Determination of DNA ploidy by fluorescence in situ hybridization (FISH) in hydatidiform moles: evaluation of FISH on isolated nuclei

In the past 20 years, the diagnosis of hydatidiform moles has become more difficult because of the widespread use of early uterine evacuation. Differentiating hydropic degeneration, partial, and complete moles is important because of their different prognosis. However, clinical diagnosis is less obvious, and the pathologist has to separate the different entities on the basis of very subtle morphologic criteria. In difficult cases, ploidy may be determined by various methods, including fluorescence in situ hybridization (FISH) on routine histological sections from paraffin-embedded specimens. However, FISH analysis is often difficult because of the presence of numerous truncated nuclei. In this context, we have tested the advantages of FISH on isolated nuclei, a well-known variant of the technique that might be more sensitive. We reviewed 24 cases of products of abortion: hydropic degenerations, complete hydatidiform moles, partial moles, and nonmolar triploidies. After histological review, FISH on isolated nuclei proved conclusive in all cases. The results could be easily interpreted thanks to the reduced number of truncated nuclei. The percentage of cells with 2 signals was always >70% in the diploid cases and >60% in the triploid cases. In conclusion, this sensitive technique seems to be a valuable tool for the diagnosis of moles.     

Chromogenic in situ hybridization (CISH) to detect HER2 gene amplification in breast and gastric cancer: Comparison with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH)

The chromogenic in situ hybridization (CISH) assay, designed to detect the amplification of the HER2 gene in formalin‐fixed, paraffin‐embedded (FFPE) breast cancer (BC) and gastric cancer (GC) tissue specimens, was evaluated in 125 FFPE BC cases and 198 FFPE GC cases for which the HER2 status had been predetermined using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the 125 BC cases and the 198 gastric cases, we found a very good concordance (98.4% and 99.0%, respectively) between CISH and FISH. In particular, we evaluated the polysomy cases, as these cases often have ambiguous treatment options in clinical practice. The polysomy of chromosome 17 was defined as the presence of three or more CEP17 signals in at least 10% of the tumor cells. In the 50 BC cases and 54 GC cases displaying chromosome 17 polysomy, the concordance between FISH and CISH was 98.0% and 98.1%, respectively. These results indicate that CISH could provide an accurate and practical alternative to FISH for the clinical diagnosis of HER2 gene amplification in FFPE BC and FFPE GC samples.     

Numerical aberrations of chromosomes 1 and 7 in renal cell carcinomas as detected by interphase cytogenetics

Alcohol-fixed single cell suspensions of 37 renal cell carcinomas (RCCs) were assessed by both flow cytometry (FCM) and the fluorescence in situ hybridization (FISH) technique, using chromosome 1- and chromosome 7-specific centromere DNA probes. DNA diploidy or near-diploidy was observed in 30 of the 37 RCCs and only 12 of these (near-)diploid tumours were disomic for both chromosomes 1 and 7. Numerical aberrations of chromosome 1 and/or chromosome 7 were present in 18 of the 30 (near-)diploid RCCs and five of these cases showed monosomy for chromosome 1 in more than 50 per cent of the tumour cells. A double target FISH, with a centromeric and a telomeric specific probe for 1p36, excluded misinterpretation on the basis of clustering of 1q12, and suggested a complete loss of chromosome 1. All these five (near-)diploid RCCs with monosomy for chromosome 1 were eosinophilic chromophilic cell carcinomas, according to the Thoenes classification of RCC. This observation is of special interest, because it was recently concluded from cytogenetic studies that the diagnosis of chromophilic renal cell carcinoma must be considered as obsolete. Monosomy for chromosome 1 seems to be a non-random numerical aberration of (near-)diploid eosinophilic chromophilic cell carcinomas, and a gain of one or more chromosomes 1 appeared to be a common phenomenon in RCCs, especially in the DNA aneuploid tumours. As these chromosomal abnormalities were not found in the earlier classical cytogenetic studies, we conclude that in situ hybridization techniques are required in addition to chromosome banding techniques to obtain a complete characterization of the chromosome imbalances in RCCs.     

