Molecular cytogenetic characterization of early and late renal cell carcinomas in von Hippel-Lindau disease

Deletions of 3p25, gains of chromosomes 7 and 10, and isochromosome 17q are known cytogenetic aberrations in sporadic renal cell carcinoma (RCC). In addition, a majority of RCCs have loss of heterozygosity (LOH) of the Von Hippel-Lindau (VHL) gene located at chromosome band 3p25. Patients who inherit a germline mutation of the VHL gene can develop multifocal RCCs and other solid tumors, including malignancies of the pancreas, adrenal medulla, and brain. VHL tumors follow the two-hit model of tumorigenesis, as LOH of VHL, a classic tumor suppressor gene, is the critical event in the development of the neoplastic phenotype. In an attempt to define the cytogenetic aberrations from early tumors to late RCC further, we applied spectral karyotyping (SKY) to 23 renal tumors harvested from 6 unrelated VHL patients undergoing surgery. Cysts and low-grade solid lesions were near-diploid and contained 1-2 reciprocal translocations, dicentric chromosomes, and/or isochromosomes. A variety of sole numerical aberrations included gains of chromosomes 1, 2, 4, 7, 10, 13, 21, and the X chromosome, although no tumors had sole numerical losses. Three patients shared a breakpoint at 2p21-22, and three others shared a dicentric chromosome 9 or an isochromosome 9q. In contrast to the near-diploidy of the low-grade lesions, a high-grade lesion and its nodal metastasis were markedly aneuploid, revealed loss of VHL by fluorescence in situ hybridization (FISH), and contained recurrent unbalanced translocations and losses of chromosome arms 2q, 3p, 4q, 9p, 14q, and 19p as demonstrated by comparative genomic hybridization (CGH). By combining SKY, CGH, and FISH of multiple tumors from the same VHL kidney, we have begun to identify chromosomal aberrations in the earliest stages of VHL-related renal cell tumors. Our current findings illustrate the cytogenetic heterogeneity of different VHL lesions from the same kidney, which supports the multiclonal origins of hereditary RCCs. Published 2001 Wiley-Liss, Inc.     

Molecular cytogenetic characterization of nasopharyngeal carcinoma cell lines and xenografts by comparative genomic hybridization and spectral karyotyping

Nasopharyngeal carcinoma (NPC) cell lines and xenografts represent valuable models for functional and therapeutic studies on this common malignancy in Southeast Asia. The karyotypic information in most NPC cell lines and xenografts, however, remains largely unclear to date. We have characterized the chromosomal aberrations in six commonly used human NPC cell lines and xenografts using the molecular cytogenetic technique of comparative genomic hybridization (CGH). Genomic imbalances identified in cell lines were further correlated with structural abnormalities indicated from spectral karyotyping (SKY) analysis. CGH revealed consistent overrepresentations of 8q (six out of six cases) with a smallest overlapping region identified on 8q21.1q22. Other common gains included 7p (4/6 cases), 7q (4/6 cases), 12q (4/6), and 20q (4/6 cases), where minimal overlapping regions were suggested on 7p15p14, 7q11.2q21, and 12q22q24.1. Common losses were detected on 3p12p21 (4/6 cases) and 11q14qter (4/6 cases). Although SKY analysis on cell lines revealed predominantly unbalanced rearrangements, reciprocal translocations that involved chromosome 2 [i.e., t(1;2), t(2;3), and t(2;4)] were suggested. Furthermore, SKY examination illustrated additional breakpoints on a number of apparently balanced chromosomes. These breakpoints included 3p21, 3q26, 5q31, 6p21.1p25, 7p14p22, and 8q22. Our finding of regional gains and losses and breakpoints represents information that may contribute to NPC studies in vitro.     

