Biologic,Immunocytochemical, and Cytogenetic Characterization of Two New Human Melanoma Cell Lines: IIB-MEL-LES and IIB-MEL-IAN

Two human melanoma cell lines, derived from metastases of two patients with epithelioid malignant amelanotic melanomas, and designated IIB-MEL-LES and IIB-MEL-IAN, have been established. Both cell lines have been in continuous culture over 2 years and were propagated continuously for 85 and 75 serial passages, respectively. Morphologically, IIB-MEL-LES is composed predominantly of spindle shaped cells, whereas IIB-MEL-IAN grows as a monolayer of cuboid and stellate shaped cells with many rounded cells in suspension. Immunocytochemical studies revealed that both cell lines express S-100 protein, vimentin, and GD₃ and GD₂ gangliosides but are negative for keratin and collagen. Both cell lines express HLA class I and HLA-DR antigens in variable proportions. The MAGE-1 gene is expressed only by the IIB-MEL-IAN cell line, as revealed by PCR analysis. Cytogenetic analysis of both cell lines revealed abnormal karyotypes; the modal chromosome numbers of IIB-MEL-LES and IIB-MEL-IAN were 48 and 81, respectively. IIB-MEL-LES cells presented rearrangements in chromosomes 1, 14 and X, gains in chromosomes 10,20, and 21 losses in chromosomes 15 and Y. The most frequent markers observed in IIB-MEL-IAN cells were 7q+, 10p+, 2p+, i(6p), 2q+, and 10q-. Clonal gains were observed in chromosomes 12 and 21, whereas losses were seen in chromosomes 1, 2, 3, 4, 6, 7, 11, and 17. Both cell lines were capable of forming colonies in soft agar and developed tumors when transplanted into nude mice, reproducing and maintaining the characteristics of the original tumors. These cell lines and their xenografts appear to provide useful systems for studying the biology, genetics and histogenesis of human malignant melanoma and could be utilized for the development of melanoma vaccines.     

Amplification at 9p in cervical carcinoma by comparative genomic hybridization.

DNA copy number changes were studied by comparative genomic hybridization on 10 tumor specimens of squamous cell carcinoma of cervix obtained from Korean patients. DNA was extracted from paraffin-embedded sections after removal of non-malignant cells by microdissection technique. Copy number changes were found in 8/10 tumors. The most frequent changes were chromosome 19 gains (n=6) and losses on chromosomes 4 (n=4), 5 (n=3), and 3p (n=3). A novel finding was amplification in chromosome arm 9p21-pter in 2 cases. Gains in 1, 3q, 5p, 6p, 8q, 16p, 17, and 20q and losses at 2q, 6q, 8p, 9q, 10p, 11, 13, 16q, and 18q were observed in at least one of the cases.     

Patterns of DNA copy number changes in sentinel lymph node breast cancer metastases

The sentinel lymph node (SLN) is considered to be the first axillary node that contains malignant cells in metastatic breast tumors, and its positivity is currently used in clinical practice as an indication for axillary lymph node dissection. Therefore, accurate evaluation of the SLN for the presence of breast metastatic cells is essential. The main aim of our study is to characterize the genomic changes present in the SLN metastatic samples with the ultimate goal of improving the predictive value of SLN evaluation. Twenty paired samples of SLN metastases and their corresponding primary breast tumors (PBT) were investigated for DNA copy number changes using comparative genomic hybridization (CGH). Non-random DNA copy number changes were observed in all the lesions analyzed, with gains being more common than losses. In 75% of the cases there was at least one change common to both PBT and SLN. The most frequent changes detected in both lesions were gains of 1pter-->p32, 16, 17, 19, and 20 and losses of 6q13-->q23 and 13q13-->q32. In the PBT group, alterations on chromosomes 1, 16, and 20 were the most frequent, whereas chromosomes 1, 6, and 19 were the ones with the highest number of changes in the SLN metastatic group. A positive correlation was found between the DNA copy number changes per chromosome in each of the groups. Our findings indicate the presence of significant DNA copy number changes in the SLN metastatic lesions that could be used in the future as additional markers to improve the predictive value of SLN biopsy procedure.     

