Interphase and metaphase detection of the breakpoint of 14q32 translocations in B-cell malignancies by double-color fluorescence in situ hybridization

The breakpoint of 14q32 translocations found in B-cell malignancies was delineated specifically in both metaphase spreads and interphase nuclei by double-color fluorescence in situ hybridization (FISH) using bacteriophage clones containing the human immunoglobulin gamma chain gene locus (Ig gamma) and a cosmid clone, CY24-68, containing VH segments. CY24-68 is more telomeric than Ig gamma, separated by approximately 1 megabase (Mb). FISH studies were performed on four patients with non-Hodgkin's lymphoma (NHL), one with acute lymphoblastic leukemia (ALL), one with plasma cell leukemia (PCL), and three cell lines. In each patient with t(8;14), t(14;18), and t(3;14), the signal of Ig gamma gene was observed on der(14) and that of CY24-68 at respective partner sites of these translocations, 8q24.1, 18q21.3, and 3q27. Interphase nuclei with a signal of Ig gamma clearly separated from that of CY24-68 were more frequently encountered in all of the patients (45% to 74%) than those in normal controls (4% to 5%). Even in cases where only interphase nuclei were available for FISH studies, 14q32 translocations are detected as shown in two patients each with NHL and t(11;14)-carrying PCL. In two cell lines, HS-1 derived from ALL carrying t(8;14) and FR4 derived from a plasmacytoma carrying a complex form of t(8;14), the signal of Ig gamma was observed at the breakpoint region 8q24.1 of the der(8) in addition to the der(14), indicating that translocation event occurred within the Ig gamma locus. Intense Ig gamma signal was found at the breakpoint region on the der(14)t(11;14) in HBL-2 derived from NHL, indicating amplification of the Ig gamma gene, and presumably the resultant chimeric DNA between Ig gamma and DNA sequences at 11q13. The present approach allowed us to unequivocally detect tumor-specific breakpoints of 14q32 translocations. Furthermore, interphase FISH provides a rapid diagnostic procedure to detect 14q32 translocations in B-cell malignancies.     

The Ig heavy chain gene is frequently involved in chromosomal translocations in multiple myeloma and plasma cell leukemia as detected by in situ hybridization

Chromosome rearrangement of 14q32.33 has recurrently occurred with variable partner sites, including 11q13.3, 8q24.1, 18q21.3, and 6p21.1 in multiple myeloma (MM). To assess the actual incidence of 14q32.33 translocation and to elucidate its implication in the pathogenesis of MM, we studied 42 patients with MM, plasma cell leukemia, or plasmacytoma and 5 with monoclonal gammopathy with undetermined significance (MGUS) by G-banding and molecular cytogenetic methods. Using double-color fluorescence in situ hybridization (DCFISH) with 2 Ig heavy chain (IgH) gene probes, a yeast artificial chromosome (YAC) clone containing variable region, and a phage clone containing gamma constant region, 14q32.33 translocation was detected as split signals of the IgH gene in 31 patients with plasma cell malignancies and 3 with MGUS. In contrast, of 40 patients who were assessed by G-banding, 3 (7.5%) showed the 14q+ chromosome. DCFISH detected a split of the IgH gene on interphase nuclei in 34 (73.9%) of 46 patients analyzed, whereas on metaphase spreads, it was in 22 (51.2%) of 43 patients analyzed. Interphase DCFISH was particularly useful to detect 14q32.33 translocation in 17 (65.4%) of 26 patients with normal karyotypes. Donor sites were identified in 11 of 22 patients demonstrated as carrying 14q32.33 translocation by metaphase FISH. Chromosome t(11;14)(q13.3; q32.33) was detected in 5 patients, t(8;14)(q24.1;q32.33) in 2, t(14;18)(q32.33;q21.3) in 2, and t(7;14)(q32.1;q32.33) in 1. A complex 14q32.33 translocation involving 3q and 16q24 was detected in 1 patient. Myeloma cells with t(7;14) showed myelomonocytoid surface antigen. Because rearrangements of 14q32.33 were closely associated with translocation of proto-oncogenes into the IgH gene, our findings indicate that 14q32.33 translocation with various partner chromosomes is a critical event in the pathogenesis of MM and MGUS.     

