Multiple recurrent chromosomal breakpoints in mantle cell lymphoma revealed by a combination of molecular cytogenetic techniques

Mantle cell lymphoma (MCL) is genetically characterized by 11q13 translocations leading to the overexpression of CCND1, and additional secondary genomic alterations that may be important in the progression of this disease. We have analyzed 22 MCL cases and 10 MCL cell lines using multicolor fluorescence in situ hybridization (M‐FISH), FISH, and comparative genomic hybridization (CGH). The 19 cases with abnormal karyotype showed the t(11;14)(q13;q32) translocation and, additionally, 89% of cases showed both numerical (n = 58) and structural (n = 77) aberrations. All but one MCL cell line showed t(11;14) and structural and numerical alterations in highly complex karyotypes. Besides 11 and 14, the most commonly rearranged chromosomes were 1, 8, and 10 in the tumors and 1, 8, and 9 in the cell lines. No recurrent translocations other than the t(11;14) were identified. However, we identified 17 recurrent breakpoints, the most frequent being 1p22 and 8p11, each observed in four cases and two cell lines. Interestingly, five tumors and four cell lines displayed a complex t(11;14), cryptic in one case and two cell lines, preferentially involving chromosome 8. In typical MCL, ATM gene deletions were significantly associated with a high number of structural and numerical alterations. In conclusion, MCL does not have recurrent translocations other than t(11;14), but shows recurrent chromosomal breakpoints. Furthermore, most MCL harbor complex karyotypes with a high number of both structural and numerical alterations affecting several common breakpoints, leading to various balanced and unbalanced translocations. © 2008 Wiley‐Liss, Inc.     

Molecular cytogenetic analysis of breast cancer: a combined multicolor fluorescence in situ hybridization and G-banding study of uncultured tumor cells

In six patients with breast cancer, uncultured tumor cells were investigated with G-banding and multicolor fluorescence in situ hybridization (M-FISH). A large number of numerical and structural aberrations could be analyzed. Among other structural abnormalities, reciprocal, hidden and complex translocations were found. Recurrent t(1;10) and t(6;16), not previously described, were identified, as well as t(15;22). The latter was also found in additional cases among our unpublished breast carcinomas. The significance of t(15;22) for breast cancer is discussed, taking into account also data drawn from the literature. Reciprocal translocations were a prominent feature in a pseudodiploid lobular carcinoma. Hidden translocations on 6p22-p24 were detected with M-FISH. Involvement of 6p22-p24 was observed in five cases. The analysis of various other translocations and different structural abnormalities revealed the following common breakpoints (according to frequency of involvement): 1p34-p36, 3p12-p13, 4p13-->q11, 14p11-->q11, 1q42, 8p11, 8q24, 10q22, 11q13, 11q23-q24, 13q13, and 18p10-p11. Loss of 3p and 1p34-p36-->pter and complete or partial loss of 13q and chromosome 17 were also found. With the combination of G-banding and M-FISH techniques, chromosome misclassification is avoided and the characterization of complex tumor karyotypes is more effective.     

Molecular cytogenetic characterization of non-Hodgkin lymphoma cell lines.

