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.     

EBV in situ hybridization study for non-Hodgkin's lymphomas.

Epstein-Barr virus(EBV) has been implicated in the pathogenesis of B-lymphoproliferative disorders, T-cell lymphomas and Hodgkin''s disease. In this report, we performed an in situ hybridization study on EBV genome in 10 cases of nasal non-Hodgkin''s lymphoma(NHL), 20 cases of Waldeyer''s ring(WR) NHL, and 20 cases of nodal NHLs to document EBV association with lymphomas in Koreans. For immunophenotyping, monoclonal antibodies for CD 20, MB 2, CD 45Ro & CD 43 were used. For in situ hybridization study, EBV DNA probe for Bam HI ''V'' fragment and EBV RNA probe for EBER and BHLF were used. Twenty two cases(44%) of malignant lymphomas were positive for EBV genome. Generally, T-cell lymphomas showed a higher positive rate(61%) than B-cell lymphomas(24%). Among T-cell lymphomas, nasal lymphomas showed a higher positive rate(80%) than WR(50%) or nodal lymphomas(50%). Of 22 EBV genome positive cases, 10 cases were positive for EBER, 10 cases for BHLF, and 2 cases for both EBER and BHLF. The histologic types by Working Formulation(WF) were not correlated with EBV genome positive rate, whereas lymphomas showing the histologic spectrum of polymorphic reticulosis(PR) showed a higher positive rate(65%) than lymphomas without PR-like features(40%). These results indicate that nasal T-cell lymphomas with the histologic spectrum of PR are strongly associated with EBV and that the anatomic site may be an important factor in this association.     

Detection of chromosome aberrations in interphase nuclei using fluorescence in situ hybridization technique.

We report here several experiences of interphase cytogenetics, using fluorescence in situ hybridization (FISH) technique, for the detection of chromosome aberrations. FISH, using alpha satellite specific probes of 18, X, Y chromosomes, was done in interphase nuclei from peripheral blood of patients with Edwards'' syndrome, Klinefelter''s syndrome and Turner''s syndrome with healthy male and female controls, respectively. The distributions of fluorescent signals in 100 interphase nuclei were well correlated with metaphase findings. Nowadays FISH plays an increasingly important role in a variety of research areas, including cytogenetics, prenatal diagnosis, tumor biology, gene amplification and gene mapping.     

Detection of three common translocation breakpoints in non-Hodgkin's lymphomas by fluorescence in situ hybridization on routine paraffin-embedded tissue sections

The receptor for advanced glycation end-products (RAGE) is a newly recognized factor regulating cancer cell invasion and metastasis. This study investigated the expression of RAGE in gastric carcinomas and its association with invasion and metastasis. Of eight gastric cancer cell lines examined, seven constitutively expressed RAGE messenger ribonucleic acid (mRNA), MKN45 being the exception. RAGE protein expression of MKN28 cells treated with RAGE antisense S-oligodeoxynucleotide was nine times less than that of sense S-oligodeoxynucleotide-treated cells. Growth of cells under RAGE antisense S-oligodeoxynucleotide treatment was not different from that seen under sense S-oligodeoxynucleotide treatment in MKN28 (a cell line producing high levels of RAGE) and MKN45 (a non-RAGE-expressing cell line). RAGE antisense S-oligodeoxynucleotide treatment suppressed the invasive activity of RAGE-positive MKN28 cells, as estimated by in vitro invasion assay. The number of MKN28 cells invading the type IV collagen-coated membrane under RAGE antisense S-oligodeoxynucleotide treatment was significantly lower than under RAGE sense S-oligodeoxynucleotide treatment (p<0.0001). In contrast, antisense and sense S-oligodeoxynucleotide-treated RAGE-negative MKN45 cells showed no difference. A wound-healing assay showed that no RAGE antisense S-oligodeoxynucleotide-treated MKN28 cells migrated into the scraped area, whereas sense S-oligodeoxynucleotide-treated cells showed many budding nests in the scraped area. Immunohistochemistry of gastric carcinoma tissue showed that 62 (65%) of the 96 cases examined were RAGE-positive and that poorly differentiated adenocarcinomas preferentially expressed RAGE protein (38/42, 90%) (p<0.0001). Strong RAGE immunoreactivity was also correlated with depth of invasion and lymph node metastasis (p<0.0001). RAGE-positive cancer cells tended to be distributed at the invasive front of primary tumours and were detected in all metastatic foci in lymph nodes. In contrast, a major RAGE ligand, amphoterin, was expressed in 82 (85%) of the 96 cases, regardless of histological type and disease progression. RAGE expression appears to be closely associated with invasion and metastasis in gastric cancer.     