DNA fiber fluorescence in situ hybridization analysis of immunoglobulin class switching in B-cell neoplasia: aberrant CH gene rearrangements in follicle center-cell lymphoma

Immunoglobulin class switching usually involves deletion of part of the immunoglobulin CH region. By DNA fiber fluorescence in situ hybridization (FISH) with a barcode of probes covering the DH, JH, and CH genes, the configuration of the entire CH region can be visualized on single DNA molecules. Using this technique, we have studied class switching in three types of B-cell neoplasia, mantle-cell lymphoma (MCL), follicular lymphoma (FL) and hairy cell leukemia (HCL), representing B cells in, respectively, pregerminal center, germinal center, and postgerminal center stages of development. In MCL and FL, simultaneous detection of the t(11;14) and t(14;18) breakpoint with probes for the BCL-1 and BCL-2 loci, respectively, allowed differentiation between productive and nonproductive alleles. In none of 10 MCL cases was class switching detected. In 21 HCL, all nonimmunoglobulin M (IgM) cases had class-switch deletion consistent with the expressed isotype on at least one allele. In FL, however, a peculiar pattern of CH rearrangement was observed. In IgM expressing FL, the translocated alleles had switched in 11 of 13 cases, and the nontranslocated allele showed complex rearrangements downstream from the Cmu-Cdelta genes in 9 of 13 cases. These downstream rearrangements may reflect tumor-specific deregulation of the class-switch machinery. All seven immunoglobulin G (IgG) expressing FL showed class switching on both alleles. Fiber FISH analysis also showed several polymorphisms. The most frequent one, present on 38% of all analyzed alleles, consisted of an extra Cgamma gene or pseudogene in the 3' cluster.     

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.     

Amplification of c-myc, K-sam, and c-met in gastric cancers: detection by fluorescence in situ hybridization

Gene amplifications of c-myc, K-sam, and c-met were examined in cancer nuclei isolated from 154 primary gastric adenocarcinomas by fluorescence in situ hybridization (FISH) using cosmid probes for 8q24 (c-myc locus) and 7q31 (c-met), as well as a DNA probe for K-sam synthesized by PCR. The results were compared with those of Southern blot analysis. Dual-color FISH using gene locus and chromosome-specific probes detected gene amplifications of c-myc in 24 tumors (15.5%), c-met in 6 tumors (3.9%), and K-sam in 3 tumors (2.9%). The six tumors with c-myc amplification had also been found to have amplified c-erbB-2 in our previous study, and coamplification of c-myc and c-met was found in two other tumors. This technique also differentiated the amplified genes on the homogeneous staining region (HSR) and on double minute chromosomes (DMs) in metaphase spreads and interphase nuclei of cell lines established from poorly differentiated adenocarcinomas, KATO III, SNU 16, and HSC 39. Examination of FISH images of these cell lines suggested that the high-level amplifications of c-myc found in primary tumors occurred mainly on DM in four tumors and on HSR in one, and those of K-sam occured on DM in two tumors and on HSR in one. No high-level amplification of c-met was found. These high-level amplifications were also detected in formalin-fixed, paraffin-embedded tissues from primary gastric tumors and metastatic lymph nodes, in some of which heterogeneity of gene amplification was demonstrated within the same tumor. We conclude that FISH is an important tool for examining the proto-oncogene aberrations in intact cells in solid tumors.     