A combination of molecular cytogenetic analyses reveals complex genetic alterations in conventional renal cell carcinoma

Here we report the complex pattern of genomic imbalances and rearrangements in a panel of 19 renal cell carcinoma cell lines detected with molecular cytogenetic analysis. Consistent heterogeneity in chromosome number was found, and most cell lines showed a near-triploid chromosome complement. Several cell lines showed deletions of the TP53 (alias p53), CDKN2A (alias p16), and VHL genes. Multiplex fluorescence in situ hybridization (M-FISH) analysis revealed chromosome 3 translocated to several other partners chromosomes, as well as breakage events commonly affecting chromosomes 1, 5, 8, 10, and 17. The most common abnormality detected with comparative genomic hybridization (CGH) was deletions of chromosome 3p, with loss of the RASSF1, FHIT, and p44S10 loci frequently involved. CGH gain of 5q showed overrepresentation of the EGR1 and CSF1R genes. Recurrent alterations to chromosome 7 included rearrangement of 7q11 and gains of the EGFR, TIF1, and RFC2 genes. Several lines exhibited rearrangement of 12q11 approximately q14 and overrepresentation of CDK4 and SAS loci. M-FISH revealed several other recurrent translocations, and CGH findings included loss of 9p, 14q, and 18q and gain of 8q, 12, and 20. Further genomic microarray changes included loss of MTAP, IGH@, HTR1B, and SMAD4 (previously MADH4) and gains of MYC and TOP1. An excellent correlation was observed between the genomic array and FISH data, demonstrating that this technique is effective and accurate. The aberrations detected here may reflect important pathways in renal cancer pathogenesis.     

Combined classical cytogenetics and microarray‐based genomic copy number analysis reveal frequent 3;5 rearrangements in clear cell renal cell carcinoma

Karyotypic analysis and genomic copy number analysis with single nucleotide polymorphism (SNP)‐based microarrays were compared with regard to the detection of recurrent genomic imbalances in 20 clear cell renal cell carcinomas (ccRCCs). Genomic imbalances were identified in 19 of 20 tumors by DNA copy number analysis and in 15 tumors by classical cytogenetics. A statistically significant correlation was observed between the number of genomic imbalances and tumor stage. The most common genomic imbalances were loss of 3p and gain of 5q. Other recurrent genomic imbalances seen in at least 15% of tumors included losses of 1p32.3‐p33, 6q23.1‐qter and 14q and gain of chromosome 7. The SNP‐based arrays revealed losses of 3p in 16 of 20 tumors, with the highest frequency being at 3p21.31‐p22.1 and 3p24.3‐p25.3, the latter encompassing the VHL locus. One other tumor showed uniparental disomy of chromosome 3. Thus, altogether loss of 3p was identified in 17 of 20 (85%) cases. Fourteen tumors showed both overlapping losses of 3p and overlapping gains of 5q, and the karyotypic assessment performed in parallel revealed that these imbalances arose via unbalanced 3;5 translocations. Among the latter, there were common regions of loss at 3p21.3‐pter and gain at 5q34‐qter. These data suggest that DNA copy number analysis will supplant karyotypic analysis of tumor types such as ccRCC that are characterized by recurrent genomic imbalances, rather than balanced rearrangements. These findings also suggest that the 5q duplication/3p deficiency resulting from unbalanced 3;5 translocations conveys a proliferative advantage of particular importance in ccRCC tumorigenesis. © 2010 Wiley‐Liss, Inc.     

Chromosomal alterations in 15 breast cancer cell lines by comparative genomic hybridization and spectral karyotyping

Breast cancer cell lines have been widely used as models in functional and therapeutical studies, but their chromosomal alterations are not well known. We characterized the chromosomal aberrations in 15 commonly used human breast carcinoma cell lines (BT-474, BT-549, CAMA-1, DU4475, MCF7, MDA-MB-134, MDA-MB-157, MDA-MB-361, MDA-MB-436, MPE600, SK-BR-3, T-47D, UACC-812, UACC-893, and ZR-75-1) by comparative genomic hybridization (CGH) and spectral karyotyping (SKY). By CGH the most frequent gains were detected at 1q, 8q, 20q, 7, 11q13, 17q, 9q, and 16p, whereas losses were most common at 8p, 11q14-qter, 18q, and Xq. SKY revealed a multitude of structural and numerical chromosomal aberrations. Simple translocations, typically consisting of entire translocated chromosome arms, were the most common structural aberrations. Complex marker chromosomes included material from up to seven different chromosomes. Evidence for a cytogenetic aberration not previously described in breast cancer, the isoderivative chromosome, was found in two cell lines. Translocations t(8;11), t(12;16), t(1;16), and t(15;17) were frequently found, although the resulting derivative chromosomes and their breakpoints were strikingly dissimilar. The chromosomes most frequently involved in translocations were 8, 1, 17, 16, and 20. An excellent correlation was found between the number of translocation events found by SKY in the individual cell lines, and the copy number gains and losses detected by CGH, indicating that the majority of translocations are unbalanced. Genes Chromosomes Cancer 28:308-317, 2000.     

