Apparently unrelated cytogenetic abnormalities among 462 probands referred for the detection of del(22q) by FISH

Laboratory-based reports of the cytogenetic abnormalities detected during the course of testing for deletion del(22q) are scant. We report our findings from the testing with FISH of 462 patients suspected to have del(22q) between 1994 and 2000. Of these, 447 had a normal karyotype. An apparently unrelated cytogenetic abnormality was detected in 15 (3.2%). Two of these abnormalities involved reciprocal translocation with chromosome 22q and one of these showed del(22q) with FISH. The other abnormalities included sex chromosome aneuploidies and unbalanced rearrangements of various chromosomal segments. There was no commonality among these abnormalities and no correlation with other reported cases. Among those with a normal karyotype, an unexpected deletion of the control arylsulphatase A (ARSA) probe was found, providing a definite frequency of 1/262 for ARSA deletions among patients suspected to have del(22q). Of the 462 referrals, 48 (10%) had one or more additional diagnoses, and in this group, 4 (8%) had del(22q) and 2 (4%) had an apparently unrelated cytogenetic abnormality. The data highlight the importance of initial cytogenetic analysis in patients suspected of del(22q) and negates the use of interphase FISH screening by itself for del(22q). The finding of 3.2% unrelated cytogenetic abnormalities is noteworthy. FISH should be used in any structural rearrangement to ascertain if the relevant locus is deleted or not. The continued reporting of patients diagnosed with del(22q) found to have an unrelated cytogenetic abnormality will expand the phenotypic spectrum and possible gene mapping may refine phenotypic specificity.     

Cytogenetic and molecular genetic characterization of papillary thyroid carcinomas.

A combined cytogenetic and molecular analysis was performed on 11 cases of papillary thyroid carcinoma. A simple karyotypic abnormality was detected in five tumors, whereas six had no apparent chromosome change. In four of five rearranged cases the presence of a specific chromosomal abnormality involving chromosome 10 (cases 1 and 2) and chromosome 1 (cases 3 and 4) was associated with the rearrangement of two protooncogenes: RET and NTRKI (formerly trk), respectively, with different donor genes. Moreover, the chromosomal localization of the involved genes and the type of chromosomal change observed suggested that RET and NTRKI activation occurred by intrachromosomal rearrangements. The six cases with normal karyotype did not show RET or NTRKI activation. These findings suggest that a combined cytogenetic and molecular approach would be useful in understanding the pathogenesis of thyroid neoplasia.     

Complex cytogenetic changes in benign neoplasms. Report of six lipomas

We report the detailed cytogenetic findings from short-term cultures of six lipomas with complex chromosomal abnormalities. In all six cases, the abnormality occurred in two stages; an initial inversion, translocation, or insertion of the involved chromosome(s) followed by a subsequent rearrangement of the resultant derivative chromosome(s). A striking feature of these rearrangements was the consistent involvement of bands q13 - q14 on chromosome 12 in all the abnormalities. This region has been shown to be specifically rearranged in most of the lipomas studied. The other chromosomes involved in the rearrangements were chromosomes 1 in four cases, 5 and 9 in two cases each, and 2, 3, 4, 7, and 10 in one case each. Our findings and published findings show that, with a few exceptions, benign tumors that were previously considered cytogenetically normal, are characterized not only by specific numerical and structural changes but may also contain complex chromosome rearrangements that are generally considered a hallmark of advanced malignancy. In benign tumors, this suggests that the genes at the region of the breakpoints may represent proliferation-related genes or that benign tumors with such complex aberrations represent neoplasms potentially capable of undergoing transformation to malignancy, or both.     

Fusion of the EWS Gene to a DNA segment from 9q22-31 in a human myxoid chondrosarcoma

Southern blot analyses revealed a rearrangement of the EWS gene in a skeletal human myxoid chondrosarcoma. Interphase fluorescence in situ hybridization (FISH) studies, using cosmid clones F7 and G9 that flank the EWS locus on 22q12, confirmed the presence of this EWS gene abnormality. Cloning the rearranged EWS DNA fragment and mapping by FISH demonstrated that the EWS gene is joined to DNA sequences localised in 9q22-31. These findings are consistent with previous cytogenetic reports of a recurrent t(9;22)(q22-31;q11-12) in the myxoid variant of chondrosarcoma and reveal involvement of the EWS gene in a fourth type of human sarcoma.     

