A new method to extract nuclei from paraffin-embedded tissue to study lymphomas using interphase fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) is difficult to accomplish using thin-sections of paraffin-embedded lymphoid tissue because of the high cellularity and truncated cells that interfere with accurate scoring of individual nuclei. We modified and tested a new technique to isolate individual nuclei from tissue cores of paraffin-embedded tissue processed with xylene, proteinase K, citric acid, and pepsin. The efficacy of this method to study paraffin-embedded tissue was investigated in six normal lymph nodes or tonsils and 32 malignant lymphomas including five mantle cell, five follicular, five Burkitt, five extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue, five anaplastic large-cell, and seven diffuse large B-cell. Fusion of CCND1 and IgH, BCL2 and IgH, c-myc and IgH, and MALT1 and API2 were detected using probes with a dual-fusion FISH strategy. Anomalies involving ALK and BCL6 were detected using break-apart FISH probes. FISH studies were successful for each of the 38 specimens. Chromosome anomalies were detected in each malignant specimen, but not in the normal lymphoid tissue. The correct chromosome anomaly was detected in 22 of 22 specimens with genetic abnormalities that were established by other genetic techniques. This FISH technique is useful to detect chromosome anomalies with high sensitivity and specificity in paraffin-embedded tissue and may provide important diagnostic and prognostic genetic information.     

Interphase cytogenetic analysis of distinct X-chromosomal translocation breakpoints in synovial sarcoma

Synovial sarcomas show a specific translocation involving chromosomes X and 18, t(X;18)(p11.2;q11.2). Two distinct X-chromosomal breakpoints occur in different synovial sarcoma tumour samples. These breakpoints are located within two related genomic regions containing ornithine aminotransferase-like sequences, termed OATAL1 and OATL2. Preliminary observations indicated the potential correlation of OATL1-associated breakpoints with biphasic tumours and OATL2-associated breakpoints with monophasic fibrous tumours. The present study uses interphase cytogenetics to investigate the nature of chromosomal aberrations in frozen synovial sarcoma tissue samples. Two-colour fluorescence in situ hybridization (FISH) was performed using probes specific for the centromeres of chromosome X or 18, along with yeast artificial chromosome probes corresponding to the distinct breakpoint regions on Xp. One monophasic epithelial and two monophasic fibrous synovial sarcomas showed an OATL2-associated breakpoint, while a biphasic tumour revealed a hybridization pattern indicating a breakpoint within the OATL1 region. These results confirm our previous suggestion of a relationship between alternative breakpoints in Xp11.2 and different histological phenotypes observed in synovial sarcomas. They also demonstrate the utility of the two-colour hybridization approach for the identification of chromosomal changes in interphase nuclei isolated from frozen tissues.     

荧光原位杂交检测在产前诊断中的临床价值

目的探讨运用羊水间期细胞开展荧光原位杂交检测在产前诊断中的临床价值。方法运用FISH技术对110例孕16～24周孕妇的羊水间期细胞进行检测,每例均进行常规染色体核型分析。结果运用FISH法,所有样本均在19h内获得检测结果,除2例羊水培养失败外,所有样本均取得染色体核型分析报告结果。两种方法均检出特氏综合征、21-三体综合征各1例,所有样本的两种方法检测结果均互相符合。常规染色体核型分析有2例异常,因缺乏相应DNA探针。FISH法未能检出。结论运用羊水间期细胞开展荧光原位杂交检测,缩短了诊断时间,缓解了孕妇及家属因等待检测结果所产生的焦虑心情。检测程序简单,结果判定明确,在产前诊断实施过程中具有重要的临床价值。     