应用间期荧光原位杂交技术进行快速产前诊断

选用１８、Ｘ着丝粒探针及２１号染色体特异重复序列探针分别对未培养的羊水和绒毛细胞进行了间期荧光原位杂交。结果表明，常染色体探针出现３个及１个信号点的比例约为１％和６％。Ｘ染色体探针出现３点的比例也在１％左右，可为常染色体单体或三体以及Ｘ三体或多体提供诊断依据。同传统的细胞遗传学方法比较，间期荧光原位杂交具有简便、迅速、灵敏等优点，有一定临床应用价值     

Paraffin section interphase fluorescence in situ hybridization in the diagnosis and classification of non-hodgkin lymphomas.

Cytogenetic data can contribute valuable information that may assist in the diagnosis and classification of non-Hodgkin lymphomas and may in some cases also provide prognostic information. Interphase fluorescence in situ hybridization (FISH) studies offer the ability to assess for characteristic cytogenetic abnormalities even when material for standard metaphase cytogenetic analysis is not available. This review discusses the use of FISH in paraffin-embedded material with particular attention paid to the use of intact thin paraffin sections. The basic principles of FISH analysis are summarized, the advantages and disadvantages of analysis of thin paraffin sections rather than intact nuclei are discussed, and the more commonly encountered artifacts are considered. Each of the well-characterized cytogenetic abnormalities that are associated with particular types of non-Hodgkin lymphoma and can be detected with commercially available FISH probes is discussed individually. In particular, their incidence in various types of lymphoma is reviewed, the types of commercially available FISH probes to detect such abnormalities are discussed, and clinical situations where such analysis can be of diagnostic utility are described.     

Detection of numerical chromosome anomalies in interphase cells of ovarian carcinomas using fluorescence in situ hybridization

Fluorescence in situ hybridization was used in interphase cells of 30 ovarian carcinomas to detect numerical changes in copy number of 13 different centromeres (1, 2, 3, 4, 6, 7, 8, 10, 11, 12, 17, 18, and X). Thirty-seven percent of samples (11/30) were near diploid and demonstrated only minor changes in centromere copy number, involving gain and/or loss of one or a few centromeres. The most common changes included loss of centromeres 4, 6, 17, and 18 and gain of centromere 1. The remaining 63% of samples were hyperdiploid and demonstrated a general increase in copy number of most or all centromeres examined. Among these samples, the centromere of chromosome 1 was most often found to be at higher copy number. Centromeres that were less often at increased copy or deleted within the hyperdiploid samples include centromeres 4, 17, 18, and X. These results suggest that tumor-suppressor genes that are located on chromosomes 4, 6, 17, and 18 may be involved in the development and progression of ovarian cancer. Genes Chromosom Cancer 16:120–129 (1996). © 1996 Wiley-Liss, Inc.     

Use of interphase fluorescence in situ hybridization as a powerful diagnostic tool in cytology.