Spectrum of genetic changes in gastro-esophageal cancer cell lines determined by an integrated molecular cytogenetic approach

Adenocarcinomas arising around the gastro-esophageal junction (GEJ) are highly malignant, and their incidence has risen rapidly in the last decades. Cell lines are the basic in vitro system for functional and therapeutic studies in GEJ tumors, but only a small number of cell lines are currently available, and none of them has been fully karyotyped. We analyzed 5 GEJ tumor cell lines using a combination of 24-color fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH) and genomic microarrays. Using CGH we demonstrated that these cell lines present imbalances similar to those we had previously observed in primary GEJ tumors, namely gains on 1q, 7q, 8q, 17q, 19q, 20, and X, and losses on 3p, 4, 5q, 9p, 18q, and 21. Multicolor FISH karyotyping revealed multiple structural rearrangements involving chromosomes 1, 5, 6, 7, 8, 9, 13, 17, 18, and 22. Rearrangements of chromosome 8 involved 10 different chromosomes, while rearrangements of chromosome 17 involved 5. Different rearrangements resulted in imbalances of similar chromosome regions, suggesting that similar genomic imbalances are constitutively important but are achieved through different pathways. The use of a commercially available genomic array excluded TOP2A (17q), and MYBL2, PTPT1, CSE1L, and ZNF217 (20q) as candidate genes for frequently amplified areas on these chromosomes, and contributed to refining the limits of chromosome regions involved in genomic imbalances.     

Multiple reciprocal translocations in salivary gland mucoepidermoid carcinomas

Mucoepidermoid carcinoma, the most common human malignant salivary gland tumor, can arise from both major and minor salivary glands, including sites within the pulmonary tracheobronchial tree. We performed comparative genomic hybridization (CGH) and spectral karyotyping (SKY) on two tumor cell lines: H3118, derived from tumor originating in the parotid gland, and H292, from tumor in the lung. In both cell lines, CGH showed a partial gain within the short arm of chromosome 7 and SKY revealed the presence of the previously reported reciprocal translocation t(11;19)(q21;p12). Additional chromosomal rearrangements were found in both cell lines, including three more reciprocal translocations in cell line H292 [t(1;16), t(6;8)x2] and three other reciprocal translocations in cell line H3118 [t(1;7), t(3;15), and t(7;15)]. A review of the literature of other reported cases of mucoepidermoid carcinomas analyzed with standard G-banding techniques, as well as distinct benign salivary gland tumors, such as pleomorphic adenomas and Warthin tumor, confirmed the presence of a karyotype dominated by reciprocal translocations. Four chromosomal bands were involved in chromosomal translocations in both cell lines: 1q32, 5p15, 7q22, and 15q22. Fluorescence in situ hybridization studies showed that the breakpoints in these four bands were often within a few megabases of each other. The involvement of similar chromosomal bands in breakpoints in these two cell lines suggests that these regions may be predisposed or selected for chromosomal rearrangements in this tumor type. The presence of multiple reciprocal translocations in both benign and malignant salivary gland tumors may also suggest a particular mechanism within mucous or serous glands mediating chromosomal rearrangements.     

Molecular cytogenetic evaluation of 10 uveal melanoma cell lines

Uveal melanoma is the most common intraocular tumor in adults and often results in unilateral blindness and/or death. Previous cytogenetic characterizations of this tumor consistently revealed chromosomal abnormalities involving chromosomes 3, 6, and 8; reports of other abnormalities vary in frequency. We defined cytogenetic abnormalities of this tumor using complementary in situ hybridization techniques on 10 uveal melanoma cell lines. Synthesis of comparative genomic hybridization (CGH) and spectral karyotyping (SKY) results revealed that chromosomal rearrangement is involved in DNA sequence copy number abnormalities throughout the genome, but monosomy 3 was not found. Monosomy 3 is thought to be a significant prognostic indicator, so its absence was investigated further. Fluorescence in situ hybridization (FISH) for chromosome 3 revealed approximately 1 centromere signal per cell, but probes for 3p and 3q revealed multiple telomere signals per cell, suggesting chromosomal rearrangement without whole-chromosome loss. Based on combined CGH, SKY, and FISH data, we propose that chromosome 3 is more frequently involved in chromosomal rearrangements than whole-chromosome loss in uveal melanoma. Future approaches should be designed to confirm and enhance the resolution of regions of imbalance in primary tumors. Once identified, conserved chromosomal alterations that contribute to uveal melanoma may reveal the underlying aspects of uveal melanoma onset, metastasis and resistance to current treatment modalities.     