Cytogenetic profile of lymphoma of follicle mantle lineage: correlation with clinicobiologic features

Conventional chromosome analysis (CCA) and interphase fluorescence in situ hybridization (FISH) was performed in 42 patients with mantle-cell lymphoma (MCL), with BCL1 rearrangement. The t(11;14)(q13;q32) or 11q abnormalities were detected by CCA in 34 cases, 20 of which had additional aberrations. A normal karyotype was observed in 8 cases. Probes detecting the chromosome aberrations that were observed in at least 3 cases by CCA, ie, +12, 13q14 deletion, and 17p deletion, were used for interphase FISH analysis. FISH detected total or partial +12, 13q14 deletion and 17p- in 28.5%, 52.4%, and 26% of the cases, respectively. The presence of these anomalies was not a function of karyotype complexity. Based on the results of CCA/FISH, three groups of increasing karyotype complexity were recognized: group 1, including 11 patients without detectable aberrations in addition to BCL1 rearrangement; group 2, including 14 patients with 1 to 2 additional anomalies; and group 3, including 17 patients with three or more additional anomalies. Clinical parameters associated with shorter survival were male sex (P =.006) and primary lymph-node involvement compared with primary bone marrow involvement (P =.015). Trisomy 12 was the only single cytogenetic parameter predictive of a poor prognosis (P =.006) and the best prognostic indicator was the derived measure of karyotype complexity (P <.0001), which maintained statistical significance in multivariate analysis (P<.0001). We arrived at the following conclusions: 13q14 deletion occurs at a high incidence in MCL; 17p deletion and total/partial +12 are relatively frequent events in MCL, the latter aberration being associated with a shorter survival; and the degree of karyotype complexity has a strong impact on prognosis in this neoplasia.     

Interphase cytogenetic diagnosis of bladder cancer on cells from urine and bladder washing

In order to determine the value of fluorescence in situ hybridization (FISH) in the diagnosis and follow-up of bladder cancer interphase cytogenetics was performed on cells from urine and bladder washings. 50 ml of urine or bladder washings were collected. FISH was carried out using centromere probes for chromosomes 7, 8, 9 and 12 according to standard protocols. In each case 100 cell nuclei were analysed. Fifty-four samples from urine and 67 samples from bladder washing were analysed by FISH in comparison with results obtained by conventional cytology. Sensitivity of detection of tumor cells by FISH was 68.5% in urine and 63% in bladder washings regardless of tumor stage and grade. Sensitivity obtained by conventional cytology was 50% in urine and 77.3% in bladder washings. FISH on cells from urine samples is an effective complement to the standard urine cytology. Using centromere probes this approach is characterized by high specificity and sensitivity in tumors with T-category higher than pTa and grade higher than G1.     

Detection of deletions in the short arm of chromosome 3 in uncultured renal cell carcinomas by interphase cytogenetics

PURPOSE: Analysis of genetic alterations may facilitate the differential diagnosis of renal cell carcinoma (RCC) subtypes. For genetic classification, deletion of the short arm of chromosome 3 (3p), the hallmark of nonpapillary/clear cell RCC, is a major diagnostic criterion. Because of the limited routine applicability of cytogenetics and molecular genetic techniques we investigated interphase fluorescence in situ hybridization (FISH) for the detection of this aberration in RCC. MATERIALS AND METHODS: Using seven chromosome 3 specific probes FISH was performed on isolated nuclei from 26 uncultured sporadic RCC. RESULTS: Alterations of chromosome 3 were identified in 19 RCC (73%). Monosomy and/or 3p-deletions were observed in 15 of 19 (79%) non-papillary/clear cell RCC but not in other morphologic subgroups. The median percentage of cells in a specimen containing loss of 3p was 45%. Deletion mapping indicated that large deletions affecting different regions in 3p are predominant. Chromosomal region 3p24 was recurrently involved in all RCC with a deletion in 3p. CONCLUSION: Interphase FISH for the detection of loss in 3p provides a sensitive and feasible method for the genetic classification of kidney tumors and the delineation of recurrently deleted regions in 3p.     

