Detection of numerical alterations of chromosome 1 in cytopathological specimens of breast tumors by chromogen in situ hybridization

To investigate the effectiveness of chromogen in situ hybridization (CISH) in the diagnosis of breast tumors, numerical alterations of chromosome 1 were examined by CISH and fluorescence in situ hybridization (FISH) methods, and the presence of der(16)t(1;16) was also examined by FISH in imprinted cytology specimens from resected tissues of 14 carcinomas and five non-malignant lesions. The modal signal counts of chromosome 1 were compared between the specimens processed by CISH and FISH for each case. Aneusomies of the long arm of chromosome 1 were detected in 10 (71%) carcinomas as the major clones by both methods. In addition, one atypical papilloma demonstrated tetrasomy of 1q12 as a major clone by CISH, but such a clone was at first overlooked by FISH. Four other benign lesions showed disomic 1q12 signals as a major clone by both CISH and FISH. As additional information from FISH, eight cancers showed structural or numerical alterations of chromosome 16, and four showed der(16)t(1;16). In total, 10 carcinomas showed chromosome 16 alterations, and all of these overlapped with the carcinomas with 1q12 aneusomies. The CISH method provided almost the same results as the FISH method, and both methods were considered applicable in supportive diagnosis of cytological specimens of breast tumors. In addition, the CISH method was superior in the detection of numerical alterations in carcinoma cells by referring to the morphology of cells and in the detection of significant clones which might be missed under dark-field microscopy.     

Detection of numerical alterations for chromosomes 7 and 12 in benign thyroid lesions by in situ hybridization. Histological implications.

Polysomies of chromosomes 7 and 12 have been frequently observed by conventional cytogenetics in a subgroup of thyroid follicular adenomas and in some cases of thyroid goiters. To further study possible cytogenetic similarities between these two types of thyroid lesions, we have used fluorescence in situ hybridization (FISH) to detect polysomies of chromosomes 7 and/or 12 in isolated nuclei from frozen and paraffin-embedded material of goiters and thyroid follicular adenomas and compared results with previous ones obtained by flow cytometry and conventional cytogenetics. With a set of two alpha-satellite DNA probes specific for the centromeric regions of chromosomes 7 and 12, used either separately (single-target fluorescence in situ hybridization) or simultaneously (double-target fluorescence in situ hybridization), we detected polysomies of chromosome 7 in 35.7% of the thyroid follicular adenomas and in 10.7% of the goiters. Polysomies of chromosome 12 were detected in 29.6% of the thyroid follicular adenomas and 6.7% of the goiters. The significantly higher frequency of adenomas with numerical alterations for chromosomes 7 and/or 12 supports the idea of a biological continuum and karyotypic evolution between both lesions. It is also noteworthy that polysomies of chromosomes 7 and/or 12 were observed only in lesions with an exclusive (or predominant) microfollicular histological component, as detected by enzymatic in situ hybridization on frozen sections.     

Evaluation of numerical abnormalities of chromosomes 1 and 17 in proliferative epithelial breast lesions using fluorescence in situ hybridization

Our aim was to investigate the putative role of chromosome abnormalities of chromosomes 1 and 17 in the process of breast carcinogenesis. Numerical abnormalities of chromosomes 1 and 17 were investigated using fluorescence in situ hybridisation (FISH) in a series of 16 primary invasive breast carcinomas associated with intraductal proliferative epithelial lesions. Chromosome 1 aneusomy was detected in 55.6% of ductal hyperplasia (DH), 81.8% of ductal carcinomas in situ (DCIS) and 87.5% of invasive ductal carcinomas (IDC). Chromosome 17 aneusomy was not detected in the cases of DH and was present in 90.9% of DCIS and in 87.5% of IDC. Simultaneous aneusomy of chromosome 1 and 17 was found in 81.8% of DCIS and in 75.0% of IDC. Our results showed that the number of chromosome 1 and 17 copies increases from normal epithelium to invasive cancer. The numerical abnormalities of chromosome 1 were already detected in DH, suggesting that a gain in the copy number of chromosome 1 may be involved early in breast carcinogenesis.     

Complex chromosome rearrangement involving chromosomes 1, 4 and 16 revealed by fluorescence in situ hybridization

A 7-year-old boy with mental retardation had apparently balanced reciprocal translocations, involving the telomeric regions of chromosomes 1p and 4q, which was detected by routine chromosome analysis. Fluorescence in situ hybridization (FISH) was used and also revealed the telomeric region of chromosome 16p to be involved in a still apparently balanced translocation-complex, impossible to discover with classical cytogenetic analysis. We want to emphasize the importance of FISH in detecting small chromosomal aberrations. We discuss whether the abnormal phenotype is caused by unbalanced karyotype with cryptic undetected translocations or small deletions or mutations in the translocation-breakpoints.     