Comparative genomic hybridization analysis identifies gains of 1p35 approximately p36 and chromosome 19 in osteosarcoma.

Osteosarcomas (OS) are aggressive tumors of the bone and often have a poor prognosis. Conventional cytogenetic analyses of OS have revealed highly complex karyotypes, with numerous abnormalities. In this study, we analyzed 18 untreated OS tumors from 17 patients of the younger incidence age group by comparative genomic hybridization (CGH), 4 tumors by spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH). Comparative genomic hybridization identified frequent copy number changes of the chromosomal region 1p (10/17) and gain of part or all of chromosome 19(8/17). In addition gains were observed at 5p(3/17), 8q(3/17), 16p(3/17), and 17p(5/17); and losses at chromosomes 2q(3/17), 10(4/17) and 13(3/17). High level gains were detected in the 8q23 approximately q24 region in two tumors as well as at 17p in one primary and a metastatic tumor. Minimal regions of gain were present at 1p35 approximately p36.3 (8/17); 5p14 approximately p15.2 (3/17), and 8q22 approximately q24.3 (3/17). SKY analysis demonstrated that OS has a complex pattern of clonal and non-clonal rearrangements and helped confirm the structural basis for the imbalances detected by CGH. Spectral karyotyping confirmed an overall pattern of chromosomal gain affecting 1p in all four tumors. Fluorescence in situ hybridization analysis from these tumors confirmed the gain of the 1p36 region in 2 tumors as determined by CGH analysis as well as the amplification of 8q.     

Definitive molecular cytogenetic characterization of 15 colorectal cancer cell lines

In defining the genetic profiles in cancer, cytogenetically aberrant cell lines derived from primary tumors are important tools for the study of carcinogenesis. Here, we present the results of a comprehensive investigation of 15 established colorectal cancer cell lines using spectral karyotyping (SKY), fluorescence in situ hybridization, and comparative genomic hybridization (CGH). Detailed karyotypic analysis by SKY on five of the lines (P53HCT116, T84, NCI‐H508, NCI‐H716, and SK‐CO‐1) is described here for the first time. The five lines with karyotypes in the diploid range and that are characterized by defects in DNA mismatch repair had a mean of 4.8 chromosomal abnormalities per line, whereas the 10 aneuploid lines exhibited complex karyotypes and a mean of 30 chromosomal abnormalities. Of the 150 clonal translocations, only eight were balanced and none were recurrent among the lines. We also reviewed the karyotypes of 345 cases of adenocarcinoma of the large intestine listed in the Mitelman Database of Chromosome Aberrations in Cancer. The types of abnormalities observed in the cell lines reflected those seen in primary tumors: there were no recurrent translocations in either tumors or cell lines; isochromosomes were the most common recurrent abnormalities; and breakpoints occurred most frequently at the centromeric/pericentromeric and telomere regions. Of the genomic imbalances detected by array CGH, 87% correlated with chromosome aberrations observed in the SKY studies. The fact that chromosome abnormalities predominantly result in copy number changes rather than specific chromosome or gene fusions suggests that this may be the major mechanism leading to carcinogenesis in colorectal cancer. Published 2009 Wiley‐Liss, Inc.     

Multicolor spectral karyotyping of serous ovarian adenocarcinoma.