Spectral karyotyping (SKY) and comparative genomic hybridization (CGH) have greatly enhanced the resolution of cytogenetic analysis, enabling the identification of novel regions of rearrangement and amplification in tumor cells. Here we report the analysis of 10 malignant non-Hodgkin lymphoma (NHL) cell lines derived at the Ontario Cancer Institute (OCI), Toronto, designated as OCI-Ly1, OCI-Ly2, OCI-Ly3, OCI-LY4, OCI-Ly7, OCI-Ly8, OCI-Ly12, OCI-Ly13.2, OCI-Ly17, and OCI-Ly18, by G-banding, SKY, and CGH, and we present their comprehensive cytogenetic profiles. In contrast to the 52 breakpoints identified by G-banding, SKY identified 87 breakpoints, which clustered at 1q21, 7p15, 8p11, 13q21, 13q32, 14q32, 17q11, and 18q21. G-banding identified 10 translocations, including the previously described recurring translocations, t(8;14)(q24;q32) and t(14;18)(q32;q21). In contrast, SKY identified 60 translocations, including five that were recurring, t(8;14)(q24;q32), t(14;18)(q32;q21), t(4;7)(p12;q22), t(11;18)(q22;q21), and t(3;18)(q21;p11). SKY also identified the source of all the marker chromosomes. In addition, 10 chromosomes that were classified as normal by G-banding were found by SKY to be rearranged. CGH identified seven sites of high-level DNA amplification, 1q31-32, 2p12-16, 8q24, 11q23-25, 13q21-22, 13q32-34, and 18q21-23; of these, 1q31-32, 11q23-25, 13q21-22, and 13q32-34 have previously not been described as amplified in NHL. This comprehensive cytogenetic characterization of 10 NHL cell lines identified novel sites of rearrangement and amplification; it also enhances their value in experimental studies aimed at gene discovery and gene function.     

Quantitative multigene FISH on breast carcinomas identifies der(1;16)(q10;p10) as an early event in luminal A tumors

In situ detection of genomic alterations in cancer provides information at the single cell level, making it possible to investigate genomic changes in cells in a tissue context. Such topological information is important when studying intratumor heterogeneity as well as alterations related to different steps in tumor progression. We developed a quantitative multigene fluorescence in situ hybridization (QM FISH) method to detect multiple genomic regions in single cells in complex tissues. As a “proof of principle” we applied the method to breast cancer samples to identify partners in whole arm (WA) translocations. WA gain of chromosome arm 1q and loss of chromosome arm 16q are among the most frequent genomic events in breast cancer. By designing five specific FISH probes based on breakpoint information from comparative genomic hybridization array (aCGH) profiles, we visualized chromosomal translocations in clinical samples at the single cell level. By analyzing aCGH data from 295 patients with breast carcinoma with known molecular subtype, we found concurrent WA gain of 1q and loss of 16q to be more frequent in luminal A tumors compared to other molecular subtypes. QM FISH applied to a subset of samples (n = 26) identified a derivative chromosome der(1;16)(q10;p10), a result of a centromere‐close translocation between chromosome arms 1q and 16p. In addition, we observed that the distribution of cells with the translocation varied from sample to sample, some had a homogenous cell population while others displayed intratumor heterogeneity with cell‐to‐cell variation. Finally, for one tumor with both preinvasive and invasive components, the fraction of cells with translocation was lower and more heterogeneous in the preinvasive tumor cells compared to the cells in the invasive component. © 2014 The Authors Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc.     

Molecular cytogenetic characterization of TCF3 (E2A)/19p13.3 rearrangements in B-cell precursor acute lymphoblastic leukemia

The t(1;19)(q23;p13.3) is one of the most common chromosomal abnormalities in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and usually gives rise to the TCF3-PBX1 fusion gene. Additional rare, and sometimes cytogenetically cryptic, translocations involving the TCF3 gene have also been described. Using a dual color split-signal fluorescence in situ hybridization (FISH) probe, we have investigated the involvement of this gene in a series of BCP-ALLs harboring 19p13 translocations, as well as an unselected patient cohort. The TCF3 gene was shown to be involved in the majority of cases with a cytogenetically visible t(1;19) translocation, while the remaining TCF3-negative ALLs demonstrated breakpoint heterogeneity. Although most "other" 19p13 translocations did not produce a split-signal FISH pattern, a novel t(13;19)(q14;p13) involving TCF3 was discovered. A prospective screen of 161 children with BCP-ALL revealed a cryptic t(12;19)(p13;p13), another novel TCF3 rearrangement, and a series of patients with submicroscopic deletions of TCF3. These results demonstrate the utility of a split-signal FISH strategy in confirming the involvement of the TCF3 gene in 19p13 rearrangements and in identifying novel and cryptic TCF3 translocations. In addition to its role as a fusion partner gene, we propose that TCF3 can also act as a tumor suppressor gene in BCP-ALL.     

Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization

Chromogenic (CISH) and fluorescent (FISH) in situ hybridization have emerged as reliable techniques to identify amplifications and chromosomal translocations. CISH provides a spatial distribution of gene copy number changes in tumour tissue and allows a direct correlation between copy number changes and the morphological features of neoplastic cells. However, the limited number of commercially available gene probes has hindered the use of this technique. We have devised a protocol to generate probes for CISH that can be applied to formalin-fixed, paraffin-embedded tissue sections (FFPETS). Bacterial artificial chromosomes (BACs) containing fragments of human DNA which map to specific genomic regions of interest are amplified with phi29 polymerase and random primer labelled with biotin. The genomic location of these can be readily confirmed by BAC end pair sequencing and FISH mapping on normal lymphocyte metaphase spreads. To demonstrate the reliability of the probes generated with this protocol, four strategies were employed: (i) probes mapping to cyclin D1 (CCND1) were generated and their performance was compared with that of a commercially available probe for the same gene in a series of 10 FFPETS of breast cancer samples of which five harboured CCND1 amplification; (ii) probes targeting cyclin-dependent kinase 4 were used to validate an amplification identified by microarray-based comparative genomic hybridization (aCGH) in a pleomorphic adenoma; (iii) probes targeting fibroblast growth factor receptor 1 and CCND1 were used to validate amplifications mapping to these regions, as defined by aCGH, in an invasive lobular breast carcinoma with FISH and CISH; and (iv) gene-specific probes for ETV6 and NTRK3 were used to demonstrate the presence of t(12;15)(p12;q25) translocation in a case of breast secretory carcinoma with dual colour FISH. In summary, this protocol enables the generation of probes mapping to any gene of interest that can be applied to FFPETS, allowing correlation of morphological features with gene copy number.     

Unusual karyotype aberrations involving 2p12, 3q27, 18q21, 8q24, and 14q32 in a patient with non-Hodgkin lymphoma/acute lymphoblastic leukemia

The t(2;18)(p12;q21), known as a rare variant of the t(14;18)(q32;q21), together with t(3;14)(q27;q32), t(8;15)(q24;q22) and two other unusual translocations involving chromosomes 6, 9, 12, and 13, were demonstrated in the bone marrow cells of a 70-year-old male with suspected non-Hodgkin lymphoma/acute lymphoblastic leukemia. The complex chromosomal aberrations were identified by chromosome banding analysis and by fluorescence in situ hybridization (FISH) with whole chromosome painting probes, centromere-specific alpha-satellite probes, and probes specific for genomic sequences of some likely to be involved candidate genes. Several but not all of the chromosomal aberrations could be proved by multicolor FISH. Possible mechanisms leading to this unusual karyotype commonly associated with different histologic lymphoma subtypes and their prognostic implications are discussed.     

Characterization of oncogene dysregulation in multiple myeloma by combined FISH and DNA microarray analyses

Chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus and various partner loci frequently are associated with multiple myeloma (MM). We investigated the expression profiles of the FGFR3/MMSET, CCND1, CCND3, MAF, and MAFB genes, which are involved in t(4;14)(p16.3;q32), t(11;14)(q13;q32), t(6;14)(p21;q32), t(14;16)(q32;q23), and t(14;20)(q32;q12), respectively, in purified plasma cell populations from 39 MMs and six plasma cell leukemias (PCL) by DNA microarray analysis and compared the results with the presence of translocations as assessed by dual-color FISH or RT-PCR. A t(4;14) was found in 6 MMs, t(11;14) in 9 MMs and 1 PCL, t(6;14) in 1 MM, t(14;16) in 2 MMs and 1 PCL, and t(14;20) in 1 PCL. In all cases, the translocations were associated with the spiked expression of target genes. Furthermore, gene expression profiling enabled the identification of putative translocations causing dysregulation of CCND1 (1 MM and 1 PCL) and MAFB (1 MM and 1 PCL) without any apparent involvement of immunoglobulin loci. Notably, all of the translocations were mutually exclusive. Markedly increased MMSET expression was found in 1 MM showing associated FGFR3 and MMSET signals on an unidentified chromosome. Our data suggest the importance of using combined molecular cytogenetic and gene expression approaches to detect genetic aberrations in MM.     