一种能精确检测人间期细胞核中21号染色体拷贝数的DNA探针

目的制备能精确检测人类间期细胞核中２１号染色体拷贝数的ＦＩＳＨ探针。方法利用万能引物ＰＣＲ法,从定位于人２１ｑ１１的ＹＡＣ克隆８８１Ｄ２分离制备ＤＮＡ探针,并用于与８例正常人和５例２１三体患者的外周血淋巴细胞中期相和间期核,及经细胞松弛素Ｂ（ｃｙｔｏｃｈａｌａｓｉｎＢ）诱导的双核细胞进行ＦＩＳＨ分析。结果该探针具有以下特点：（１）长度集中于３５０～７５０ｂｐ;（２）其靶序列位于２１号染色体长臂上且紧靠着丝粒;（３）特异性强;（４）杂交信号明亮,容易辨认;（５）对中期相及间期细胞核中２１号染色体的检出率分别高达９９．６０％和９８．４０％。结论制备的ＤＮＡ探针能精确检测人类间期细胞核中２１号染色体的拷贝数,且适用于细胞分裂时２１号染色体分离情况的研究     

应用荧光原位杂交检测人喉癌中EGFR基因扩增

目的 检测人喉癌Ｈｅｐ－２细胞系和５个喉癌组织中表皮生长因子受体（ｅｐｉｄｅｒｍａｌ ｇｒｏｗｔｈ ｆａｃｔｏｒ ｒｅｃｅｐｔｐｒ,ＥＧＦＲ）基因扩增。方法 荧光原位杂交技术。结果 在Ｈｅｐ－２细胞和２例喉癌组织中期染色体和间期细胞核中,检测到明显的集中成簇和多个斑点分散排布的杂效信号,另３例喉癌组织间期细胞核中杂交信号未见增强或数目增加。结论 以正常二倍休 色体和间期细胞核为对照,在喉癌Ｈｅｐ－     

实体瘤间期细胞制备技术对荧光原位杂交效果的影响

目的将改进的实体瘤间期细胞制备方法应用于荧光原位杂交,为实体瘤间期细胞的制备建立技术平台。方法采用随机引物法标记3号染色体着丝粒探针,分析实体瘤不同间期细胞制备技术对荧光原位杂交效果的影响。结果6种制片方法各具特点,根据其在应用方面有不同侧重,可采用不同制片方法。胶原酶法制片的荧光原位杂交效果最佳。对于冰冻组织或较小的组织,可采用印片法制片。结论适当的制片方法,结合荧光原位杂交,为实体瘤的染色体研究和诊断应用奠定了技术基础。     

Interphase FISH analysis on histologic sections of fixed tissues for t(11;14) (q13;q32) detection in mantle cell lymphoma and t(8;14)(q24;q32) in Burkitt's lymphoma

We describe an interphase FISH analysis for formol-fixed, paraffin-embedded tissue sections with commercial probes detecting the t(11;14)(q13;q32) in mantle cell lymphoma and the t(8;14)(q24;q32) in Burkitt's lymphoma. Staining of an adjacent section allowed identification of tumoral areas. The cut-off value was evaluated in reactive lymph nodes at 5% for both probes. An incomplete hybridization pattern was found in 18 to 26% of the nuclei but was always lower than the percentages of translocated cells in tumoral regions. A t(11;14) was detected in 4/4 mantle cell lymphomas and a t(8;14) in 2/3 Burkitt's lymphomas. Interphase FISH analysis of fixed-tissue sections is a reliable technique for the direct detection of lymphoma-associated translocations on routine histological material.     

Visualization of interphase chromosomes in postmitotic cells of the human brain by multicolour banding (MCB)

Molecular cytogenetics offers the unique possibility of investigating numerical and structural chromosomal aberrations in interphase nuclei of somatic cells. Previous fluorescence in-situ hybridization (FISH) investigations gave hints of numerical chromosomal imbalances in the human brain, present as low-level mosaicism. However, as precise identification of aneuploidy rates in somatic tissues faces major difficulties due to the limitations of FISH using whole chromosome painting or centromeric probes, in this study low-level mosaicism in the human brain was addressed for the first time using microdissection-based multicolour banding (MCB) probe sets. We demonstrated that MCB is suitable for this application and leads to more reliable results than the use of centromeric probes in parallel on the same samples. Autosomes and the active X chromosome appear as discrete metaphase chromosome-like structures, while the inactive X chromosome is condensed in more than 95% of interphase nuclei. The frequency of stochastic aneuploidy was found to be 0.2–0.5% (mean 0.35%) per autosome pair, 2% for the X chromosome in the female brain, and 0.4% in the male brain, giving a cumulative frequency of aneuploidy of approximately 10% in the adult brain. Moreover, MCB as well as multi-probe FISH using centromeric probes revealed associated signals in a large proportion of brain cells (10–40%). While co-localized signals could not be discriminated from numerical chromosome imbalances after FISH using centromeric probes, interphase MCB allows such differentiation. In summary, MCB is the only approach available at present that provides the possibility of characterizing the chromosomal integrity of arbitrary interphase cell populations. Thus, cytogenetics is no longer limited in its application to dividing cells, which is a great step forward for brain research.     