Detection of genetic alterations in primary bladder carcinoma with dual-color and multiplex fluorescence in situ hybridization

Cytogenetic studies of bladder cancer have shown several nonrandom aberrations. Numerical aberrations of both sex chromosomes were investigated in 32 primary bladder tumors with bicolor fluorescence in situ hybridization (FISH). Loss of chromosome Y and overrepresentation of chromosome X were observed in subgroups of male patients. Chromosome X was represented normally in female patients. Two of the above primary bladder tumors, a transitional cell carcinoma (TCC) and an adenocarcinoma, were further analyzed with both multiplex FISH (24-color M-FISH) and G-banding. Both cases exhibited 1) common breakpoints on 5q11 approximately q12 and 15q24; 2) involvement of the pericentromeric area of chromosome 13; 3) structural abnormalities of chromosomes 8 and 17, with loss of material on the short arm; 4) structural abnormalities involving chromosome 11; and 5) loss of chromosome Y. The TCC case also exhibited structural abnormalities of chromosome 9, resulting in loss of 9q. The combined G-banding and M-FISH findings could help reveal regions potentially involved in bladder tumorigenesis.     

Genetic heterogeneity of primary and metastatic breast carcinoma defined by fluorescence in situ hybridization.

Breast carcinoma is frequently associated with nonrandom chromosomal aberrations, but their identification by standard cytogenetics (SC) is often limited by technical difficulties. Fluorescence in situ hybridization (FISH) studies of interphase nuclei can circumvent some of these difficulties and has the potential to identify nonrandom molecular cytogenetic events occurring in breast cancer. FISH was performed on tumor nuclei isolated from 15 formalin-fixed, paraffin-embedded archival breast carcinomas using a panel of chromosome-specific alpha-satellite probes for enumerating chromosomes in interphase nuclei. Freshly isolated cells from these same cases had previously been studied by standard cytogenetics and FISH. In addition to archival primary carcinoma, archival metastases and normal tissue were also studied by FISH. Genetic numerical alterations were identified by standard cytogenetics or FISH in 14 of 15 carcinomas. Numeric alterations initially identified by standard cytogenetics were confirmed by FISH in 9 of 10 cases. Results of FISH performed on nuclei isolated from paraffin-embedded material were in agreement with FISH performed on freshly isolated cells. Clonal numeric alterations were observed in the archival primary tumor as well as in metastases. Archival normal tissue was consistently disomic.     

荧光原位杂交检测卵巢癌8号染色体畸变

目的：探讨卵巢癌染色体改变与卵巢癌发生、发展及预后的关系。方法：应用荧光原位杂交技术对卵巢癌、卵巢良性肿瘤及正常卵巢标本各12例进行8号染色体检测。结果：本组12例卵巢癌8号染色体出现单体、三体或四体畸变，畸变率100％（12／12）；卵巢良性肿瘤8号染色体出现三体畸变，畸变率25％（3／12）；正常卵巢8号染色体无畸变，卵巢癌与卵巢良性肿瘤及正常卵巢比较，差异有显著性（P＜0．001）。结论：卵巢癌第8号染色体畸变与卵巢癌发生、发展密切相关，其改变发生在卵巢癌早期，与患者临床分期、病理分化相关。     

A fluorescence in situ hybridization study of ETV6-NTRK3 fusion gene in secretory breast carcinoma

The translocation t(12;15)(p13;q25), in which the ETV6 gene from chromosome 12 is rearranged with the NTRK3 gene from chromosome 15, has recently been identified in secretory breast carcinoma (SBC). This fusion gene was initially described in congenital fibrosarcoma and congenital mesoblastic nephroma. The biological consequence of this translocation is the expression of a chimeric protein tyrosine kinase with potent transforming activity. To assess the frequency of t(12;15)(p13;q25) in breast cancer, we developed complementary probe sets (fusion and split-apart probes) for the detection of this translocation by fluorescence in situ hybridization (FISH) in paraffin-embedded, formalin-fixed tissue sections. We tested four histologically confirmed cases of SBC for the presence of the ETV6-NTRK3 gene fusion and then applied the FISH assay to tissue microarrays (TMAs) in order to screen 481 cases of formalin-fixed, paraffin-embedded invasive breast carcinomas of various histologic subtypes. Three of the four cases of SBC revealed fusion signals. Of the 481 cases in the TMAs, 202 gave signals of sufficient quality for screening by FISH, and only one case showed fusion signals in most or all of the tumor cells. On review of the histology of this case, SBC was confirmed. On the other hand, none of the fusion-negative breast cancers revealed SBC histology. In all cases, the results from the fusion and split-apart FISH assays for the ETV6-NTRK3 fusion genes were concordant. Our data suggest that the ETV6-NTRK3 fusion gene is a specific genetic alteration in SBC.     