Assessment of a HER2 scoring system for gastric cancer: results from a validation study

Aims:  Human epidermal growth factor receptor 2 (HER2) overexpression/amplification is implicated in the development of various solid tumour types. Validated methods and scoring systems for evaluating HER2 status exist in breast cancer, but not in gastric cancer. The aim was to establish a HER2 scoring system for gastric cancer to identify suitable patients for enrolment in a trial of trastuzumab (Herceptin^®) in advanced metastatic gastric cancer.
Methods and results:  Formalin-fixed paraffin-embedded gastric cancer samples were tested for HER2 status using the fluorescence in situ hybridization (FISH) pharmDx™ kit (Dako Denmark A/S). Immunohistochemistry (IHC) was performed using the HercepTest™ (Dako). Concordance between FISH and IHC was 93.5% in 168 evaluable samples. Eleven samples were scored as FISH+ but IHC− or equivocal.
Conclusions:  IHC/FISH discrepancies were attributed to basolateral membranous immunoreactivity of glandular cells resulting in incomplete membranous reactivity and/or a higher rate of tumour heterogeneity in gastric cancer compared with breast cancer. With modifications to the IHC scoring system, the HercepTest™ is considered valid for the identification of HER2+ gastric tumours for this clinical trial. Correlation of HER2 scores with clinical outcomes will be needed to determine which patients might benefit from trastuzumab therapy.     

应用荧光原位杂交技术检测卵巢浆液性囊腺癌中ZNF217基因拷贝数

目的探讨锌指蛋白217（zinc finger protein 217，ZNF217）基因在卵巢浆液性囊腺癌中20号染色体基因拷贝数改变情况及其临床意义。方法应用荧光原位杂交技术及LSI ZNF217探针对2种卵巢癌细胞株SKOV3、HO-8910和23例卵巢浆液性囊腺癌、10例浆液性囊腺瘤及7份正常卵巢组织进行检测。结果两种卵巢癌细胞株及12例卵巢癌出现ZNF217基因扩增，浆液性囊腺瘤有1例发生了基因的扩增，其余的及正常卵巢组织未出现基因扩增，卵巢癌与卵巢浆液性囊腺瘤、正常卵巢细胞相比较，ZNF217基因拷贝数改变差异有统计学意义（P〈0．05），而低分化的卵巢癌比高分化发生ZNF217基因扩增的几率明显增高（P〈0．05），Ⅲ～Ⅳ期比Ⅰ期的卵巢癌ZNF217基因拷贝数明显增多（P〈0．05）。结论ZNF217基因很可能是卵巢癌发生的促进因子之一，并与卵巢癌分化及预后不良有关。     

Application of the crypt isolation technique to the flow cytometric analysis of DNA content in gastric carcinoma

An inevitable limitation of conventional flow cytometric analysis of gastric cancer DNA content is that the preparations of tumor cell nuclei are contaminated with stromal cell nuclei. Using the crypt isolation technique, we separated tumor tissues from stromal tissues and analyzed the DNA content in samples of pure gastric cancer cells (64 intestinal-type and 46 diffuse-type) by flow cytometry. Morphologically, crypts from well-differentiated and moderately differentiated adenocarcinomas usually showed large tube-like or sheet structures, whereas tumor tissues isolated from poorly differentiated adenocarcinomas usually exhibited small tumor cell clumps or clusters of varying sizes. Tumor ploidy was divided into diploid, aneuploid, and multiploid subgroups. Aneuploidy and multiploidy were observed in 12% (13 of 110) and 64% (71 of 110) of gastric cancers, respectively. A high frequency of DNA aneuploidy or multiploidy was associated with intestinal-type tumors, but not with any of the other clinicopathologic variables tested. In contrast, high S-phase fraction values demonstrated a close association with tumors with abnormal ploidy, advanced stage, intestinal type, and late TNM stage. Our results suggest that S-phase fraction may be a more useful indicator of aggressive behavior in gastric cancers than DNA aneuploidy. To our knowledge, the present study is the first to report flow cytometric DNA content in a large number of gastric cancer samples obtained using the crypt isolation technique.     

Metastatic mode of gastric carcinoma by flow cytometric and clinicopathologic parameters

We used flow cytometric and conventional clinicopathologic parameters to analyse the metastatic mode of cancer in 113 stomach cancer patients. Liver metastasis was frequent in cases with intestinal-type cancer, cancer located in the distal stomach, positive venous invasion and aneuploid cancer. Lung and pleural metastasis (excluding nodular lung metastasis), however, were frequent in cases with serosal invasion and diploid cancer. Peritoneal metastasis was frequently seen with tumors located in the proximal or whole stomach, diffuse-type cancer and cancer with serosal invasion. All cases developing bone metastasis were positive for lymph node metastasis. DNA ploidy was partially related to the metastatic mode of stomach cancer, but this was not the sole parameter for predicting metastasis. However, prediction may be possible if based on both DNA ploidy and clinicopathologic parameters, suggesting the possibility of the prophylaxis of recurrence by appropriate postoperative adjuvant therapy.     

胃癌细胞DNA含量和倍体分析的研究进展

利用流式细胞分析仪和图像细胞分析仪检测胃癌细胞核中DNA含量和倍体已成为病理诊断中的重要手段，DNA含量和倍体分析可用于胃癌前病变及早期胃癌的诊断，与胃癌的临床病理有密切的联系，可用于患者治疗效果的评估和预后的估计。     

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.     