Interphase fluorescence in situ hybridization (I-FISH) using labeled nucleic acid probes detects chromosomal and genetic aberrations at a cellular level. I-FISH is a relatively fast and sensitive technique for evaluating a large number of cells and revealing more specific information than other techniques. It has been proven to be an invaluable molecular test in cytologic analyses for the detection of subtle genetic alterations that correlate with disease progression. In this postgenomic era, with the draft of the human genome available and expansion of the knowledge of tumor-specific genetic changes, the application of I-FISH probes in cytologic analysis should be of great value in the early detection, risk assessment, and monitoring of therapy efficacy in cancer. Here, we outline the principle of the I-FISH procedure, present suggestions to efficiently analyze cytologic materials, provide examples of practical applications, and discuss new aspects of the technique.     

Chromosomal gains and losses are uncommon in hairy cell leukemia: a study based on comparative genomic hybridization and interphase fluorescence in situ hybridization.

In contrast to other subtypes of lymphoproliferative malignancies, the genetic mechanisms underlying the pathogenesis of hairy cell leukemia (HCL) are unknown. We studied densely infiltrated splenic tissue of 14 cases of HCL for the presence of chromosomal gains and losses by comparative genomic hybridization (CGH). Chromosomal imbalances were detected in only four of the 14 cases. Chromosomal gains involved the regions 5q13-q31 (two cases) and 1p32-p36.2 (one case). A loss of the region 11q14-q22 was found in one additional patient. The imbalances affecting the regions 5q and 11q were confirmed by interphase fluorescence in situ hybridization (FISH) using PAC clone 144G9 (5q31) and YAC clones 755B11 (11q22.3-q23.1) and 801E11 (11q22.3-q23.1 spanning the ATM gene) and occurred in 61% to 75% of analyzed nuclei. The latter DNA probes and probes hybridizing to chromosomal regions, which are frequently deleted in other subtypes of non-Hodgkin lymphomas (NHL), namely 9p21/ P16(INK4A), 13q14/D13S25, and 17p13/P53 were subsequently applied to all 14 cases of HCL, but no additional abnormalities were found. We conclude that overrepresentation of chromosome 5 represents a recurrent aberration in HCL and that the commonly overrepresented region resides in 5q13-q31. Chromosomal imbalances including deletions of the tumor suppressor gene loci 9p21/P16(INK4A), 13q14/D13S25, and 17p13/P53 rarely occur in HCL in contrast to some other subtypes of B-cell NHL. The pathogenetic role of 11q/ATM alterations in HCL remains to be determined.     

Detection of karyotype changes in interphase cells: oligonucleotide-primed in situ labelling versus fluorescence in situ hybridization

Interphase cytogenetics is a rapidly developing technique which is usually performed by fluorescence in situ hybridization (FISH). Recently, oligonucleotide-primed in situ synthesis (PRINS) has become established as a method of labelling centromeric regions of chromosomes in metaphase spreads. We tested the suitability of PRINS in detecting the exact copy number of chromosomes 1, 3, 7 and 8 in intact interphase cells of 17 cytological preparations derived from normal and neoplastic tissues. Control procedures consisted in preparation of metaphase spreads of lymphocytes of healthy donors, conventional cytogenetics in some of the specimens, and omission of the primers or Taq polymerase from the reaction mixture. All specimens were additionally examined by FISH and analysed blind by two experienced observers. Both PRINS and FISH revealed a corresponding distribution of hybridization signals for all chromosomes examined in specimens of normal bone marrow (n = 5), normal liver cells (n = 5), three samples of acute nonlymphocytic leukaemia in which conventional chromosome analyses had shown monosomy 7 or trisomy 8, and in four hepatocellular carcinomas that displayed trisomy 1. Overall, statistical analysis revealed no significant difference in the signal distribution between the two techniques. Our results demonstrate that PRINS is as reliable as FISH for detecting chromosome copy numbers in interphase nuclei of intact cells. The PRINS method, however, is easier to perform, faster and less expensive, holding great potential for future applications in diagnostic pathology.     