Chromosomal imbalances in primary and metastatic pancreatic carcinoma as detected by interphase cytogenetics: basic findings and clinical aspects

To date, cytogenetic studies on pancreatic carcinoma are rare, and little is known about the frequency of cytogenetic aberrations in primary carcinomas compared with metastatic tumour cells. We therefore evaluated the frequency of chromosomal aberrations in 12 primary pancreatic carcinomas and in effusion specimens from 25 patients with pancreatic cancer by using interphase fluorescence in situ hybridization (FISH) and a panel of four centromeric probes. Hyperdiploidy and chromosomal imbalances, predominantly affecting chromosome 8, were a constant finding in metastatic effusion cells, whereas concordant gain of chromosomes or relative loss of chromosome 18 characterized primary pancreatic carcinomas. The potential role of oncogenes located on chromosome 8 for pancreatic cancer progression was further investigated by double-hybridization studies of aneuploid effusion cells with a probe to 8q24 (MYC) and a centromeric probe to chromosome 8, which demonstrated amplification of the MYC oncogene in two of ten cases (20%). Finally, a potential application of basic findings in the clinical setting was tested by searching for micrometastatic cells in effusions from pancreatic cancer patients primarily negative by FISH. Two-colour FISH in combination with extensive screening (>10,000 nuclei) seems to be a useful tool to unequivocally identify micrometastatic cells by demonstrating hyperdiploidy and intranuclear chromosomal heterogeneity.     

Prevention of maternal and developmental toxicity in rats via dietary inclusion of common aflatoxin sorbents: potential for hidden risks

The chromosomal translocation t(11;14)(q13;q32) fuses the IGH and CCND1 genes and leads to cyclin D1 overexpression. This genetic abnormality is the hallmark of mantle cell lymphoma (MCL), but is also found in some cases of atypical chronic lymphocytic leukemia (CLL), characterized by a poor outcome. For an unequivocal assessment of this specific chromosomal rearrangement on interphase cells, we developed a set of probes for fluorescence in situ hybridization (FISH). Northern blotting was performed for analysis of the cyclin D1 expression in 18 patients. Thirty-eight patients, with either a typical MCL leukemic phase (17 patients) or atypical CLL with an MCL-type immunophenotype, i.e., CD19-, CD5+, CD23-/low, CD79b/sIgM(D)++, and FMC7+ (21 patients), were analyzed by dual-color interphase FISH. We selected an IGH-specific BAC probe (covering the JH and first constant regions) and a commercially available CCND1 probe. An IGH-CCND1 fusion was detected in 28 of the 38 patients (17 typical MCL and 11 cases with CLL). Cyclin D1 was not overexpressed in two patients with typical MCL and an IGH-CCND1 fusion. In view of the poor prognosis associated with MCL and t(11;14)-positive CLL, we conclude that this set of probes is a valuable and reliable tool for a rapid diagnosis of these entities.     

Comparison of fluorescence in situ hybridization with flow cytometry and static image analysis in ploidy analysis of paraffin-embedded prostate adenocarcinoma

Nuclear DNA ploidy has been shown to have an important prognostic association for patients with adenocarcinoma of the prostate. Flow cytometry and static image analysis are ploidy methods that have been used in prostate carcinoma. Fluorescence in situ hybridization (FISH) using chromosome-specific probes can be used to evaluate the ploidy of interphase nuclei. In this study FISH was compared with flow cytometry and static image analysis in determining ploidy in paraffin-embedded tissue from 34 prostatic adenocarcinomas. Ploidy status using FISH was determined by enumerating centromeres of two chromosomes (8 and 12) by use of directly-labeled alpha-satellite DNA probes in isolated whole nuclei obtained by the Hedley technique. All three methods identified 11 of 34 cases as diploid and 17 of 34 cases as nondiploid (82% concordance). Six cases were discordant; two cases had discrepant results by each method. Ploidy classification as determined by FISH had an 88% concordance with ploidy classification by either flow cytometry or static image analysis. In conclusion, FISH was found to be a sensitive method of ploidy analysis in isolated paraffin-embedded nuclei from prostate adenocarcinomas. When the chromosomes commonly involved in aneuploidy have been identified in prostate adenocarcinoma, FISH has the potential to provide greater sensitivity for aneuploidy detection compared with currently available methods.     

