A cytogenetic and fluorescence in situ hybridization evaluation of interferon-alpha in the treatment of chronic myeloid leukemia

The effect of interferon-alpha (IFN) for chronic myeloid leukemia (CML) in the chronic phase (CP) was retrospectively evaluated in comparison with that of busulfan (BU) or hydroxyurea (HU) given alone. Among 107 patients diagnosed with CML between 1982 and 1997, 72 CP cases evaluable for long-term follow-up included 13 patients treated with BU alone, 18 with HU alone, and 41 with IFN-based therapy. Complete cytogenetic response (CCR) was achieved in 4/41 IFN cases (10%), and partial or minimal cytogenetic response occurred in 18/41 IFN cases (44%). In contrast, no cytogenetic response (NCR) was achieved in any BU or HU case. IFN treatment for 6 to 60 months was needed to achieve CCR. Overall survival curves revealed that the IFN group had significantly better survival than BU and HU groups (p=0.008 and 0.04, respectively). A significant correlation was found between karyotypic findings and fluorescence in situ hybridization (FISH) analyses in IFN-treated cases (r=0.739, p=0.0001). In some cases, however, the two methods showed discrepancy; BCR/ABL-positive cells represented only 20-75% of interphase bone marrow cells in NCR cases, although all metaphases examined were positive for the Philadelphia chromosome (Ph). A discrepancy was also seen in CCR cases; up to 22% of cells assessed were BCR/ABL-positive. These findings suggest that IFN is a useful therapy for CML in CP, and may have a suppressive effect on the CML clone even in NCR cases. The results also indicate that a combination of FISH and cytogenetic analyses may provide more detailed information for evaluating the efficacy of IFN than conventional cytogenetics alone.     

多探针荧光原位杂交检测急性髓系白血病常见细胞遗传学异常

目的:评价多探针荧光原位杂交(FISH)在检测急性髓系白血病(AML)常见细胞遗传学异常中的价值,探讨细胞遗传学异常与临床诊断、治疗、预后的关系。方法:采用针对AML/MDS的FISH多探针诊断系统,即以针对AML1/ETO融合基因、PML-RARα融合基因、CBFβ/MYH11融合基因、MLL基因、P53基因、Del(5q)、Del(7q)、Del(20q)8种DNA探针对40例患者进行多探针FISH检测,同时联合染色体核型、临床资料进行研究。结果:40例AML中,共22例多探针FISH检出了细胞遗传学改变,包括:AML1/ETO、PML-RARα、MLL基因断裂重排、Del(5q)、Del(7q)、P53基因缺失、8号染色体三体7种细胞遗传学异常。而常规染色体核型分析仅检出11例遗传学异常。多探针FISH与染色体核型分析的总阳性率分别为57.50%及27.50%。AML1/ETO、PML/RARα阳性者首次诱导化疗效果较理想;而Del(7q)、MLL基因断裂重排阳性、伴复杂细胞遗传学改变者可能预示不良预后。结论:FISH多探针诊断系统检测AML患者常见遗传学异常更省时、准确、高效,有利于完善白血病的分层诊断及指导临床个体化治疗。     

荧光原位杂交技术与细胞遗传学分析在慢性淋巴细胞白血病检测中的评价应用

目的比较常规细胞遗传学分析（CCA）及荧光原位杂交（FISH）两种技术在慢性淋巴细胞白血病（CLL）染色体异常检测中的灵敏度和特异性。方法CCA法采用骨髓短期培养法,核型分析采用R带技术。FISH法采用间期FISH。结果采用CCA法,CLL患者的染色体异常检出率为35．71％,而采用FISH技术染色体异常检出率为64．2％。结论采用组合探针的FISH更为敏感和特异。     

Combined cytogenetic testing and fluorescence in situ hybridization analysis in the study of chronic lymphocytic leukemia and multiple myeloma

We investigated the usefulness of fluorescence in situ hybridization (FISH) panels when testing for chromosomal aberrations of known prognostic significance in chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). Our CLL panel included probes for 11q, 12 centromere, 13q, 14, and 17p. Karyotype and FISH were abnormal in 13 of 60 (21.6%) cases, two (3.3%) abnormal by karyotype alone, and 25 (41.6%) by FISH alone. Karyotype and FISH were normal in 16 (27%) patients, and 4 samples were unsuitable for karyotype analysis. One patient had an abnormality not included in the panel (20q deletion). FISH was abnormal in 19 cases (31.6%) with a normal karyotype and in 6 cases with no analyzable metaphases. Thirteen CLL cases with abnormal karyotypes were either confirmed or clarified by FISH. The MM panel probes were 11q, 13q, 17p, and t(11;14). Karyotype and FISH were abnormal in 18 of 139 (13%) MM cases. Twenty patients (14.4%) had a normal karyotype and abnormal FISH. Two samples not suitable for metaphase analysis were abnormal by interphase FISH. Karyotype and FISH were normal in 94 (68%) patients. Five patients (3.6%) had chromosomal abnormalities not included in the panel. Compared to karyotyping alone, the FISH panels improved the detection rate of recurrent chromosomal aberrations in CLL from 22-63% and in MM from 15-29%.     