Detection by dual color fluorescence in situ hybridization of in vivo chromosome damage in rat hepatocyte nuclei

Our previous study using a multicolor fluorescence in situ hybridization (FISH) technique revealed that region-specific DNA probes for rat chromosome 1 enabled the detection of structural chromosome damage in rat interphase nuclei from peripheral blood and bone marrow cells. In the present study, this FISH technique was modified for application to a non-hematopoietic organ (liver) and the usefulness of the system was tested using diethylnitrosamine (DEN) as a model hepatocarcinogen. Male Sprague-Dawley rats were orally treated once with DEN at 200 mg/kg. Their livers were removed at 4, 7 or 14 days after treatment and homogenized with a tissue grinder to isolate hepatocyte nuclei. The nucleus suspension was fixed in methanol:acetic acid and air dried. Dual color FISH with two probes, one labeled with tetramethylrhodamine and one with digoxigenin, demonstrated that the maximum increase in the frequency of nuclei with spatially abnormal signals was observed 7 days after treatment. A dose-response relationship for induction of abnormal nuclei was observed. This improved dual color FISH system is potentially valuable for assessing in vivo clastogenicity in all rat organs.     

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.     

Complementarity of interphase and metaphase chromosome analysis in human renal tumors

Fluorescence in situ hybridization (FISH) was used as a complement to earlier cytogenetic studies of human renal tumors. Chromosome-specific para-centromeric probes were applied to cells disaggregated from tissue blocks of tumors, fresh samples from which had yielded cytogenetic results after short-term culture. Cells were dissociated from thick sections of paraffin-embedded, formalin-fixed tissues. Biotin-labeled probes specific for chromosomes 1, 3, 7, 11, 12, 15, 17, and the Y chromosome were applied in individual cases and were detected by fluorescence. Probes for chromosomes 1, 7, and 17 yielded clean signals with disomic control frequencies near 90%, and 97% of controls showed a single bright spot for the Y chromosome. The 9 cases selected all provided ample numbers of dissociated cells which were hybridized successfully, although some chromosomal signals were poor in individual cases. Abnormal copy numbers of chromosomes 1 and/or 17, not identified in culture, were observed in 7 cases. Trisomy 7 observed in culture was substantiated by FISH on the original tissues, as was loss of the Y, in each of 4 cases, respectively. Our results include limited validation of culture cytogenetics, evidence of selection in culture of tumor subpopulations, and demonstration that interphase cytogenetics by FISH is applicable to archival tissue blocks after prolonged periods of storage. Conditions of culture before harvest and inherent heterogeneity within tumors permit selection for nonrepresentative subgroups within tumors and emphasize the need for multiple approaches to evaluation. © 1993 Wiley-Liss, Inc.     

Generation of locus-specific probes for interphase fluorescence in situ hybridisation--application in Barrett's esophagus

Despite the wide range of probes commercially available for interphase fluorescence in situ hybridisation (FISH), the supply of locus-specific probes is limited to genes or chromosomal regions commonly altered in genetic diseases or during carcinogenesis. Generation of these probes is therefore desirable to accommodate individual research requirements. Hence, we detail the methodology required to design and produce custom locus-specific interphase FISH probes for any human genomic region of interest and their application was illustrated in cytogenetic investigations of Barrett's tumourigenesis. Previously utilising FISH, we observed that Barrett's tissues demonstrated chromosome 4 hyperploidy [Gut 52 (2003) 623], but as centromeric probes were used in this analysis, it was not known if the whole chromosome was amplified. We consequently generated single-copy sequence probes for the 4p16.3 and 4q35.1 subtelomeric loci. Multicolour FISH was subsequently performed on interphase preparations originating from patients with Barrett's esophagus at varying histological grades, thus demonstrating the whole region of chromosome 4 was amplified within the tissues. Additionally, probes for the DNA methyltransferase genes were produced to determine if gene dosage alterations were responsible for increasing methylation activity during Barrett's neoplastic progression. No significant alterations at the DNMT1 and DNMT3a loci were detected. An increased copy number of these genes is therefore not the basis for the hypermethylation commonly observed in this premalignant lesion.     