Rapid induction of B-cell lymphomas in mice carrying a human IgH/c-mycYAC

Activation of the c-myc proto-oncogene by one of the immunoglobulin (Ig) loci after chromosomal translocation is a consistent feature of Burkitt's lymphoma. Different subtypes of this tumor vary in the molecular architecture of the translocation region. In most cases there are no known regulatory elements of the Ig locus neighboring the oncogene and this considerably obscures the mechanism of its deregulation. In order to assess possible oncogene activation signals, we produced an experimental translocation region by insertion of a c-myc gene about 50 kb from the IgH intron enhancer in a yeast artificial chromosome (YAC) containing a 220 kb region of the human Ig heavy chain (IgH) locus. Single copy integration of this YAC into the genome of mouse embryonic stem (ES) cells was achieved by spheroplast fusion. Chimeric mice derived from these ES cells developed monoclonal B-cell lymphomas expressing surface IgM by 8-16 weeks of age. The IgH/c-myc translocus showed different V(h)DJ(H) rearrangement in almost all tumors without any alterations of the distance between c-myc and the IgH intron enhancer. This mouse model can be used for the in vivo analysis of c-myc deregulation and the tumor formation capacity of the IgH locus in aberrant rearrangements.     

Poly-FISH: a technique of repeated hybridizations that improves cytogenetic analysis of fetal cells in maternal blood

Prenatal diagnosis of fetal chromosomal abnormalities using interphase fetal nucleated erythrocytes (FNRBCs) separated from maternal peripheral blood can be technically challenging due to the limited number of FNRBCs available for analysis, the limited number of probes that can be used simultaneously, and low FISH efficiency on the formaldehyde-fixed and immunohistochemically stained interphase FNRBCs. We developed a technique of sequential FISH analysis that involves removal of the previous hybridized probe under denaturing conditions, and rehybridization with different probes to improve FISH efficiency. This technique facilitates the analysis of multiple chromosome-specific probes on the same nuclei. Results from our experiments show that FISH can be performed at least nine times on the same interphase nucleus and at least three different probes can be used simultaneously. Thus, theoretically, at least 24 different chromosomes can be analysed on a single interphase fetal cell isolated from maternal blood. We have termed this technique 'Poly-FISH', and have successfully diagnosed trisomy 21, triploidy, and other chromosome abnormalities in FNRBCs using this technique.     

Prognosis of operable squamous cell carcinoma of the esophagus. Relationship with clinicopathologic features and DNA ploidy

Fluorescence in situ hybridization (FISH) was performed on human interphase sperm nuclei to determine the utility of this technique for aneuploidy detection. Repetitive DNA sequences specific for chromosomes 1, 12 and X were biotinylated and hybridized with mature sperm, which had been treated with cetyltrimethylammonium bromide and dithiothreitol to render them accessible to the probes. Detection of bound probe was accomplished with fluoresceinated avidin and antiavidin. For each of the chromosomes studied, chromosome number was determined by counting the fluorescent signals, representing hybridized regions, within the sperm nuclei. The frequencies for disomy, that is for nuclei containing two signals, for chromosomes 1, 12 and X were 0.06%, 0.04% and 0.03%, respectively. The congruence of these results with those determined by the cross-species hamster oocyte-human sperm assay, and the high efficiency of hybridization indicate that FISH is a sensitive and reliable tool for aneuploidy detection in human sperm.     

Possibilities for false-negative findings in trisomy 21 screening with FISH

In approximately 5% of individuals with Down syndrome aneuploidy results from a chromosomal rearrangement. FISH analysis on chromosome metaphases and interphase nuclei of 5 individuals with Down syndrome carrying different types of chromosome 21 translocations demonstrated the diagnostic efficiency of this method. By use of different commercially available chromosome 21 specific probes we were able to show that only the cosmid probe specific for the Down syndrome critical region (DCR) in 22qll gave reliable results for interphase analysis of trisomy 21, while the use of chromosome 21 centromere- or of painting probes carry a high risk of a false-negative diagnosis in translocation trisomy 21.     