Molecular cytogenetic characterization of pancreas cancer cell lines reveals high complexity chromosomal alterations

Karyotype analysis can provide clues to significant genes involved in the genesis and growth of pancreas cancer. The genome of pancreas cancer is complex, and G-band analysis cannot resolve many of the karyotypic abnormalities seen. We studied the karyotypes of 15 recently established cell lines using molecular cytogenetic tools. Comparative genomic hybridization (CGH) analysis of all 15 lines identified genomic gains of 3q, 8q, 11q, 17q, and chromosome 20 in nine or more cell lines. CGH confirmed frequent loss of chromosome 18, 17p, 6q, and 8p. 14/15 cell lines demonstrated loss of chromosome 18q, either by loss of a copy of chromosome 18 (n = 5), all of 18q (n = 7) or portions of 18q (n = 2). Multicolor FISH (Spectral Karyotyping, or SKY) of 11 lines identified many complex structural chromosomal aberrations. 93 structurally abnormal chromosomes were evaluated, for which SKY added new information to 67. Several potentially site-specific recurrent rearrangements were observed. Chromosome region 18q11.2 was recurrently involved in nine cell lines, including formation of derivative chromosomes 18 from a t(18;22) (three cell lines), t(17;18) (two cell lines), and t(12;18), t(15;18), t(18;20), and ins(6;18) (one cell line each). To further define the breakpoints involved on chromosome 18, YACs from the 18q11.2 region, spanning approximately 8 Mb, were used to perform targeted FISH analyses of these lines. We found significant heterogeneity in the breakpoints despite their G-band similarity, including multiple independent regions of loss proximal to the already identified loss of DPC4 at 18q21.     

Detection of DNA copy number changes in human endometriosis by comparative genomic hybridization

Endometriosis is characterized by infertility and pelvic pain in 10-15% of women of reproductive age. The genetic events involved in endometriotic cell expansion remain in large part unknown. To identify genomic changes involved in development of this disease, we examined a panel of 18 selected endometriotic tissues by comparative genomic hybridization (CGH), a molecular cytogenetic method that allows screening of the entire genome for chromosomal gains and/or losses. The study was performed on native, nonamplified DNA extracted from manually dissected endometriotic lesions. Recurrent copy number losses on several chromosomes were detected in 15 of 18 cases. Loss of chromosome 1p and 22q were detected in 50% of the cases. Additional common losses occurred on chromosomes 5p (33%), 6q (27%), 7p(22%), 9q (22%), 16 (22%) as well as on 17q in one case. Gain of DNA sequences were seen at 6q, 7q and 17q in three cases. To validate the CGH data, selective dual-color FISH was performed using probes for the deleted regions on chromosomes 1, 7 and 22 in parallel with the corresponding centromeric probes. Cases showing deletion by CGH all had two signals at 1p36, 7p22.1 and 22q12 in less than 30% of the nuclei in comparison to the double centromeric labels found in more than 85% of the cells. These findings indicate that genes localized to previously undescribed chromosomal regions play a role in development and progression of endometriosis.     

Comparative genomic hybridization study of nasal-type NK/T-cell lymphoma

BACKGROUND: Nasal-type NK/T-cell lymphoma is a rare type of non-Hodgkin's lymphoma. The genetic changes associated with pathogenesis have not been well defined. This study investigates the nonrandom genetic alteration of nasal-type NK/T-cell lymphoma. METHODS: Nine cases were studied. Comparative genomic hybridization (CGH) was carried out using fresh tumor tissues of seven nasal-type NK/T-cell lymphomas. To complement the data by CGH, loss of heterozygosity (LOH) of chromosomes 6q, 1p, and 17p using polymorphic markers and p53 gene mutation by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) were analyzed. RESULTS: The DNA copy number changes of seven nasal-type NK/T-cell lymphomas were gains on chromosomes 2q(5), 13q(4), 10q(3), 21q(2), 3q(2), 5q(2), and 17q(2), and losses involving chromosomes 1p(4), 17p(4), 12q(3), 13q(2), and 6q(1). One of six cases informative for at least two markers for chromosome 6q showed LOH at D6S300, D6S1639, D6S261, D6S407, and D6S292. Two cases showing loss of 1p and 17q by CGH revealed LOH at D1S214, D1S503, and D17S559. P53 mutation was detected in exon 8 in one of nine cases. CONCLUSIONS: Frequent DNA losses at 1p, 17p, and 12q and gains at 2q, 13q, and 10q suggested that these regions could be targets for further molecular genetic analysis to investigate tumor suppressor genes or oncogenes associated with tumorigenesis of NK/T-cell lymphoma. Infrequent alteration of 6q contrary to previous studies raises doubt about an implication of 6q loss in the pathogenesis of early-stage NK/T-cell lymphoma. Further studies on more defined cases are required to verify their association.     