Chromosome 21 abnormalities with AML1 amplification in acute lymphoblastic leukemia

Fluorescence in situ hybridization (FISH) studies were performed in three cases of acute lymphoblastic leukemia (ALL) with marker chromosomes to analyze the contribution of chromosome 21 in these markers. FISH with a chromosome 21 painting probe confirmed that chromosome 21 was involved in all three cases. FISH with YAC probes showed that the number of extra copies varied according to their location on chromosome 21. Attention was focused on the AML1 gene, which was present as five copies in most of the cells exhibiting the marker chromosomes. As controls, 11 cases of childhood ALL were studied with PAC probes covering AML1. The results agreed with the banded karyotypes in 10 patients. FISH uncovered a clone with four copies of AML1 which were only observed by FISH analysis of interphase nuclei in one patient. No point mutation was detected in exons 3-5, encoding the runt domain of AML1, in the three cases, suggesting an oncogenic role of wild-type AML1 amplification.     

The applications of FISH in tumor pathology

The current FISH technology was greatly improved during the past 10 years. A large number of cosmids and yeast (YACs), bacterial (BACs), phage P1 derived (PACs) artificial chromosomes have been rapidly mapped and are useful as probes. In parallel, methods were established to specifically "paint" entire chromosomes or chromosome segments. Using these chromosome libraries as probes, complex rearrangements and marker chromosomes can be identified irrespective of their banding pattern. Ripetitive DNA probes specific for each chromosome centromere (alpha satellite sequences), are also available and may be used to identify specific aneuploidies. The use of sensitive digital imaging systems on the basis of "colour" rather than morphology increased the improvement of new FISH techniques. In particular, colour karyotyping results in the differential colour display of all human chromosomes. Another recent development of FISH technology is comparative genome hybridization (CGH), a genome-scanning technique that allows to identify and map chromosomal and subchromosomal gains and losses. FISH techniques may be used to investigate chromosome abnormalities not only on metaphasic chromosomes but also on interphasic nuclei. Any given tissue or cell source, such as sections of frozen tumors, imprinted cells, cultured cells, paraffin-embedded sections may be hybridized. The interphasic FISH may be extremely informative in tumor pathology even if the results are dependent on a good technical quality and adequate controls.     

Fluorescence in situ hybridization on formalin-fixed and paraffin-embedded tissue: optimizing the method.

Fluorescence in situ hybridization (FISH) is widely used to study numerical and structural genetic abnormalities in both metaphase and interphase cells. The technique is based on the hybridization of labeled probes to complementary sequences in the DNA or RNA of the cells. Interphase FISH is most often applied on cytologic material such as hematologic smears or imprints, but the method is also used to study genetic changes in tissue sections when morphology is important or when cytologic material is not available. In cases in which the presence of intact nuclei is of importance, such as quantitation of signals as in triploidy, it is possible to isolate nuclei from paraffin-embedded tissue. However, using formalin-fixed paraffin-embedded tissue, either in thin sections or as isolated nuclei, one encounters a range of technical problems, paralleling those met in immunohistochemistry. Variations in time lapse between removal of tissue and fixation, duration of fixation, enzymatic pretreatment, hybridization conditions, and posthybridization washing conditions are important factors in the hybridization. In this study, we have listed the results of a systematic approach to improve FISH on isolated nuclei and tissue sections from formalin-fixed, paraffin-embedded tissue.     

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.     