Interphase cytogenetics of gastric carcinoma: Fluorescence in situ hybridization (FISH) applied to cells obtained from formalin-fixed paraffin-embedded tissues

The interphase cytogenetics in formalin-fixed and paraffin-embedded gastric cancer tissues were examined by fluorescence in sku hybridization (FISH) with α-satellite DNA probes. Two gastric carcinoma cell lines, TMK-1 and MKN-28, were first analyzed cytogenetically. Of 25 TMK-1 cell karyotypes, chromosome 7 showed trisomy and chromosome 17 showed disomy in 18 cells. Most MKN-28 cells showed disomy of both chromosomes 7 and 17. Suspensions of singly isolated TMK-1 and MKN-7 cells were obtained from the cultured cells, and from paraffinembedded tissue specimens fixed with formalin for 0, 1, 3 and 5 days obtained from xenotrans-planted tumors in nude mice. The numbers of chromosomes 7 and 17 analyzed with the karyotypic preparations coincided well with those determined by FISH, even in the paraffin-embedded specimens. The number of tumor cells showing no signals, however, increased in the specimens after 5 days formalin fixation. In 10 surgically removed gastric carcinomas, the predominant signal number for chromosomes 7 and 17 in the cells of paraffinembedded tissues was two (disomy), except in one papillary carcinoma, which was trisomic for chromosome 7. Large subpopulations (more than 20%) showing trisomy were found in four cases for chromosome 7 and in five cases for chromosome 17. A higher frequency of trisomy was found in well differentiated than in poorly differentiated carcinomas. These findings suggest that the FISH technique is a useful tool for detecting chromosomal aberrations in gastric adenocarcinoma cells, even in paraffinembedded specimens, as long as the tissues are fixed with formalin for an appropriate time.     

胃癌中HER-2表达异质性的病理学分析

目的检测胃癌组织中HER-2蛋白表达水平和基因扩增情况,定量分析HER-2表达和分布特征的异质性,探讨其在判读中的影响。方法应用免疫组化检测并观察胃癌组织中HER-2蛋白表达水平及分布特征,应用荧光原位杂交(fluorescence in situ hybridization,FISH)技术检测HER-2基因扩增情况,并运用欧式距离等进行统计学处理。结果 373例胃癌手术切除标本中HER-2蛋白阳性率(3+)为12.33%,与基因扩增情况显著相关(P0.001)。98例有HER-2蛋白表达的标本总异质性、组内异质性及组间异质性大小差异均有统计学意义(P值分别为0.025、0.001、0.040);组内异质性大小明显低于组间异质性。根据标准判读为HER-2蛋白为2+及3+的病例异质性最大,1+病例最小。HER-2蛋白表达分布较均一的病例近乎为零,而同时具有阴阳脸及花斑样特征的病例最多。根据标准判读HER-2蛋白为0的病例中,有7例具有明显异质性。结论胃癌组织中HER-2蛋白表达的异质性较大且类型多样,判读时首先要增加观察视野,选择至少2个组织块进行HER-2蛋白检测;其次无论HER-2蛋白表达水平高低,均需同时检测基因扩增情况,以确保判读结果的准确性,更好的指导临床用药。     

Duplication Xp22.2 and pseudoisodicentric Yq detected by FISH and PCR in a sterile male

A chromosome mosaicism with two cell lines was diagnosed in a sterile man. One cell line had a 45,—Y, dup (X) (p22.2) karyotype and accounted for 83% of lymphocytes analyzed. Fluorescence in situ hybridization (FISH) analysis with specific X and Y probes excluded a translocation between the short arms of the X and Y chromosomes and showed that Xp duplication involved a region containing the DXS85 locus, distal to the ZFX and DSS sites. The other cell line consisted of a diploid karyotype with a rearranged Y chromosome, which was shown to be a pseudoisodicentric Yq by FISH. Moreover, FISH with a specific probe for the AZF locus and polymerase chain reaction using Yq SY108 and SY121 primers showed no signals for this region, possibly accounting for the azoospermia in this patient.     

DNA aneuploidy in prostatic adenocarcinoma: A frequent event as shown by fluorescence in situ DNA hybridization

Fluorescence in situ hybridization (FISH) using specific DNA probes for chromosomes 1, 7, 10, and Y was performed on 53 prostatic tissue samples obtained from 33 radical prostatectomy specimens and two benign control specimens. The 53 samples from carcinomatous prostates included 33 cancerous and 20 noncancerous samples. Additionally, four metastatic lymph node specimens were examined. Clonal chromosome abnormalities were observed in 78% of the tumors studied. They were detected in a higher proportion in stage pT2 and pT3 tumors (86% and 88%, respectively) compared with stage pT1 tumors (25%). No stage pT4 tumor was analyzed. There was evidence of remarkable focal intratumoral heterogeneity documented by the study of two samples from the same tumor in three of six cases. Comparing FISH determined ploidy patterns with DNA flow cytometry (FCM) in 22 samples, FISH showed aneuploidy whereas FCM showed none.     