Amplification of 4q21-q22 and the MXR gene in independently derived mitoxantrone-resistant cell lines

Molecular cytogenetic studies were conducted on three multidrug-resistant cancer sublines which are highly resistant to the chemotherapeutic agent mitoxantrone, an anthracenedione. The three independently selected sublines were derived by exposure to mitoxantrone or Adriamycin and do not overexpress MDR1 or MRP. Two sublines, MCF-7 AdVp3000 and MCF-7 MX, showed an amplification peak at 4q21-q22, as demonstrated by comparative genomic hybridization (CGH), while the third, S1-M1-80, did not. FISH using a whole chromosome 4 paint demonstrated multiple rearrangements involving chromosome 4 in MCF-7 AdVp3000 and MCF-7 MX, while S1-M1-80 contained only a simple reciprocal translocation. The parental cell lines had no chromosome 4 rearrangements and no copy number gain or amplification of chromosome 4. Spectral karyotyping (SKY) analysis revealed a balanced translocation, t(4;17)(q21-q22;p13) in S1-M1-80 and multiple clonal translocations involving chromosome 4 in MCF-7 AdVp3000 and MCF-7 MX. A novel cDNA, designated MXR, which encodes an ABC half-transporter and is highly overexpressed in the three sublines, was localized to chromosome 4 by somatic cell hybrid analysis. Southern blot analysis demonstrated amplification of the MXR gene in MCF-7 AdVp3000 and MCF-7 MX, but not in S1-M1-80. FISH studies with a BAC probe for MXR localized the gene to 4q21-22 in the normal chromosome 4 and revealed in both MCF-7 AdVp3000 and MCF-7 MX amplification of MXR at one translocation juncture, shown by SKY to be t(4;5)(4qter-->4cen-->4q21-22::5q13-->5qter++ +) in MCF-7 AdVp3000 and t(6;4;6;3)(6pter-->6q15::4q21-q22::hsr::6q?::3q?27-->+ ++3qter) in MCF MX; neither of the breakpoints in the partner chromosomes showed amplification by CGH. The data are consistent with the hypothesis of a transporter, presumably that encoded by the MXR gene, mediating mitoxantrone resistance. The MXR gene encodes a half-transporter and the absence of cytogenetic evidence of coamplification of other regions suggests that a partner may not be overexpressed, and instead the MXR half-transporter homodimerizes to mediate drug transport. Genes Chromosomes Cancer 27:110-116, 2000. Published 2000 Wiley-Liss, Inc.     

Comparative genomic hybridization detects multiple chromosomal amplifications and deletions in undifferentiated embryonal sarcoma of the liver

Undifferentiated embryonal sarcoma (UES) is the third most common hepatic malignancy in children. Previous reports have described a broad range of complex cytogenetic abnormalities in individual cases of hepatic UES. Herein we report the cytogenetic findings of six cases of hepatic UES at our institution analyzed by conventional cytogenetic methods and comparative genomic hybridization (CGH). The CGH demonstrated several chromosomal gains and deletions in each case, but there was no specific abnormality seen in every case. Patterns of chromosomal changes included gains of chromosome 1q (four cases), 5p (four cases), 6q (four cases), 8p (three cases), and 12q (three cases), and losses of chromosome 9p (two cases), 11p (two cases), and chromosome 14 (three cases). The three cases in which CGH showed gains in the 12q region were studied specifically for amplifications of MDM2 and CDK4, two genes that have been shown to be amplified in other soft tissue sarcomas. However, Southern analysis showed no amplification of MDM2 or CDK4 in these three cases. Further analysis will be needed to determine the critical events in the pathogenesis of these malignant pediatric liver tumors.     

Involvement of chromosomes 17 and 22 in dermatofibrosarcoma protuberans

Literature on the cytogenetics of dermatofibrosarcoma protuberans (DFSP) is limited; only I0 cases with chromosome aberrations have been reported. They are karyotypically characterized by the presence of supernumerary ring(s), either as the sole cytogenetic abnormality or together with a few additional structural or numerical changes. We report the cytogenetic and fluorescence in situ hybridization (FISH) analysis of three new DFSP, one primary and two recurrent tumors. In two cases we found a supernumerary ring as the sole change, whereas the third had two copies of a marker chromosome and monosomy of chromosome 22. Sequences of chromosomes I7 and 22 were identified by FISH in the supernumerary rings and in the markers. The fluorescence pattern suggested that additional sequences were present in the two rings, but showed that the marker chromosomes were entirely painted by chromosome 17 and 22 probes. The findings indicate that juxtaposition and/or amplification of chromosome 17 and 22 sequences could be crucial in the pathogenesis of DFSP. © 1995 Wiley-Liss, Inc.     

Cytogenetic and molecular cytogenetic analyses in diffuse astrocytomas

Diffuse astrocytomas are highly variable tumors and show complex biologic behavior that is based on multi-step oncogenesis. We report cytogenetic and molecular cytogenetic investigations in 23 cases of diffuse astrocytomas. The results of conventional karyotyping, interphase fluorescence in situ hybridization (FISH), comparative genomic hybridization, multicolor FISH, and spectral karyotyping are reported. Various numerical and structural chromosomal aberrations were identified. Clustering of structural alterations in the short arm of chromosome 2 (2p) and the long arm of chromosome 7 (7q) were detected. Using spectral karyotyping, additional chromosome rearrangements not detectable by conventional methods were found. Some of these anomalies have not been previously described in diffuse astrocytomas. An independent validation of these discrepant findings is required.     