High incidence of cryptic translocations involving the Ig heavy chain gene in multiple myeloma,as shown by fluorescence in situ hybridization

Cytogenetic studies have shown rearrangements of the Ig heavy chain (IGH) gene at 14q32 in 10–60% of patients with multiple myeloma (MM) or primary plasma cell leukemia (PCL). Analysis of MM patients and human myeloma cell lines (HMCL) using interphase fluorescence in situ hybridization (FISH) and molecular techniques has shown IGH rearrangements in 75% of MM cases and in up to 100% of HMCL. A review of the literature revealed at least 18 different partner chromosomal regions. To investigate whether some of these translocations were recurrent and possibly to identify new partner regions, we developed a set of FISH probes to detect every IGH recombination. We analyzed 28 MM and 4 primary PCL patients with abnormal karyotypes and 12 HMCL. Whereas conventional cytogenetics detected a 14q32 abnormality in only 15% of the patients, FISH detected it in 47% of patients and in 75% of HMCL. The partner chromosome was identified in 10 of 15 patients with a 14q32 rearrangement. Interestingly, the same t(4;14)(p16;q32) was detected in five patients and three HMCL, i.e., 33% of patients and HMCL with an IGH rearrangement. New partner chromosomal regions have also been identified, i.e., 9p13, 12p11, 12p13, and Xq28, besides the previously reported 8q24, 11q13, 12q24, and 16q24 rearrangements. The genes involved in these new translocations are not known, except for 9p13, where PAX5 was shown to be the partner gene. We conclude that: 1) IGH recombinations are frequent but not constant in MM, 2) these rearrangements often occur through cryptic translocations, and 3) the t(4;14)(p16;q32) is one of the most frequent translocations, but many other chromosomal regions may be involved. Genes Chromosomes Cancer 24:9–15, 1999. © 1999 Wiley‐Liss, Inc.     

Comprehensive molecular cytogenetic characterization of cervical cancer cell lines

We applied a combination of molecular cytogenetic methods, including comparative genomic hybridization (CGH), spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH), to characterize the genetic aberrations in eight widely used cervical cancer (CC) cell lines. CGH identified the most frequent chromosomal losses including 2q, 3p, 4q, 6q, 8p, 9p, 10p, 13q, and 18q; gains including 3q, 5p, 5q, 8q, 9q, 11q, 14q, 16q, 17q, and 20q; and high-level chromosomal amplification at 3q21, 7p11, 8q23-q24, 10q21, 11q13, 16q23-q24, 20q11.2, and 20q13. Several recurrent structural chromosomal rearrangements, including der(5)t(5;8)(p13;q23) and i(5)(p10); deletions affecting chromosome bands 5p11, 5q11, and 11q23; and breakpoint clusters at 2q31, 3p10, 3q25, 5p13, 5q11, 7q11.2, 7q22, 8p11.2, 8q11.2, 10p11.2, 11p11.2, 14q10, 15q10, 18q21, and 22q11.2 were identified by SKY. We detected integration of HPV16 sequences by FISH on the derivative chromosomes involving bands 18p10 and 18p11 in cell line C-4I, 2p16, 5q21, 5q23, 6q, 8q24, 10, 11p11, 15q, and 18p11 in Ca Ski, and normal chromosome 17 at 17p13 in ME-180. FISH analysis was also used further to determine the copy number changes of PIKA3CA and MYC. This comprehensive cytogenetic characterization of eight CC cell lines enhances their utility in experimental studies aimed at gene discovery and functional analysis.     