双色荧光原位杂交检测苯系物接触者精子7、8号染色体数目畸变

目的　检测苯系物接触者精子 7、8号染色体数目畸变率。方法　用生物素标记 7号染色体探针 (D7Z1)和地高辛标记 8号染色体探针 (D8Z1)与精子核进行双色荧光原位杂交 ,测定精子染色体畸变率。结果　车间空气中苯的时间加权浓度为 42 .2 9mg/m3 ,高于国家卫生标准 ( 6mg/m3 )。苯系物接触者尿粘康酸浓度 ( 0 .861mg/g肌酐 ) ,高于对照组 ( 0 .44 4mg/g肌酐 )。杂交效率 99.77%～ 99.97% ,共计数15例苯系物接触者 15 5 72 1条精子核和 12名对照组者 12 3 771条精子核型。接触组二倍体精子率 ,平均7、8号染色体双体精子率 (分别为 0 .12 9%、0 .170 %和 0 .0 78% )均高于对照组 (分别为 0 .0 5 5 %、0 .0 5 3 %、0 0 3 3 % )。接触组 7号、8号染色体缺体精子率 (分别为 0 .165 %和 0 .0 88% ) ,也分别高于对照组 ( 0 .0 5 6%、0 0 2 9% )。总数目畸变精子率接触组为 0 .745 % ,高于对照组的 0 .2 89%。结论　较高浓度的苯系物暴露可引起接触者精子 7、8号染色体非整倍体率和二倍体精子率增高。     

Analysis of chromosome aneuploidy in breast carcinoma progression by using fluorescence in situ hybridization

Nonisotopic fluorescence in situ hybridization by using alpha satellite centromeric probes was performed on intact tissue sections of 12 breast carcinomas to compare the pattern of aneuploidy for chromosomes 7, 8, 16, and 17 between foci of residual in situ carcinoma (DCIS) and a representative area of coexisting invasive neoplasm. Most hybridization pairs (58%) showed a gain in chromosomal copy number between the in situ and corresponding invasive area, whereas 29% showed no apparent change and 13% showed loss in copy number. Hybridizations from areas of invasive carcinoma, thus, were more frequently characterized by tumor cells with trisomy/polysomy (78%) than neoplastic cells from residual DCIS (50%) and less frequently characterized by cells with monosomy (10% versus 16%, p = 0.01). Even when DCIS cells exhibited chromosome trisomy, 65% of hybridizations demonstrated a significantly greater proportion of trisomic cells in the corresponding invasive population. The hybridization pairs (n = 7) initially showing apparent loss in chromosome copy number from in situ to invasive growth were all from two cases that demonstrated morphologic heterogeneity. Enumeration of cells from histologically distinct areas of these cases revealed different patterns of aneusomy, in keeping with karyotypic diversity. However, comparison of histologically similar areas of DCIS and invasive neoplasm demonstrated a pattern of chromosome copy gain with invasive growth, similar to morphologically homogeneous tumors. We conclude that invading cells in breast carcinomas differ from residual in situ populations with respect to degree of chromosome aneuploidy and that tumor progression from preinvasive to an invasive phenotype in human breast carcinoma is characterized by a significant increase in the degree of genetic instability. The observed pattern of chromosome copy number gain, moreover, is consistent with a common cellular level genetic mechanism underlying early breast tumor progression.     