Karyotypic abnormalities and immunoglobulin gene rearrangements in Hodgkin's disease

Biopsy samples from seven patients with Hodgkin's disease (HD) were examined for cytogenetic abnormalities and rearrangement of the genes encoding the immunoglobulin chains and T-cell receptor chains. Three samples demonstrated clonal rearrangements of both IgH and IgL genes. No rearrangements of the TCR beta genes were detected in any of the samples. Karyotypic abnormalities were also found but only in the three cases where a clonal rearrangement of the immunoglobulin genes was shown. Two of these three cases had multiple karyotypic abnormalities, with the remaining patient being trisomic for chromosome 16 as the sole abnormality. These results are discussed and compared with previous reports in the literature concerning HD.     

Diffuse large B-cell lymphoma with TEL/ETV6 translocation

Cytogenetic abnormalities of chromosome 12p involving the TEL/ETV6 gene are observed in a variety of hematopoietic neoplasms including acute leukemias, myelodysplastic syndromes, and myeloproliferative disorders. Karyotypic aberrations, including rearrangements, deletions, and amplifications of chromosome 12p, have been documented in B-cell non-Hodgkin lymphoma; however, rearrangements targeting TEL have rarely been reported. Here we describe a diffuse large B-cell lymphoma that had a complex karyotype including t(9;12)(q22;p13), which was confirmed by fluorescence in situ hybridization to represent rearrangement of TEL. Additional cytogenetic abnormalities included t(3;14)(q27;q32) involving the variant, alternative breakpoint region of the BCL6 gene and del(6)(q13q23), resulting in the loss of 1 allele of BLIMP1. This case reiterates the importance of correlating morphologic and phenotypic findings with the results of cytogenetic analysis to avoid errors in diagnosing hematologic neoplasms and highlights the rare association of B-cell non-Hodgkin lymphoma with aberrations of TEL.     

Chromosomal in situ hybridization and Southern blot analyses using c-abl, c-sis, or bcr probe in chronic myelogenous leukemia cells with variant Philadelphia translocations

The Philadelphia (Ph) chromosome is a cytogenetic hallmark of chronic myelogenous leukemia (CML). Whereas the majority of Ph-positive CML patients show the standard Ph translocation involving chromosomes 9 and 22, t(9;22)(q34;q11), the minority of cases exhibit a variant type of Ph translocation involving these two and other chromosomes (complex type) or those involving #22 and chromosomes other than #9 (simple type). To get an insight into the nature of variant Ph translocations and the process of their formation, we examined the localization of the c-abl and c-sis oncogenes and the breakpoint cluster region (bcr) gene by chromosomal in situ hybridization in ten variant Ph translocations of CML including five simple and five complex ones as initially interpreted. In situ hybridization showed that c-abl localized to band 9q34 and c-sis localized to band 22q12-q13 were translocated on the Ph and on one of the rearranged chromosomes other than #9, respectively, in all the variant translocations examined. On the other hand, bcr localized to band 22q11 was translocated on various chromosomes but mostly on chromosome 9. Parallel Southern blot analyses on DNA from leukemic cells of five patients including two with simple translocations and three with complex ones revealed rearrangements of bcr with breakpoints occurring mostly in a 5' portion of 5.8-kb BamHI/BglII sequences, which are quite similar to those detected so far in CML cases with the standard Ph translocation. The present findings strongly suggest that variant Ph translocations of CML are all complex, and some of them are formed stepwisely from the standard translocation.     

A fluorescence in situ hybridization study of complex t(9;22) in two chronic myelocytic leukemia cases with a masked Philadelphia chromosome

The t(9;22)(q34;q11) is evident in more than 90% of patients with chronic myelocytic leukemia (CML) and gives rise to the Philadelphia chromosome (Ph). Approximately 5%-10% of CML patients show variant translocations involving other chromosomes in addition to chromosomes 9 and 22. In some variant translocations, additional material is transferred on der(22), resulting in a masked Ph chromosome. In this paper, we report two apparently Ph-negative (Ph-) CML cases showing a t(7;9;22)(q22;q34;q11) and a t(8;9;22)(q12;q34;q11), respectively. A detailed molecular cytogenetic characterization was performed by fluorescence in situ hybridization (FISH), which disclosed the presence of the 5'BCR/3'ABL fusion gene on the der(7) and der(8) chromosomes, respectively. Derivative (22) appeared as a masked Ph chromosome in both cases. FISH analysis with appropriate BAC/PAC clones allowed us to precisely characterize the complex chromosomal rearrangements that were not detected by conventional cytogenetic analysis.     