Chromosomal changes in prostate cancer: a fluorescence in situ hybridization study

The incidence of prostate cancer (PC) is increasing steadily with the aging population in Singapore. As the pattern of chromosomal aberrations in Asian men with PC is poorly understood, we investigated the numerical aberrations for chromosomes 7, 8, 11, and 17 by fluorescence in situ hybridization (FISH). FISH was performed on standard sections and tissue microarrays of 54 PC and 33 benign prostatic hyperplasia (BPH) specimens. Among the 54 PC specimens, FISH detected 44 cases as aneusomy and two as disomy and was unsuccessful for eight cases. Cytogenetic alterations of two or more chromosomes per tumor were detected in 33/46 (72%) PCs. The most frequent alteration was aneusomy of chromosome 8 detected in 34/46 (74%) cases followed by numerical aberrations in chromosome 7 (61%). Gain of 8q24, loss of chromosome 7, and gain of 11q13 were associated with higher Gleason score and were statistically significant. Gain of chromosome 7 was more common in locally advanced disease, while gain of chromosome 11q13 and chromosome 7 was more common in metastatic disease.     

Confirmation of a cryptic unbalanced translocation using whole chromosome fluorescence in situ hybridization.

We report on a 7-year-old boy with minor anomalies, growth retardation, and developmental delay with an initial 46,XY,der(18)t(18;?)(q23;?) chromosome constitution. To determine the origin of the additional chromosome segment, several candidate regions were identified including 4q and 18q. Clinical comparison showed more similarities to individuals with partial dup(4q) than to those with a dup(18q). Whole chromosome fluorescence in situ hybridization (FISH) was used to demonstrate the correct origin of the translocated region, clarifying the karyotype as 46,XY,der(18)t(4;18)(q28.2;q22.2), thus generating information of clinical importance. This illustrates the use of whole chromosome FISH to identify chromosome regions that cannot be determined conclusively using standard cytogenetic banding techniques.     

Incidence of chromosome numerical changes in multiple myeloma: fluorescence in situ hybridization analysis using 15 chromosome-specific probes.

The presence of complex karotypes with frequent numerical and structural abnormalities has been reported in 20 to 50% of multiple myeloma (MM) patients. This variability is mainly due to the difficulty of conventional cytogenetics to obtain tumor metaphases representative of all possible neoplastic clones in MM. To gain insight into the real incidence of numerical chromosome changes in MM we have studied by fluorescence in situ hybridization technique 15 different human chromosomes, 1, 3, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, X, and Y, in a series of 52 MM patients. In all cases, the DNA index assessed by a propidium iodide/CD38 double-staining technique with flow cytometry was simultaneously investigated for correlation, with fluorescence in situ hybridization results. Additional aims of this study were 1) to analyze whether the abnormalities detected were common to all plasma cells or were present in only a subpopulation of tumor cells, 2) to explore changes caused by disease progression, and 3) to establish possible associations among the altered chromosomes. Although the overall incidence of numerical abnormalities was 67%, this frequency increased to 80% in the 41 cases in which 7 or more chromosomes were analyzed. Trisomies were significantly more common than monosomies (84% versus 16%). Chromosomes 9 and 15 were the most frequently altered (52% and 48% of cases, respectively), with all of their abnormalities corresponding to trisomies. The most frequent losses involved chromosomes 13 (26%) and X in females (32%). Other common numerical changes corresponded to chromosomes 1 (39%), 11 (37%), 6 (32%), 3 (31%), 18 (29%), 7 (28%), and 17 (22%). By contrast, chromosomes 8(13%), 10(8%), and 12(3%) were rarely altered. DNA aneuploidy by flow cytometry was detected in 67% of patients, and a high degree of correlation was observed between the DNA index obtained by flow cytometry and the chromosome index derived from fluorescence in situ hybridization studies, calculated according to two mathematical formulas (coefficient of correlation of 0.82 and 0.91 when at least 7 or 12 chromosomes were considered, respectively). The frequency of numeric chromosome aberrations was higher in those patients with progressive disease and, interestingly, trisomy of chromosome 8 was exclusively detected in this latter group of patients. Our study shows that, with the exception of chromosome 8, a possible marker of clonal evolution, the numeric chromosome changes are present in nearly all malignant plasma cells (r > 0.84). Finally, frequent associations between chromosomal aberrations were observed (ie, chromosomes 6, 7, 9, and 17; 7 and 15; and 11 and 17). By excluding them, it was found that two triple combinations of chromosome-specific probes, chromosomes 1 and 9 together with either chromosome 13 or 15, could be a useful marker for detection of residual disease, as it permits the identification of most MM patients displaying numerical changes.     