We applied multicolor spectral karyotyping (SKY) to decipher the chromosomal complexity of a panel of seven cell lines and four primary tumors derived from patients with high‐grade serous adenocarcinoma of the ovary. By this method we identified a total of 188 unbalanced translocations, nine reciprocal translocations [t(2;15)(q13;q23), t(7;17) (q32;q21), t(8;22)(p11;q11), t(8;22) (q24;q13), t(10;19) (q24;q13.2), t(11;19) (q13;p11), t(12;21)(q13;q22),t(18;20) (q?11;q?11), t(18;22)(q?11;q?13)], 6 isochromosomes [i(1q), i(7q), i(8q), i(9p), i(17q), i(21q)], and 23 deletions. By detailed mapping of rearrangement breakpoints, it was possible to identify several recurring breakpoint clusters at chromosomal bands 1p36, 2p11, 2p23, 3p21, 3q21, 4p11, 6q11, 8p11, 9q34, 10p11, 11p11, 11q13, 12p13, 12q13, 17q21, 18p11, 18q11, 20q11, and 21q22. Recurrent interstitial deletion of chromosomal bands 8p11, 11p11, and 12q13 and a recurrent unbalanced translocation—der(6)t(6;8)(q11;q11)—were also identified. In addition, a homogeneously staining region localized in one cell line to 11q13 was found using SKY to be derived from genetic material originating from chromosome 12. Subsequent comparative genomic hybridization (CGH) studies on this tumor revealed the amplification of DNA sequences derived from the short arm of chromosome 12 at the 12p11.2 region. These studies demonstrate the power of SKY, CGH, and G‐banding to resolve the full spectrum of chromosomal rearrangements in serous ovarian adenocarcinoma. © 2002 Wiley‐Liss, Inc.     

Molecular cytogenetic analysis of oral squamous cell carcinomas by comparative genomic hybridization, spectral karyotyping, and fluorescence in situ hybridization

We investigated relationships between DNA copy number aberrations and chromosomal structural rearrangements in 11 different cell lines derived from oral squamous cell carcinoma (OSCC) by comparative genomic hybridization (CGH), spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH). CGH frequently showed recurrent chromosomal gains of 5p, 20q12, 8q23 approximately qter, 20p11 approximately p12, 7p15, 11p13 approximately p14, and 14q21, as well as losses of 4q, 18q, 4p11 approximately p15, 19p13, 8p21 approximately pter, and 16p11 approximately p12. SKY identified the following recurrent chromosomal abnormalities: i(5)(p10), i(5)(q10), i(8)(q10), der(X;1)(q10;p10), der(3;5)(p10;p10), and der(3;18)(q10;p10). In addition, breakpoints detected by SKY were clustered in 11q13 and around centromeric regions, including 5p10/q10, 3p10/q10, 8p10/q10 14q10, 1p10/1q10, and 16p10/16q10. Cell lines with i(5)(p10) and i(8)(q10) showed gains of the entire chromosome arms of 5p and 8q by CGH. Moreover, breakages near the centromeres of chromosomes 5 and 8 may be associated with 5p gain, 8q gain, and 8p loss in OSCC. FISH with a DNA probe from a BAC clone mapping to 5p15 showed a significant correlation between the average numbers of i(5)(p10) and 5p15 (R(2) = 0.8693, P< 0.01) in these cell lines, indicating that DNA copy number of 5p depends upon isochromosome formation in OSCC.     

Characterization of the human myeloid leukemia-derived cell line GF-D8 by multiplex fluorescence in situ hybridization, subtelomeric probes, and comparative genomic hybridization.

The human myeloid leukemia cell line GF-D8 was established from the peripheral blood blasts of a patient with acute myeloid leukemia FAB subtype MI (AML-MI). The karyotype, which has not changed significantly over several years of culture, was described initially as 44,XY,-5,del(7q),inv(7q),add(8q),add(11q),del(12p),-15,-17,+mar. With the advent of multicolor fluorescence in situ hybridization (FISH) techniques, the prospect of accurately characterizing this complex karyotype became feasible. In the present study, we applied 24-color whole-chromosome painting and analyzed the results using a filter-based detection system and proprietary software for multiplex FISH (M-FISH). This resulted in the refinement of the karyotype and the identification of hitherto unsuspected chromosome rearrangements. M-FISH identified the origin of the add(8q) and add(11q) as well as the small marker chromosome. Both the del(7q) and del(12p) were redefined as unbalanced translocations and an apparently normal chromosome 11 was shown to be t(11;17). Importantly, the del(12p) was shown to be a der(12)t(7;12). Single-color whole-chromosome painting studies confirmed these findings, but also identified a cryptic t(Y;12) not seen in the original M-FISH analysis. We then carried out a FISH screening assay using a complete set of chromosome-specific subtelomeric probes. This allowed the identification of p and q subtelomeric regions involved in the translocations and indicated amplification of the 8q subtelomeric region. Comparative genomic hybridization (CGH) revealed a highly unbalanced karyotype, as deletions accompanied the majority of translocations, and identified the regions of amplification as 8q22.3-qter and 11q21-qter. Finally, conventional FISH with centromeric and unique sequence probes was necessary to elucidate all of the rearrangements.     