Y chromosome loss in chronic myeloid leukemia detected in both normal and malignant cells by interphase fluorescence in situ hybridization

Loss of the Y chromosome in bone marrow (BM) cells is a normal age-associated event. Y chromosome loss is also observed in the Philadelphia chromosome (Ph) positive BM cells of some patients with chronic myeloid leukemia (CML) in chronic phase, but at a younger age than in normal individuals. While the significance of loss of the sex chromosome in normal males is uncertain, -Y marrow cells are not believed to be of clonal origin. However, because CML is a clonal disease, CML sub-populations with Y loss may constitute a disease-related sub-clone. We used a PCR-amplified yeast artificial chromosome containing the BCR gene region for single color interphase analysis of BCR rearrangement by fluorescence in situ hybridization (FISH). Then, using two color FISH, with one fluorochrome detecting the BCR gene region and the other detecting Y chromosome repeat sequences, we surveyed peripheral and BM Y loss in both normal Ph- (BCR not disrupted) and CML Ph+ (BCR rearranged) interphase nuclei of two patients with Y loss in Ph positive cells observed by metaphase analysis. -Y was seen in a proportion of Ph+ cells in both cases, and the proportion matched that seen in Ph- cells, indicating that Y loss is probably sporadic in both normal and CML populations, and that the propensity for Y loss in normal BM cells may be a phenotype that can be retained by malignant cells in CML.     

Clinical implications of fluorescence in situ hybridization analysis in 13 chronic myeloid leukemia cases: Ph-negative and variant Ph-positive.

Thirteen chronic myeloid leukemia (CML) patients, 10 with variant Philadelphia (Ph) translocations and 3 Ph negative cases, were analyzed by fluorescence in situ hybridization (FISH) with the use of BCR and ABL cosmid probes and a chromosome 22 painting probe. In the variant Ph translocations, the BCR-ABL fusion gene was located on the Ph chromosome; in 1 CML Ph-negative patient, the BCR-ABL fusion gene was located on the Ph chromosome; and, in 2 patients, it was located on chromosome 9. The chromosome 22 painting probe was detected on the third-party chromosome of the variant translocation, and in none of the variant translocations was there any detectable signal on chromosome 9. In CML patients with clonal evolution of a simple Ph, a signal of the chromosome 22 painting probe was detected on the der(9) of the Ph translocation. It was concluded that the variant Ph translocations evolved simultaneously in a three-way rearrangement. The clinical parameters of the 13 patients were similar to those of a large group of CML patients with a simple Ph translocation. It is suggested that, to determine the prognosis of CML patients with a complex karyotype, FISH analysis with a chromosome 22 painting probe be performed.     

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.     

Application of multiplex fluorescence in situ hybridization in the cytogenetic analysis of primary gastric carcinoma

The different genetic alterations observed in diffuse and intestinal types of gastric cancer suggest that these two pathological types may represent different disease entities. We present two cases of primary gastric carcinoma, a well-differentiated intestinal type adenocarcinoma and a poorly differentiated diffuse type adenocarcinoma, both studied by a 24-color multiplex fluorescence in situ hybridization technique (M-FISH). The well-differentiated intestinal type adenocarcinoma exhibited fewer structural abnormalities with five noncomplex translocations, deletions of chromosomes 5q, 6q, and 17q and an i(8q). In the case of poorly differentiated diffuse carcinoma, structural abnormalities predominated and normal homologues were mostly absent. But there were also similarities between the two cases: translocations on 1p and 9p; structural abnormalities of chromosome 8 with consistent loss of 8p; structural abnormalities of 12q; partial loss of chromosome 17 and 18; and polysomy of chromosome 20. This study shows that M-FISH is valuable in identifying hidden structural abnormalities and could, therefore, be useful in the investigation of primary solid tumors.     