Analysis of BCL2 and MYC expression in non-Hodgkin's lymphomas by in situ hybridization: correlation with chromosome translocations.

We have used an in situ hybridization method for analysis of expression of BCL2 and MYC on cytospun preparations of normal and malignant lymphoid cell lines and tissue sections of normal and malignant lymph nodes. The probes comprised 50-mer antisense oligonucleotides starting at the ATG codons of exon 3 of BCL2 and exon 2 of MYC. We studied the expression of these two genes in frozen tissue sections of biopsy specimens derived from normal and hyperplastic lymph nodes, B-cell lymphomas carrying the t(14;18)(q32;q21) and t(8;14)(q24;q32) translocations, and T-cell lymphomas with clonal chromosome abnormalities. While all proliferating cells expressed both genes, BCL2 expression was increased two- to threefold in follicular lymphomas with t(14;18) and MYC expression was increased two- to four-fold in high-grade lymphomas with t(8;14). These results are consistent with previous data on deregulated expression of these genes obtained from study of lymphoma cell lines carrying the relevant translocations.     

用荧光原位杂交在石蜡切片上检测t(11;18)和涉及bcl-10基因染色体易位的方法

荧光原位杂交（fluorescence in situ hybridisation,FISH）是在石蜡包埋组织中检测细胞染色体变化及基因异常的一种敏感性强、特异性高的分子生物学方法。对石蜡包埋组织进行FISH染色所采用的方法有两种,在提取的细胞核上进行和直接在石蜡包埋组织切片上进行。然而,从石蜡包埋组织中提取细胞核进行FISH的方法复杂、耗时,而且所需组织较多,     

间期双色双融合荧光原位杂交技术检测成人B细胞急性淋巴细胞白血病费城染色体

目的 探讨成人B细胞急性淋巴细胞白血病(B-lineage acute lymphoblastic leukemia.BALL)中费城染色体(Philadelphia chromosome,Ph染色体)的发生率.方法 应用针对BCR-ABL的双色双融合探针和间期荧光原位杂交(dual-color dual-fusion interphase fluorescence in situ hybridization,DDFISH)技术前瞻性检测112例初诊成人B-ALL患者染色体标本中Ph染色体.并与常规细胞遗传学(conventional cytogenetics,CC)结果 进行比较.结果 成功进行CC检测的89例患者中有16例(17.98%)检出Ph染色体,而DD-FISH检测112例发现35例(31.25%)Ph染色体,阳性细胞率为18.50%～99.00%(平均66.23%).35例DD-FISH检测出Ph+的成人B-ALL患者中,10例未能成功进行CC检测,5例核型正常,16例检出Ph染色体,20例核型异常患者中13例为复杂异常.结论 Ph染色体在成人B-ALL中以DD-FISH检测发生率为31.25%;用BCR-ABL探针进行DD-FISH为检测Ph染色体提供了有效的方法 .是细胞遗传学检查的重要补充;对成人B-ALL患者应常规进行DD-FISH检测.     