Combined 24-color karyotyping and comparative genomic hybridization analysis indicates predominant rearrangements of early replicating chromosome regions in neuroblastoma

Neuroblastoma is characterized by several distinct genetic alterations including MYCN amplification, chromosome 1p deletion and gain of chromosome 17. Although these alterations are thought to play a crucial role in oncogenesis, to date little is known about their underlying mechanisms. In order to more precisely document these genetic alterations, we have performed a combined study of 27 neuroblastoma cell lines using 24-color karyotyping (24-CK) and comparative genomic hybridization (CGH). 24-CK detected balanced translocations in 13 cases with recurrent involvement of chromosome 8. More importantly, 144 nonreciprocal translocations were observed in the 27 cell lines, with chromosome 1 as the most frequent recipient and chromosome 17 the most frequent donor. Each cell line exhibited at least one unbalanced translocation involving 17q, with 14 cell lines demonstrating more than one such translocation. Other recurrent alterations were amplification of the 2p24 chromosome region, which encodes the MYCN oncogene, losses of 1p, 3p and 11q, and gains of 1q and 7. In most cases, CGH profiles were directly linked to the presence of unbalanced translocations with gain of the donor fragment and loss of the replaced region on the recipient chromosome. Strikingly, over 60% of the chromosome breakpoints mapped to early replicating chromosome bands, which represent around 13% of the genome. Altogether these data suggest that neuroblastoma is characterized by rearrangements that predominantly involve chromosome fragments replicating early in the S-phase.     

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.     

A comprehensive karyotypic analysis on a newly developed hepatocellular carcinoma cell line, HKCI-1, by spectral karyotyping and comparative genomic hybridization

A continuously growing human hepatocellular carcinoma (HCC) cell line was established from a Chinese male, carrier of the hepatitis B virus (HBV). This cell line, designated HKCI-1, grows as an adhering monolayer of polygonal epithelial cells that embody one or more nuclei. HKCI-1 secretes alpha-fetoprotein but shows no evidence of HBV carriage. Conventional banding analysis of the short-term cultured primary tumor and the propagated HKCI-1 revealed a chromosome modal number of near-triploidy. It was, however, impossible to derive their complete karyotype due to the complex nature of chromosomal rearrangements and many marker chromosomes of uncertain origin. Spectral karyotyping (SKY) is a newly developed molecular cytogenetic technique that allows the unprecedented discernment of chromosomal abnormalities. Spectral karyotyping analysis on HKCI-1 and the primary tumor elucidated all aberrant chromosomes and revealed complex karyograms. Recurring aberrations detected in both primary tumor and HKCI-1 included der(X)t(X;11)(q10;p10), der(1)t(1;10)(q10;?pq), der(4)t(4;16)(p10;q10), i(5p), del(5)(q13), der(7)t(7;21)(q32q10::q10), der(8)t(8;17)(q10;p10), and der(9)t(9;22)(q34;?pq). Comparative genomic hybridization (CGH) was employed to monitor the culture evolution in vitro. Genomic imbalances in HKCI-1 involved chromosomal losses on 4q, 5q13-qter, 8p, 9pter-q33, 10q, 11q, 13q, 16q, 17q12-qter, and 22, and low-level gains on 6pter-q22, 7p, 8q, 9q34, 10p, 11p, 12, 17pter-q11.2, 18, 19, 20, 21, and Y. High-level amplifications were also detected on 5pter-q12, 7q11.2-qter, and Xq. The corresponding CGH finding on the primary tumor indicated similar imbalances. TP53 mutational analysis showed that both HKCI-1 and the primary tumor had the aflatoxin-associated mutation in codon 249 and an additional TP53 polymorphism in codon 72. Our present study demonstrates the value of combined SKY and CGH study in defining complex rearrangements and identifying cryptic translocations, and provides a comprehensive analysis on the chromosomal abnormalities in HKCI-1.     