FISH技术检测TERC基因在子宫颈上皮内瘤变1级与2级鉴别诊断中的实用价值

目的：采用FISH技术检测TERC基因在子宫颈上皮内瘤变( cervical intraepithelial neoplasia, CIN)不能明确1级或2级(CIN1/2)病变组织中的扩增,探讨其在CIN分级中的临床病理实用意义。方法选取子宫颈组织标本42例,其中正常子宫颈鳞状上皮20例,CIN1/2级22例(其中初步诊断1级8例,2级14例)。采用FISH技术检测石蜡包埋组织中TERC基因的扩增情况。结果正常子宫颈鳞状上皮中TERC基因无扩增,CIN1/2级组织中TERC基因的扩增率为22．7%(5/22),77．3%(17/22)的TERC基因无扩增;其中初步诊断1级8例,1例TERC基因扩增;初步诊断2级14例,4例TERC基因扩增,差异有统计学意义(P<0．05)。结论在石蜡包埋组织切片难以确定CIN1/2级时,应用FISH技术进行TERC基因检测,TERC基因扩增者可考虑为CIN2级,建议临床积极处理;无扩增者,考虑为CIN1级,可保守处理;FISH技术具有临床病理实用价值。     

Marker chromosome 21 identified by microdissection and FISH

A child without Down syndrome but with developmental delay, short stature, and autistic behavior was found to be mosaic 46,XX/47,XX,+mar(21) de novo. The marker was a small ring or dot-like chromosome. Microdissection of the marker was performed. The dissected fragments were biotinylated with sequence-independent PCR as a probe pool for fluorescence in situ hybridization (FISH). FISH results suggested an acrocentric origin of the marker. Subsequent FISH with α-satellite DNA probes for acrocentric chromosomes, and chromosome-specific 21 and 22 painting probes confirmed its origin from chromosome 21. © 1995 Wiley-Liss, Inc.     

TERC基因检测在不同级别宫颈病变中的诊断价值

目的检测不同级别宫颈病变中TERC基因的表达情况,探索其在不同宫颈病变中的诊断价值。方法采用荧光原位杂交（fluorescence in situ hybridization,FISH）技术检测20例正常对照和100例患者（CINⅠ14例、CINⅡ35例、CINⅢ36例、宫颈癌15例）的宫颈脱落上皮细胞TERC基因的表达情况。并以组织病理学结果做对照。结果随宫颈病变级别增加,TERC基因扩增的阳性率、异常细胞数和异常核型的复杂性均显著增加。宫颈癌／CINⅢ该基因扩增阳性率显著高于CINⅡ／Ⅰ及正常对照（P〈0．01）,CINⅡ病变者显著高于CINⅠ者（P〈0．01）。结论TERC基因扩增与宫颈病变的发展关系密切,FISH检测TERC基因扩增在不同级别宫颈病变的诊断中有重要意义。     

Detection of the Epstein-Barr virus in primary gastric lymphoma by in situ hybridization

The Epstein-Barr virus (EBV) has been shown to be associated with numerous human malignancies including Burktt's lymphoma and nasopharyngeal lymphoepithelioma. In addition, some typical gastric adenocarcinomas were also recently reported to demonstrate EBV relevance. The present study was designed to detect EBV in primary gastric lymphoma, using the in situ hybridization (ISH) method, in which oligo-nucleotide probes for the EBERl RNA and the EBV DNA W region have been used. Of the 49 cases of primary gastric lymphoma studied, which all showed B cell immunopheno-type, EBER1 sequences could only be found in four cases, including two low-grade cases and two high-grade cases of histological subtypes while the number of positive cells was less than 50% of the tumor cells. In one case of low-grade mucosa associated lymphoid tissue (MALT) lymphoma, the EBER1 -positive neoplastic cells were found in the regional lymph node, but the primary site of the stomach showed no positive signals. The EBV presence was further confirmed by the EBV DNA ISH. Using the ISH method, rare or occasional positive lymphoid cells (probably non-tumorous bystander cells) could be detected in 10 other cases including all histological subtypes. The present study shows that only a small proportion of primary gastric lymphoma is associated with EBV, and such positive cases could be found in both high- and low-grade histological subtypes. It is also suggested that the EBV presence in the neoplastic cells of some cases of primary gastric lymphoma is most likely a secondary phenomenon.