63-year-old man with chronic myeloid leukemia (CML) was found to have a new complex Philadelphia translocation. All of the bone marrow cells had a rearrangement of a five-way translocation, t(9;22;10;12;1), involving a single chromosome 9. The patient went into blast crisis two years after initial diagnosis and the karyotype remained unchanged. He died in blast crisis 10 months later. We believe this case is a unique 5-way translocation in which four chromosomes were translocated to a single chromosome.

High incidence of derivative chromosome arm 9q deletions in Asian chronic myelogenous leukemia

In a recent issue of Genes, Chromosomes and Cancer, Lee et al. (2003) reported deletions of the residual ABL gene on the derivative chromosome 9 in 16 of 59 (27%) chronic myelogenous leukemia (CML) patients from Korea. In addition, their findings showed that patients with ABL deletions progressed to blast crisis in a significantly shorter time than did those without such deletions, suggesting that ABL deletion may be an indicator of poor prognosis in CML, confirming previously published studies (Sinclair et al., 2000; Huntly et al., 2001). We have analyzed bone marrow samples from 54 consecutive cases of CML to determine the frequency of deletions of the ABL gene on derivative chromosome 9 in Singaporean patients. Twentyfive patients were female, and 28 were male, ranging in age from 16 to 70 years (median, 41.2 years). Forty-four were Chinese, 5 were Malay, and 5 were Indian. The frequency of deletions on derivative chromosome 9 in these patients was determined by fluorescence in situ hybridization (FISH) using the LSI BCR/ABL dual-color extrasignal (ES) FISH probe set (Vysis, Downers Grove, IL), as previously described (Lee et al., 2003). Dual-color dual-fusion FISH (D-FISH) using the BCR/ABL probe (Appligene Oncor, UK) was done on all cases with aberrant ES-FISH signals. Of the 54 cases, 5 (9%) had variant Philadelphia (Ph) translocations and 3 (6%) had masked Ph chromosomes, as confirmed by ES-FISH on metaphase cells. The remainder of the cases (85%) had the classical Ph chromosome (Table 1). A deletion of the 9q region proximal to the ABL gene and encompassing the ASS region, with deletion of chromosome 22–derived material on the derivative 9q, was present in 24% (13/54) of the cases in the current study (Table 1). This is a higher proportion compared to reports on Western populations including Canada (9%, 9/250; Kolomietz et al., 2001), Belgium (10%, 5/51; Herens et al., 2000), America (12%, 17/141; Dewald et al., 1999), Spain (12%, 16/135; Primo et al., 2003), Israel (15%, 14/94; Cohen et al., 2001), and the United Kingdom and Australia (15%, 38/253; Huntly et al., 2001). This frequency of 24% is comparable, however, to that found in Korea, of 27% (Lee et al., 2003). Statistical analysis of the pooled results from non-Asian countries, which were based on biological and geographic grounds, compared with those from Singapore and Korea gave a significant difference between the two groups (chi-square test, P < 0.0001). Similarly, the comparison of nonAsian versus Korean populations showed a significant difference (chi-square test, P < 0.001), but not that of non-Asian and Singaporean populations. In cases with classical Ph alone, the frequency of deletions remains high (24%, 11 of 46 cases). Only two of the five cases with variant Ph translocations showed a 9q deletion compared to 48% (10/21) in a study from the United Kingdom (Sinclair et al., 2000), which had suggested that deletions were more prevalent in patients with variant Ph chromosomes. Thus, the high frequency of deletion in our cases was not caused by a high proportion of cases with variant Ph translocations. Geographic variation of chromosome abnormalities in hematological malignancies has been described, including variation in the frequencies of secondary chromosome abnormalities seen in CML in accelerated phase and/or blastic transformation (Johansson et al., 1991). Such geographic variation was observed in a recent cytogenetic study, from Singapore, of acute myeloid leukemia (Enjeti et al., 2004). A gradient from Western Europe to eastern Asia of increasing frequency of chromosome 9 deletions is observed when taking into account the different frequencies reported in various populations. The higher frequencies in the United Kingdom and Australia may reflect the migration of Asians to those countries. A markedly high incidence (24%) of chromosome 9 deletions was found in this study. We recommend that the deletion status of CML patients should be assessed in all cases, particularly those of Asian origin, as these deletions can be used as an independent prognosticator. The incidence and importance of submicroscopic chromosome