应用FISH技术检测肺腺癌,鳞癌细胞部分染色体数量的改变

目的研究肺癌细胞７号，１７号及Ｙ染色体的数目改变及其意义。方法应用荧光原位杂交技术（ＦＩＳＨ）技术，对１７例腺癌和鳞癌的细胞印片进行检测。结果７号染色体：三体细胞占细胞总数的３１．７３％。１７号染色体：二体细胞占细胞总数的４３％。Ｙ染色体：单体细胞占细胞总数的５８．０４％。结论在肺腺癌和肺鳞癌中，７号染色体数目均有明显增加，并以三体所占比例最高。肺腺癌与肺鳞癌７号染色体数目分布差异明显。     

The numerical aberrations of chromosome 7 detected by fluorescence in situ hybridization in human breast cancers

The relationship between the numerical aberrations of chromosome 7 in interphase cells and the clinicopathological behavior of breast tumors was investigated in 51 touch imprinted preparations of breast tumors. Using fluorescence in situ hybridization with a chromosome 7-specific DNA probe, the fluoresceinisothiocyanate (FITC) spots mean and the representative copy number of each breast tumor were examined. The FITC spots mean (2.34) of 40 breast cancers increased compared with that of 11 benign lesions (1.98) (P < 0.02). The FITC spots mean tended to increase with the advancing stage and tumor size of the breast cancer. The FITC spots mean in the case with metastasis was also of a higher value than that without metastasis (P < 0.01). Furthermore, the existence of trisomy or over-trisomy of the copy number was related to the advancing stage and tumor size (P < 0.05 and P < 0.01, respectively). These findings suggest that the FITC spots mean and polysomy of the number of chromosome 7 may be highly predictive for breast tumor aggressiveness.     

原位杂交检测HPV-16E6,-18E6在喉癌组织中的表达

目的 检测HPV L1和16/18DNA在喉癌组织中的表达,探讨HPV在喉癌发生中的作用.方法 用原位杂交(ISH)检测123例喉癌组织及123例"正常人"的喉粘膜上皮组织中HPV-16/18 mRNA.结果 在123例喉癌标本中,ISH检出HPV-16E6的阳性率为46.34%,HPV-18E6的阳性率为35.77%.123例"正常人"的喉粘膜ISH结果表明:低危型HPV的阳性率与喉癌相近;高危型HPV的阳性率虽远远低于喉癌,但随着组织病变程度加重阳性率逐渐升高.对上述结果用SPSS 10.0软件进行case-control X2分析,HPV-16感染会增加喉上皮病变的风险(OR=14.58),感染HPV-18时,喉上皮病变的风险(OR=10.77).结论 HPV-16感染是重庆地区喉癌的重要发病因素;HPV-16感染后E6片段的保留并持续表达与喉组织的癌变进程密切相关;HPV-16感染会增强喉上皮病变的风险,而HPV-18有协同作用.     

用荧光原位杂交技术检测乳腺癌细胞系第6号染色体长臂缺失

目的 研究１０例乳腺癌细胞系的６号染色体第臂缺失。方法 运用荧光原位杂交（ｆｌｕｏｒｅｓｃｅｎｃｅ ｉｎ ｓｉｔｕ ｈｙｂｒｉｄｉｚａｔｉｏｎ,ＦＩＳＨ）技术,并采用３７个分布于６ｑ１２－６ｑ２７的ＹＡＣ探针和６号染色体着丝粒特异性探针,检测了１０例乳腺癌细胞系的６号染色体长臂。结果 在５例细胞系中发现６ｑ１２－６ｑ２７的大段缺失,１例发现有６ｑ２５－ｑ２７的小段缺失。在含有多个细胞亚群的两侧细胞     

胃癌c-erbB-2基因扩增的荧光原位杂交检测

目的：研究胃癌ｃｅｒｂＢ２ 基因扩增。方法：使用荧光原位杂交技术（ＦＩＳＨ） 检测５５ 例胃癌。结果：胃癌肿瘤区ｃｅｒｂＢ２基因扩增阳性率为３６－４％ 。在胃癌移行区内阳性率为１０－９％ ,在正常粘膜区阳性率为３－６ ％ ,相互比较,三个区域之间差异有高度显著性（ Ｐ＜ ０－０１）。在肠型和弥漫型胃癌,ｃｅｒｂＢ２ 基因扩增阳性率为５１－６％ 和１６－７％ ,两者差异有显著性（ Ｐ＜０－０５）。ｃｅｒｂＢ２ 基因扩增阳性与淋巴结内胃癌转移有明显相关性（ Ｐ＜ ０－０５）。结论：ｃｅｒｂＢ２ 基因扩增与胃癌的发生,发展有明显关联,有助于判断胃癌预后。     

Detection of subtle reciprocal translocations by fluorescence in situ hybridization

Three different subtle reciprocal translocations were detected on long, well-banded chromosomes. The same translocations were examined using fluorescence in situ hybridization (FISH) with chromosome-specific libraries and unique DNA sequences. Our findings show that FISH allows rapid and unequivocal detection and characterization of this type of chromosome rearrangement. This approach is especially useful for prenatal diagnosis when one of the parents is a balanced carrier of such small fragment translocations.     