A der(13)t(7;13)(p13;q14) with monoallelic loss of RB1 and D13S319 in myelodysplastic syndrome

Deletions or translocations of chromosome band 13q14, the locus of the retinoblastoma gene (RB1), have been observed in a variety of hematological malignancies including myelodysplastic syndrome (MDS). We describe here a novel unbalanced translocation der(13)t(7;13)(p13;q14) involving 13q14 in a patient with MDS. A 66-year-old woman was diagnosed as having MDS, refractory anemia with excess of blasts (RAEB-1) because of 7.4% blasts and trilineage dysplasia in the bone marrow cells. G-banding and spectral karyotyping analyses showed complex karyotypes as follows: 46,XX,der(6)t(6;7)(q11;?),der(7)del(7)(?p13)t(6;7)(q?;q11)t(6;13)(q?;q?),der(13)t(7;13)(p13;q14). Fluorescence in situ hybridization (FISH) analyses demonstrated that one allele of the RB1 gene and the microsatellite locus D13S319, located at 13q14 and telomeric to the RB1 gene, was deleted. Considering other reported cases, our results indicate that submicroscopic deletions accompanying 13q14 translocations are recurrent cytogenetic aberrations in MDS. The RB1 gene or another tumor suppressor gene in the vicinity of D13S319, or both, may be involved in the pathogenesis of MDS with 13q14 translocations by monoallelic deletion.     

Molecular cytogenetic study of a mantle cell lymphoma with a complex translocation involving the CCND1 (11q13) region

We describe a progressive mantle cell lymphoma (MCL) in which multicolor fluorescence in situ hybridization (M-FISH) on metaphases did not detect the characteristic t(11;14)(q13;q32), although translocations of chromosomes 11 with 15, and 14 with 15 were observed. When CCND1/IGH probes were hybridized to metaphases, however, cryptic fusion signals were detected on the der(11) and der(14) sites of CCND1 (11q13) and IGH (14q32), revealing a complex translocation involving chromosomes 11, 14, and 15. Interphase FISH with CCND1/IGH probes revealed varying patterns with one or two fusion signals, and some with no clear evidence of fusion. Loss of 17p and gain of 3q, known to be associated with disease progression in MCL, were detected with M-FISH and confirmed with the use of p53 and BCL6 probes together with comparative genomic hybridization, which detected also an interstitial deletion on 7p21. This case further illustrates the value of M-FISH in combination with fusion probes in elucidating complex cytogenetic abnormalities.     

Rearrangement of the MLL gene in acute myeloblastic leukemia: report of two rare translocations

Band 11q23 is known to be involved in translocations and insertions with a variety of partner chromosomes. They lead to MLL rearrangement, resulting in a fusion with numerous genes. We report here 2 male adults in whom a diagnosis of acute myelomonoblastic leukemia (FAB M4) and acute monoblastic leukemia (FAB M5) was made. Conventional cytogenetic techniques showed a 45,XY,t(1;11)(p32;q23),-7 karyotype in the first case and a 46,XY, t(11;17)(q23;q21) in the second case. Fluorescent in situ hybridization (FISH) with a specific MLL probe showed the gene to be disrupted, the 3' region being translocated on the derivative chromosomes 1 and 17, respectively. Fourteen and 24 patients, including ours, with acute myeloblastic leukemia associated with a t(1;11)(p32;q23) and a t(11;17)(q23;q21), respectively have been reported in the literature. Several patients with the latter translocation have also been identified to have acute lymphoblastic leukemia (ALL). Although both translocations are preferentially associated with monocytic differentiation, the t(11;17)(q23;q21) is more common in adults and has been reported in many patients with ALL, compared to the t(1;11)(p32;q23).     