Interphase FISH detection of BCL2 rearrangement in follicular lymphoma using breakpoint-flanking probes

Rearrangement of the BCL2 gene is an important parameter for the differential diagnosis of non-Hodgkin lymphomas. Although a relatively large proportion of breakpoints is clustered, many are missed by standard PCR. A FISH assay is therefore desired. Up to now, a lack of probes flanking the BCL2 gene has limited the possibilities for a FISH assay to an approach based on colocalization of probes for BCL2 and the immunoglobulin heavy chain (IGH) locus. Intrinsically high rates of false positive nuclei and high interobserver variability make such assays unsuitable for use on lymphoma tissue samples, where tumor cells often form only a minority of the cell population. Using YAC end cloning techniques and screening of a PAC library, we have isolated PAC clones flanking the BCL2 gene. Using these PACs, and several cosmid clones in the second BCL2 intron, we developed a segregation-based interphase FISH assay with two probe combinations enabling separate detection of 5' and 3' (mbr/mcr) breakpoints. The assay was applied to a series of 40 follicular lymphomas. To evaluate the results, the same lymphomas were analyzed by DNA fiber FISH with a 600-kb set of BCL2 DNA clones labeled in alternating colors in combination with a color barcode covering the IGH locus. This approach allowed precise mapping of BCL2 breakpoints, and simultaneously showed juxtaposition of IGH genes to BCL2. Comparison of the results of interphase and fiber FISH showed complete correlation. Five cases were negative with both FISH techniques as well as with Southern blotting. Interestingly, all of these 5 cases lacked BCL2 overexpression as determined by immunohistochemistry, against 3 of 35 rearrangement-positive follicular lymphomas. Furthermore, absence of t(14;18) seemed to be correlated with a higher histologic grade (grades 2 and 3 according to Berard). These data indicate that the segregation-based interphase FISH assay detects 100% of BCL2 rearrangements. Because interpretation of the results is straightforward and requires no extensive experience, this assay may be the best available diagnostic test for BCL2 rearrangement. Genes Chromosomes Cancer 27:85-94, 2000.     

应用荧光原位杂交产前诊断未培养羊水细胞非整倍体

目的探讨荧光原位杂交(fluorescenceinsituhybridization,FISH)诊断未培养羊水细胞非整倍体的临床应用价值。方法对55例孕16～32周未培养羊水细胞进行FISH快速产前诊断,应用多色FISH对另4条染色体(X、Y、13号和18号)进行检测。以经母腹穿刺取胎血常规核型分析作为FISH检测结果对照。结果被检55例羊水未培养细胞均获得诊断结果,发现两例异常胎儿。1例为标准型21三体;另1例为21三体嵌合体。FISH检测与常规核型分析结果一致。结论FISH检测未培养羊水细胞非整倍体具有快速、简便、所用样本量少的优势,结果准确可靠,可达到产前诊断要求,有较大临床应用价值。     

Detection of chromosome 11q13 breakpoints by interphase fluorescence in situ hybridization. A useful ancillary method for the diagnosis of mantle cell lymphoma

We assessed cytologic specimens from 11 mantle cell lymphomas (MCLs) and 32 other B-cell non-Hodgkin lymphomas (NHLs) for 11q13 breakpoints using a 2-color fluorescence in situ hybridization (FISH) assay that uses an 11q13 probe centered on the CCND1 gene and a centromeric chromosome 11 probe (CEP11). The number of nuclei in 200 cells were counted, and results were expressed as an 11q13/CEP11 ratio. All MCLs showed a high percentage of interphase nuclei with 3 or more 11q13 signals (mean, 74.8%; range 57%-90%). In contrast, in other B-cell NHLs the mean percentage of cells with 3 or more 11q13 signals was 9.2%. All MCLs had an elevated 11q13/CEP11 ratio (mean, 1.38). The mean ratio for other B-cell NHLs was 0.99. Two non-MCL cases, 1 large B-cell and 1 B-cell unclassified NHL, had high 11q13/CEP11 ratios of 1.15 and 1.30, respectively. Conventional cytogenetic analysis performed on the former case revealed a t(5;11)(q31;q13). Interphase FISH analysis using 11q13 and CEP11 probes is a convenient ancillary method for assisting in the diagnosis of MCL. This commercially available assay is simple to use on cytology or imprint specimens, and results can be obtained within 24 hours.