Hyponatremia of cirrhosis: role of vasopressin and decreased ''effective'' plasma volume

Interphase FISH analysis, utilizing dual color XY probes, was performed on 27 patients following allogeneic sex-mismatched bone marrow transplantation and on 31 controls. Of the 123 167 examined interphase nuclei, 63 318 were from 19 of the 21 patients (54 specimens) who engrafted, 31 827 from five of the six patients (29 specimens) who relapsed (four) or failed to engraft (one) and 24 703 from the 31 control specimens. In patients who engrafted, the mean percentage of host cells was 0.26% between day 29 and 5 years following BMT. Microchimerism of 0.7% or less than 1-5 years following BMT was not predictive of relapse. Interphase FISH analysis predicted relapse or failure of engraftment in five of the six evaluable patients. In three of five patients both conventional cytogenetics and interphase FISH of bone marrow cells provided important information regarding engraftment status and degree of chimerism.     

多发性骨髓瘤中14q32易位与13q14缺失的相关性研究

目的 研究多发性骨髓瘤(MM)常见的分子遗传学异常14q32易位与13q14缺失及其与临床指标的关系.方法 采用间期荧光原位杂交(I-FISH)技术应用RB1、D13S319和LSI IGHC/IGHV探针检测49例MM患者骨髓标本中RB1基因、13q14.3缺失及14q32易位,结合临床资料作统计分析.结果 49例MM患者有26例(53.1%)检测到14q32易位,25例(51.02%)存在13q14缺失(其中18例检测到13q14.3缺失,9例存在RB1缺失).Spearman相关分析显示,14q32易位多见于浆细胞比例高的患者(r=0.316,P=0.27),与患者年龄、国际分期系统(ISS)分期、免疫球蛋白分型、β2微球蛋白及肾损害无相关性(P＞0.05).结论 13q14缺失及14q32相关的易位在MM中发生率均较高,两者有密切相关性;14q32易位的MM患者浆细胞百分比明显升高,14q32易位的检测可作为预测MM预后的指标.     

Interphase cytogenetics in pathology: principles, methods, and applications of fluorescence in situ hybridization (FISH)

Characteristic chromosome aberrations have been identified in various tumors. Fluorescence in situ hybridization (FISH) using specific probes that are generated by vector cloning or in vitro amplification and labeled with fluorescent dyes allow for the detection of these genetic changes in interphase cells. This technique, that is also referred to as "interphase cytogenetics", can be performed in cytological preparations as well as in sections of routinely formaldehyde-fixed and paraffin-embedded tissue. In cancer research and diagnostics, interphase cytogenetics by FISH is used to detect numerical chromosome changes and structural aberrations, e.g., translocations, deletions, or amplifications. In this technical overview, we explain the principles of the FISH method and provide protocols for FISH in cytological preparations and paraffin sections. Moreover, possible applications of FISH are discussed.     

Tilt table testing in the evaluation and management of athletes with recurrent exercise-induced syncope

The development and application of a highly sensitive double-target fluorescence in situ hybridization (FISH) method in combination with immunohistochemistry for detection of chromosome 1 abnormalities in interphase nuclei of neuroblastoma samples is reported. An alpha-satellite probe specific for chromosome 1 and a VNTR probe that hybridizes to chromosome band 1p36.3 were hybridized to GD2 prestained neuroblastoma cells in double-target FISH experiments. The ratio of intact to deleted chromosome 1 homologs in the neuroblastoma cells was assessed. To demonstrate the reliability of the method described, four selected samples derived from different neuroblastoma stages are presented. FISH results correlated well with data obtained by conventional cytogenetic procedures. The technique described allows sensitive detection of chromosome 1 abnormalities in interphase nuclei and enables partial cytogenetic analysis of nondividing cells with a defined immunological phenotype.