Molecular cytogenetic characterization of human papillomavirus16-transformed foreskin keratinocyte cell line 16-MT

Anogenital cancers are closely associated with human papillomavirus (HPV), and HPV-infected individuals, particularly those with high-grade dysplasias, are at increased risk for cervical and anal cancers. Although genomic instability has been documented in HPV-infected keratinocytes, the full spectrum of genetic changes in HPV-associated lesions has not been fully defined. To address this, we examined an HPV16-transformed foreskin keratinocyte cell line, 16-MT, by GTG-banding, spectral karyotyping (SKY), and array comparative genomic hybridization (array CGH); these analyses revealed multiple numerical, complex, and cryptic chromosome rearrangements. Based on GTG-banding, the 16-MT karyotype was interpreted as 78-83,XXY,+add(1)(p36.3),+3,+4,+5,+5,+7,+8,+i(8)(q10)x2,+10,?der(12),der(13;14)(q10;q10),+15,+16,add(19)(q13.3),+21,+21,-22[cp20]. Multicolor analysis by SKY confirmed and further characterized the anomalies identified by GTG banding. The add(1) was identified as a der(1)(1qter-->1q25::1p36.1-->1qter), the add(19) as a dup(19), and the der(12) interpreted as a der(11) involving a duplication of chromosome 11 material and rearrangement with chromosome 19. In addition, previously unidentified der(9)t(9;22), der(3)t(3;19), and der(4)t(4;9) were noted. The 16-MT cell line showed losses and gains of DNA due to unbalanced translocations and complex rearrangements of regions containing known tumor suppressor genes. Chromosomal changes in these regions might explain the increased risk of cancer associated with HPV. Also, array CGH detected copy-number gains or amplifications of chromosomes 2, 8, 10, and 11 and deletions of chromosomes 3, 4, 11, and 15. These results provide the basis for the identification of candidate oncogenes responsible for cervical and anal cancer in amplified regions, and for putative tumor suppressor genes in commonly deleted regions like 11q22-23. Furthermore, these data represent the first full characterization of the HPV-positive cell line 16-MT.     

A novel tumorigenic human prostate epithelial cell line (M2205): molecular cytogenetic characterization demonstrates C-MYC amplification and jumping translocations

The paucity of cell lines from early-stage prostate cancer tumors has hindered the recognition of genetic and cellular changes that are associated with the acquisition of tumorigenesis. We describe the chromosomal complement of a novel tumorigenic prostate epithelial cell subline, called M2205, that acquired only three new, consistent chromosomal changes (from those present in the SV40T antigen immortalized parental cell line, P69SV40TAg) when it attained tumor-forming potential. The consistent changes, which were fully characterized using GTG-banding, CBG-banding, silver staining, fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY), involved segmental jumping translocations and resulted in gains in the copy number of genes located on the distal long arm of chromosome 8 (8q22 to 8q24.3), including c-myc. Furthermore, the jumping translocations also resulted in ribosomal genes being present in multiple, tandem copies next to the chromatin from 8q. Given the relatively small number of cytogenetic changes present, this subline provides a means for better understanding the cellular changes associated with the acquired chromosomal imbalances. Further studies of this subline could also provide insight as to the mechanism or mechanisms leading to the formation of jumping translocations, as well as potential position effects resulting from the relocation of ribosomal genes next to other cellular genes or oncogenes.     

Jumping translocations are common in solid tumor cell lines and result in recurrent fusions of whole chromosome arms

Jumping translocations (JTs) and segmental jumping translocations (SJTs) are unbalanced translocations involving a donor chromosome arm or chromosome segment that has fused to multiple recipient chromosomes. In leukemia, where JTs have been predominantly observed, the donor segment (usually 1q) preferentially fuses to the telomere regions of recipient chromosomes. In this study, spectral karyotyping (SKY) and FISH analysis revealed 188 JTs and SJTs in 10 cell lines derived from carcinomas of the bladder, prostate, breast, cervix, and pancreas. Multiple JTs and SJTs were detected in each cell line and contributed to recurrent unbalanced whole-arm translocations involving chromosome arms 5p, 14q, 15q, 20q, and 21q. Sixty percent (113/188) of JT breakpoints occurred within centromere or pericentromeric regions of the recipient chromosomes, whereas only 12% of the breakpoints were located in the telomere regions. JT breakpoints of both donor and recipient chromosomes coincided with numerous fragile sites as well as viral integration sites for human DNA viruses. The JTs within each tumor cell line promoted clonal progression, leading to the acquisition of extra copies of the donated chromosome segments that often contained oncogenes (MYC, ABL, HER2/NEU, etc.), consequently resulting in tumor-specific genomic imbalances. Published 2001 Wiley-Liss, Inc.