Molecular cytogenetic analysis of breast cancer: a combined multicolor fluorescence in situ hybridization and G-banding study of uncultured tumor cells

In six patients with breast cancer, uncultured tumor cells were investigated with G-banding and multicolor fluorescence in situ hybridization (M-FISH). A large number of numerical and structural aberrations could be analyzed. Among other structural abnormalities, reciprocal, hidden and complex translocations were found. Recurrent t(1;10) and t(6;16), not previously described, were identified, as well as t(15;22). The latter was also found in additional cases among our unpublished breast carcinomas. The significance of t(15;22) for breast cancer is discussed, taking into account also data drawn from the literature. Reciprocal translocations were a prominent feature in a pseudodiploid lobular carcinoma. Hidden translocations on 6p22-p24 were detected with M-FISH. Involvement of 6p22-p24 was observed in five cases. The analysis of various other translocations and different structural abnormalities revealed the following common breakpoints (according to frequency of involvement): 1p34-p36, 3p12-p13, 4p13-->q11, 14p11-->q11, 1q42, 8p11, 8q24, 10q22, 11q13, 11q23-q24, 13q13, and 18p10-p11. Loss of 3p and 1p34-p36-->pter and complete or partial loss of 13q and chromosome 17 were also found. With the combination of G-banding and M-FISH techniques, chromosome misclassification is avoided and the characterization of complex tumor karyotypes is more effective.     

Molecular cytogenetic analysis of oral squamous cell carcinomas by comparative genomic hybridization, spectral karyotyping, and fluorescence in situ hybridization

We investigated relationships between DNA copy number aberrations and chromosomal structural rearrangements in 11 different cell lines derived from oral squamous cell carcinoma (OSCC) by comparative genomic hybridization (CGH), spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH). CGH frequently showed recurrent chromosomal gains of 5p, 20q12, 8q23 approximately qter, 20p11 approximately p12, 7p15, 11p13 approximately p14, and 14q21, as well as losses of 4q, 18q, 4p11 approximately p15, 19p13, 8p21 approximately pter, and 16p11 approximately p12. SKY identified the following recurrent chromosomal abnormalities: i(5)(p10), i(5)(q10), i(8)(q10), der(X;1)(q10;p10), der(3;5)(p10;p10), and der(3;18)(q10;p10). In addition, breakpoints detected by SKY were clustered in 11q13 and around centromeric regions, including 5p10/q10, 3p10/q10, 8p10/q10 14q10, 1p10/1q10, and 16p10/16q10. Cell lines with i(5)(p10) and i(8)(q10) showed gains of the entire chromosome arms of 5p and 8q by CGH. Moreover, breakages near the centromeres of chromosomes 5 and 8 may be associated with 5p gain, 8q gain, and 8p loss in OSCC. FISH with a DNA probe from a BAC clone mapping to 5p15 showed a significant correlation between the average numbers of i(5)(p10) and 5p15 (R(2) = 0.8693, P< 0.01) in these cell lines, indicating that DNA copy number of 5p depends upon isochromosome formation in OSCC.     

In situ fluorescence hybridization of Y translations: cytogenetic analysis using probes Y190 and Y431

Two moderately repetitive DNA probes (Y190 and Y431) and a fluorescent in situ hybridization technique, using a biotin, avidin, anti-avidin system, were employed to investigate a group of patients with Y chromosome abnormalities. In normal male subjects, a bright fluorescent spot could be detected in cells in interphase and on the short arm of the Y chromosome in metaphase spreads. Translocations of DNA fragments of the short arm of the Y chromosome to autosomes 10, 13 and 15 were observed in five patients. In a 45,XX male subject the translocation involved one of the X chromosomes. With this in situ hybridization procedure, bright fluorescent spots were also noticed in uncultured amniotic cells and chorionic cellular elements from male fetuses, thus allowing a rapid and reproducible approach to prenatal fetal sexing.     

Molecular cytogenetic analysis of two primary squamous cell carcinomas of the lung using multicolor fluorescence in situ hybridization

To fully characterize the numerous chromosomal aberrations in two human squamous cell carcinomas (SCCs) of the lung, molecular cytogenetic characterization was carried out utilizing conventional banding analysis and multicolor fluorescence in situ hybridization (mFISH), providing simultaneous color discrimination of all 24 human chromosomes. Both tumors displayed complex aneuploid karyotypes with a host of numerical and structural chromosome abnormalities. Structural aberrations common to both SCCs included rearrangements of chromosomes 1, 3p, 7q, and 8q, contributing to net loss of chromosomal sequences on 1p, 3p, and 8p, and a net gain of 8q. The recently introduced mFISH technique enabled the disclosure of cryptic translocations and the chromosomal composition of previously unrecognized marker chromosomes. Furthermore, mFISH greatly enhanced the ability to delineate chromosomal breakpoints when integrating banding information from conventional banding analysis. Eventually, the application of mFISH as a powerful approach to refine complex tumor karyotypes is expected to result in a more detailed and complete picture of cytogenetic events associated with the development and progression of solid tumors.     