Cytogenetic and molecular analysis of 6q deletions in burkitt's lymphoma cell lines

Although abnormalities of chromosome 6 have frequently been observed in Burkitt's lymphoma (BL), they have 50 far not been defined by modern cytogenetic and molecular methods. By a combination of high-resolution chromosome banding, fluorescence in situ hybridization (FISH), and loss of heterozygosity (LOH) analysis, we have examined the nature of aberrations affecting chromosome 6 in 7 previously established BL cell lines. All cell lines exhibited the characteristic translocations associated with BL; 5 had t(814)(q24;q32) and 2 had t(8;22)(q24;q11). Three cell lines had deletions of 6q; 3 others had rearrangements affecting 6q, whereas one cell line had apparently normal chromosomes 6. FISH analysis of the three deletions established that they were interstitial. LOH analysis with probes mapped to the 6q26-27 region confirmed the sub-telomeric interstitial deletion in cell line BL-108, which had a del(6)(q23q27). All informative loci mapped to 6q26-27 (5/7) were deleted in BL-74, which had no apparent cytogenetic abnormality in chromosome 6, thus documenting a sub-microscopic deletion. These data define the cytogenetic and molecular limits of 6q deletions in BL and are consistent with our previous demonstration of LOH analysis of the site of a candidate tumor suppressor gene in the 6q25-27 region. Genes Chrom Cancer 9:13-18 (1994). ©1994 Wiley-Liss, Inc.     

Molecular and cytogenetic analysis of tumors in von Recklinghausen neurofibromatosis

Von Recklinghausen neurofibromatosis (NF1) is a common autosomal dominant disorder mapped to 17q11.2 and typically characterized by the occurrence of neural crest-derived tumors. The gene has recently been cloned using reverse genetics or "positional cloning" approaches. Its function, however, remains unknown. We have performed cytogenetic and molecular analyses on 9 malignant tumors from NF1 patients to look for loss of alleles or chromosome rearrangements involving chromosome 17 to test the hypothesis that the NF1 gene acts as a recessive "tumor suppressor" gene. Loss of alleles on this chromosome was detected for 3 of 9 malignant tumors. Two peripheral nerve sheath tumors showed allele loss at informative loci on both the long and short arms of chromosome 17. In contrast, a glioblastoma with focal gliosarcoma showed loss of heterozygosity on the short arm of chromosome 17 only, and not at loci on the long arm. One nerve sheath tumor was previously shown by direct sequence analysis to have a point mutation at the TP53 locus at 17p13. These data support a role for the TP53 gene or other genes on the short arm of chromosome 17 in at least some malignancies in NF1. Six other neurofibrosarcomas showed no allele loss at informative loci on chromosome 17. Cytogenetic analysis was performed on 7 tumors, including 2 with allele loss. The two tumors with allele loss showed abnormal karyotypes while all others were normal. Southern blot and pulsed-field gel analysis using probes within or closely linked to the NF1 locus detected no gross deletions or rearrangements in the tumors studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Abnormally banded region in a poorly differentiated sarcoma is not correlated with amplification of c-MYC or c-MOS protooncogenes

A poorly differentiated sarcoma in a 32-year-old female revealed a large, abnormally banded region in one chromosome #8 in all metaphases. The modal karyotype was 46,XX, -8, +mar. Southern blot hybridization was performed with probes for two protooncogenes located in chromosome 8q (c-MYC and c-MOS). No amplification or rearrangement was observed to account for the cytogenetic abnormality.     

Transposition of breakpoint cluster region (3' bcr) in CML cells with variant Philadelphia translocations

A probe derived from the 3' end of the CML breakpoint cluster region (bcr) was localized in chronic myelocytic leukemia (CML) cases with complex Philadelphia translocations, [t(8;9;22)(q13;q34;q11) and t(12;9;22)(p11;q34;q11)], and with "masked" Ph chromosomes, [t(9;5;22)(q34;q31;q11) and t(9;22)(q22;q34)], by a chromosomal in situ hybridization technique. In some cases, the 3' bcr rearrangements in the DNA were examined with Southern blot analysis. In each case, a significant accumulation of grains hybridized to the 3' bcr probe was observed at chromosomal segments derived from the long arm of a chromosome #22. In some cases, the accumulation of grains was detected on both translocated segments derived from the 22q and terminal portions of Ph chromosomes; Southern blot analysis revealed that breakage in chromosome #22 occurred within DNA sequences of the 3' bcr probe involved in the Ph translocations. In a CML cell line, K562, no accumulation of grains hybridized to the 3' bcr probe was detected, except at 22q11 of the normal chromosomes #22; Southern blot analysis of this cell line revealed that the 3' bcr sequences were missing. Thus, the data presented here suggest a complexity in the formation of "masked" Ph chromosomes.