Cytogenetic and fluorescence in situ hybridization analysis of breast fibroadenomas.

We report cytogenetic and fluorescence in situ hybridization (FISH) analysis findings in 7 patients with breast fibroadenomas (FA). Three patients were cytogenetically abnormal. One patient had a translocation t(3;5)(p22;q13), the second had trisomy 8, and the third two clones, 47, XX, +11 and 47,XX, +10.     

Chromosomal alterations detected by fluorescence in situ hybridization in urothelial carcinoma and rarer histologic variants of bladder cancer

Fluorescence in situ hybridization (FISH) with the UroVysion probe set (Abbott Molecular, Des Plaines, IL) was used to assess 31 bladder cancers for chromosomal abnormalities, including 4 adenocarcinomas, 5 urachal adenocarcinomas, 6 small cell carcinomas, 7 squamous cell carcinomas, and 9 typical urothelial carcinomas. FISH was also used to assess the benign urothelium in 4 cases. There was a significant increase (P < .001) in the mean number of chromosome 3 (2.64 vs 1.51), chromosome 7 (2.61 vs 1.48), and chromosome 17 (2.41 vs 1.41) centromeric signals observed in cells from patients with cancer compared with patients without cancer. Of the 31 tumors, 29 (94%) demonstrated polysomic signal patterns in more than 10% of cells. In the 2 remaining tumor specimens, there was a high percentage of cells (>75%) demonstrating homozygous 9p21 deletion. The data from this study suggest that chromosomal abnormalities detectable by FISH in urothelial carcinoma are also common in rarer histologic variants of bladder cancer.     

Detection of numerical chromosome aberrations in bladder cancer by in situ hybridization.

The nuclear DNA content of 53 transitional cell carcinomas (TCCs) of the urinary bladder, as determined by flow cytometry (FCM), was compared with chromosome ploidy as detected by nonradioactive in situ hybridization (ISH). For this purpose, probes for repetitive DNA targets in the (peri) centromeric region of chromosomes 1 and 18 were used. Hybridization results with both probes of 35 TCCs, which had a DNA index of approximately 1.0 as concluded from FCM, showed evident chromosome 1 aberrations in approximately 25% of the tumors, and in a few cases an aberration for chromosome 18 was detected. Comparison of the ISH spot numbers for both chromosomes showed in most cases a higher number for chromosome 1 than for chromosome 18. ISH on 18 cases of TCCs, which showed a single peak in FCM with a DNA-index of 1.2 to 3.2, exhibited a profound heterogeneity. In these TCCs the ratio between chromosomes 1 and 18 varied over a wide range, resulting in cases showing more hybridization signals for chromosome 1 than for chromosome 18 or the opposite. Furthermore, using ISH minor cell populations showing polyploidization and giant cells containing numerous ISH signals could occasionally be detected. Results showed that interphase cytogenetics by ISH enable a fast screening of numerical chromosome aberrations and detection of different cell populations within one tumor, which was apparently homogeneous according to FCM.     

Numerical chromosome alterations in colorectal carcinomas detected by fluorescence in situ hybridization

This study concerns DNA ploidy, numerical changes of chromosomes 7, 8, 10, 17 and 18, and allelic losses at chromosomes 17p13.3 (flanking the p53 gene) and 18q21 (location of the DCC gene) in 31 freshly resected colorectal tumours. Cytological smears were used to determine DNA ploidy by image analysis, and chromosome numbers by fluorescence in situ hybridization (FISH) using chromosome-specific pericentromeric -satellite DNA probes. Allelic losses were assessed by Southern blotting and by the polymerase chain reaction loss of heterozygosity method. Approximately 50% of the tumours were aneuploid. There was heterogeneity with respect to chromosome numbers, but gains and losses of chromosomes, or both, were detected in all carcinomas examined, including 10 that were nonaneuploid by image analysis. Trisomy 7 was found in 74% of the tumours, and monosomy of chromosome 18 in 32%. Allelic loss at chromosome 17p13.3 was evident in 13 of 26 informative cases, and only one case exhibited monosomy 17. In comparison monosomy 18 was found in 10 cases; 7 of them corresponded to approximately half of the cases with allelic loss within the DCC gene, and the other three were noninformative. These findings indicate that the loss of one chromosome 18 is an important mechanism producing allelic deletion of the DCC gene in colorectal carcinomas. Our data also suggest that monosomy 18 is a useful indicator for studying colorectal cancer progression on a cell by cell basis.