Detection of numerical chromosome anomalies in interphase cells of benign and malignant thyroid lesions using fluorescence in situ hybridization

Benign and malignant thyroid tumors constitute a wide range of neoplasias showing recurrent chromosome abnormalities. In an attempt to characterize specific numerical chromosome abnormalities in thyroid tissues, we present here the findings from a study of archival samples depicted by 10 malignant tumors, 30 benign lesions, and 10 normal thyroid tissues. Fluorescence in situ hybridization was performed on noncultured samples using biotinylated centromere-specific probes for chromosomes 7, 10, and 17. Trisomy or tetrasomy 7 were present in 19 benign and in 7 malignant tumors. Trisomy 10 or 17 were observed in 18 adenomas or goiters and in 9 carcinomas, and monosomy 17 was seen in 2 carcinomas. Our findings suggest that such abnormalities are an in vivo phenomenon and may be important in the neoplastic proliferation of thyroid gland.     

Analysis of ovarian borderline tumors using comparative genomic hybridization and fluorescence in situ hybridization.

It is unclear whether ovarian borderline tumors (tumors of low malignant potential) are independent entities or whether they are part of a continuum of tumor progression that culminates in ovarian carcinoma. Little is known about genetic abnormalities in borderline tumors because of the difficulty of growing them in culture for chromosome studies, and because the low ratio of tumor to nontumor cells can interfere with molecular genetic examination. To circumvent these problems, we performed comparative genomic hybridization (CGH) on 10 serous borderline tumors from nine patients, using microdissection to enrich the samples for tumor DNA and reduce contamination from stromal and inflammatory cells. CGH analysis revealed that three of the tumors had detectable chromosomal imbalances, whereas seven were in a balanced state. In those tumors with imbalances, the number of abnormalities ranged from 3-6 per tumor. Additional studies by fluorescence in situ hybridization (FISH) on disaggregated nuclei confirmed the imbalances detected by CGH, revealed one tumor to be hypertriploid, and indicated that the remaining tumors were diploid and in a balanced state. All abnormalities observed in the aneuploid cases are consistent with chromosomal aberrations previously reported for ovarian carinomas, providing further evidence that some borderline tumors are part of a continuum of tumor progression. These results also suggest that there may be different mechanisms leading to borderline tumor formation, including one associated with multiple chromosomal imbalances, and others that do not involve imbalances detectable by CGH. Genes Chromosomes Cancer 25:307-315, 1999.     

Macrophage infiltration in non-Hodgkin's lymphomas: a quantitative in situ study

The frequency and distribution pattern of macrophages within 93 non-Hodgkin's lymphomas (NHL) were evaluated in situ by immunomorphometry using stereological methods. For the identification of macrophages (M phi), several antibodies (Mono 1, Mono 2, OKM 1) reactive with surface antigens on cells of the monocyte-macrophage series and cytochemical staining for acid phosphatase were applied. The average number of macrophages within lymph node tissue of NHL was 6,299 +/- 760 cells/microliter (similar to reactive lymphatic tissue: 6,559 +/- 1,027). The highest number of infiltrating macrophages was detected in immunoblastic NHL (17,306 +/- 2,773), differing significantly from other histological subtypes and reactive lymphatic tissue (p less than 0.005). The possible impact of tumor-infiltrating macrophages on lymphoma cell proliferation and differentiation is discussed.     

Confirmation of a cryptic unbalanced translocation using whole chromosome fluorescence in situ hybridization

We report on a 7-year-old boy with minor anomalies, growth retardation, and developmental delay with an initial 46,XY,der(18)t(18;?)(q23;?) chromosome constitution. To determine the origin of the additional chromosome segment, several candidate regions were identified including 4q and 18q. Clinical comparison showed more similarities to individuals with partial dup(4q) than to those with a dup(18q). Whole chromosome fluorescence in situ hybridization (FISH) was used to demonstrate the correct origin of the translocated region, clarifying the karyotype as 46,XY,der(18)t(4;18) (q28.2;q22.2), thus generating information of clinical importance. This illustrates the use of whole chromosome FISH to identify chromosome regions that cannot be determined conclusively using standard cytogenetic banding techniques. © Wiley-Liss, Inc.