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.     

Hematologic malignancies with the t(10;11)(p13;q21) have the same molecular event and a variety of morphologic or immunologic phenotypes

Previous studies described the t(10;11)(p13-14;q14-21) as a recurring translocation associated with T-cell acute lymphoblastic leukemia (ALL). This translocation has also been reported in monocytic leukemia or ALL with a very early pre-B phenotype. However, whether these cytogenetically similar translocations involve the same molecular breakpoint is unknown. Using fluorescence in situ hybridization (FISH) with a series of probes on 11q, we mapped the 11q breakpoint of the U937 cell line, which was derived from a patient with diffuse histiocytic lymphoma and was shown by FISH to have the t(10;11)(p13-14;q14-21). Subsequently, we identified a yeast artificial chromosome (YAC) clone, y960g8, that included the breakpoint on 11q. From this YAC, we isolated a P1 clone, P91B1, that was split by the 10;11 translocation. We studied four patients with a t(10;11), one of whom had acute monocytic leukemia (AMoL), one had acute lymphoblastic leukemia (ALL), one had lymphoblastic lymphoma (LBL), and one had granulocytic sarcoma, by using FISH with y960g8 and P91B1. Y960g8 and P91B1 were split by the translocation in each patient. We showed that P91B1 included a recently identified gene, CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene), and that AF10 was also rearranged in each patient by FISH when we used y807b3, which contains the AF10 gene. These findings indicate that hematologic malignant diseases with fusion of AF10 and CALM show various morphologic and immunologic phenotypes, suggesting that this fusion occurs in multipotential or very early precursor cells. Genes Chromosomes Cancer 20:253–259, 1997. © 1997 Wiley-Liss, Inc.     

Molecular characterization of der(15)t(11;15) as a secondary cytogenetic abnormality in acute promyelocytic leukemia with cryptic PML-RAR alpha fusion on 17q

A case of acute promyelocytic leukemia (APL) with cryptic PML-RAR alpha fusion on 17q and add(15p) as a secondary abnormality was characterized using molecular cytogenetic techniques. Spectral karyotyping (SKY) showed that chromosome 11 material was added to 15p, forming a der(15)t(11;15), which was refined to der(15)t(11;15)(q13.2;p13) with information obtained by comparative genomic hybridization (CGH). Interstitial insertion of chromosome 15 material into chromosome 17q was found by fluorescence in situ hybridization (FISH) with whole chromosome painting (WCP) probes. This study illustrates the necessity of a combination of molecular cytogenetics to decipher complex karyotypic abnormalities and cryptic translocations in leukemia.     

Molecular characterization of the t(3;9) associated with immortalization in the MCF10A cell line

The t(3;9)(p14;p21) in the MCF10A human mammary gland epithelial cell line was the single cytogenetic event that accompanied the transition from primary culture to immortalized cell line, suggesting that it is related to the development of the immortalization phenotype. To study the molecular consequences of the breakpoints in this rearrangement, we used a combination of fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (CGH). The 3p14 translocation breakpoint occurs within BAC RP11-795e22, which accommodates only the TAFA1 gene, a novel cysteine-rich secreted protein thought to be involved in cytokine signaling. TAFA1 is expressed in normal breast tissue, not in MCF10A, and shows differential expression in a range of breast cancer cell lines. The 9p translocation breakpoint results in a deletion of approximately 4 megabases on the derivative chromosome 9, which includes the CDKN2A (p16) gene. Array CGH and FISH analysis demonstrated that BAC 149i22, which contains the CDKN2A/B genes, is also deleted specifically on the apparently normal copy of chromosome 9, making MCF10A null for the p16/p15 genes. The exact extent of gains and losses of chromosome regions resulting from rearrangements involving chromosomes 1q, 5q, and 8q have also been characterized using the BAC arrays.