Tetrasomy 21 as a sole chromosome abnormality in acute myeloid leukemia. fluorescence in situ hybridization and spectral karyotyping analyses

We report a case of acute myeloid leukemia with tetrasomy 21 as the sole chromosome abnormality in a constitutionally normal patient. Tetrasomy 21 was observed at presentation, disappeared in remission, but reappeared in recurrence of the disease. Fluorescence in situ hybridization analysis using a probe specific for the AML1 gene showed four distinct signals in 82.4% and three signals in 10.8% of interphase nuclei, although conventional G-banding revealed tetrasomy 21 alone in mosaicism with normal karyotype. Spectral karyotyping further confirmed the presence of extra copies of chromosome 21. Tetrasomy 21 as the only anomaly is relatively rare in patients with hematologic disorders other than Down syndrome, and to our knowledge has been reported previously in only seven cases. In a review of the literature, tetrasomy 21 as the only anomaly may be associated with myeloid disorders, although simultaneous numeric abnormalities other than chromosome 21 have been reported in acute lymphoblastic leukemia with hyperdiploid karyotype.     

Dual-colour fluorescence in situ hybridization analysis of synovial sarcoma

Mounting cytogenetic evidence indicates that synovial sarcomas, regardless of histological conformation, share the specific reciprocal chromosomal translocation t(X;18)(p11.2;q11.2). Application of dual-colour fluorescence in situ hybridization (FISH) on interphase nuclei isolated from archival paraffin-embedded material to identify the specific translocation is of diagnostic importance for pathological practice and retrospective study. Five cases of well-characterized biphasic synovial sarcomas, two monophasic fibrous synovial sarcomas, one embryonal rhabdomyosarcoma, one fibrosarcoma, and one malignant peripheral nerve sheath tumour were analysed. To visualize the translocated chromosomal fragments and their topographic relationships with centromeres of chromosomes X and 18, nuclei from each case were hybridized concurrently with chromosome X centromeric and chromosome 18 painting probes, and chromosome 18 centromeric and chromosome X painting probes, respectively. Six out of seven synovial sarcomas showed chromosomal alterations consistent with t(X;18). One biphasic synovial sarcoma had trisomy 18 and lacked the chromosomal translocation t(X;18). The other three spindle cell sarcomas and the normal control tissues showed the normal numerical and structural composition for chromosomes X and 18. It is indicated from the present study that when histological differential diagnosis is difficult, FISH would be a crucial aid in detecting a known specific chromosomal alteration and that dual-colour FISH is an efficient stable diagnostic tool for pathological research and daily diagnosis. The results also suggest that rare synovial sarcomas may lack the chromosomal translocation t(X;18). © 1998 John Wiley & Sons, Ltd.     

荧光原位杂交检测慢性粒细胞白血病

目的 探讨对慢性粒细胞白血病进行荧光原位杂交(fluorescence in situ hybridization,FISH)检测的意义.方法 对158例慢性粒细胞白血病标本采用24 h短期培养法制备染色体,然后应用双色双融合BCR/ABL探针进行FISH检测,部分标本同时采用R显带技术进行染色体核型分析.结果 158例中共检出Ph阳性标本98例,其中69例(70.4%)为典型双色双融合BCR/ABL探针信号模式(1R1G2F),其余29例(29.6%)为3类12种非典型模式.各种非典型信号模式中出现频率较高的依次为:1R1G1F7例(7.1%)、2R1G1F 5例(5.1%)、1R1G2F&1R1G3F 4例(4.1%)、2R2G1F 3例(3.1%).对18例有核型资料的非典型信号的病例分析显示:其中3例特殊信号系由变异Ph易位引起;2例中出现的3个融合信号来源于附加的Ph染色体;4例核型与FISH结果不吻合,提示染色体分析存在错漏之处;3例染色体为典型Ph易位,而FISH结果为单个融合信号,系由der(9)号的部分缺失所致;3例核型中未发现Ph染色体.但FISH显示40%～64%的细胞中存在一个融合信号,从而明确慢性粒细胞白血病诊断;3例是移植或经格列卫治疗后的患者,染色体均为正常核型,而FISH检测到极小比例的阳性细胞.结论 FISH在慢性粒细胞白血病诊断、判断变异易位、隐匿Ph易位、衍生9号缺失、干扰素及格列卫的疗效观察以及移植后监测等诸多方面均具有重要价值.