Copy number variations (CNVs) in human pluripotent cell-derived neuroprogenitors

Results from the analysis of copy number variations (CNVs) in human pluripotent cell-derived neuroprogenitor cell lines (hiPSC and hESC-derived NPC) are presented. Two different types of CNVs were detected: a) CNVs inherited from the original source of pluripotent cells (hESC and hiPSC) and b) CNVs detected either in the original source of pluripotent cells or in the derived NPC cell lines but not in both at the same time. Our data suggest that submicroscopic chromosomal changes happened during culture and manipulation of cells and those differentiation procedures could result in gains and losses of genomic regions in pluripotent cell-derived neuroprogenitors. Overall, the results indicate that even chromosomally stable stem cell lines would need to be analyzed in detail by high resolution methodologies before their clinical use.     

Genetic stability of human endometrial mesenchymal stem cells assessed with morphological and molecular karyotyping

The aim of this study was to monitor the genetic stability of endometrial mesenchymal stem cells (eMSCs) by G-banding and molecular karyotyping. We evaluated the sensitivity of each method to assess the genetic stability of eMSCs. G-banding karyotyping performed on passages 6 and 15 showed that more than 80% cells had normal karyotype. Random karyotypic changes were found in a small part of the cell population: aneuploidy, isochromosomes, chromosome breakages, interchromosomal association. Molecular karyotyping carried out on the 6th and 14th passages revealed genomic stability, except for in the case of chromosomes 7 and 14. Microduplications 7q36.3 (62 kb) and 14q11.2 (165kb) were found in these chromosomes. We interpreted these aberrations as being derived from the donor of these cells. The morphological and molecular karyotyping complemented each other. Using these methods, we can analyze karyotypic stability at different levels of the genomic organization.     

Genomic imbalances in 61 renal cancers from the proximal tubulus detected by comparative genomic hybridization

Comparative genomic hybridization (CGH) has been applied to characterize 61 primary renal cell carcinomas derived histogenetically from the proximal tubulus. The tumor samples comprised 46 clear-cell renal cell carcinomas (ccRCCs) and 15 papillary renal cell carcinomas (pRCCs). Changes in the copy number of entire chromosomes or subregions were detected in 56 tumors (92%). In ccRCCs, losses of chromosome 3 or 3p (63%); 14q (30%); 9 (26%); 1 and 6 or 6q (17% each); 4 and 8 or 8p (15% each); 22 (11%); 2 or 2q and 19 (9% each); 7q, 10, 16, 17p, 18, and Y (7% each); and 5, 11, 13, 15, and 21 (4% each) were detected. Most frequent genomic gains in ccRCC were found on chromosome 5 (63%); 7 (35%); 1 or 1q (33%); 2q (24%); 8 or 8q, 12, and 20 (20% each); 3q (17%); 16 (15%); 19 (13%); 6 and 17 or 17q (11% each); and 4, 10, 11, 21, and Y (9% each). In pRCCs, gains in the copy number of chromosomes 7 and 17 (7/15, each) and 16 and 20 (6/15, each) were frequent. One pRCC showed amplification of subchromosome regions 2q22-->q33, 16q, 17q and the entire X chromosome. In pRCC, losses were less frequently seen than gains. Losses of chromosomes 1, 14, 15, and Y (3/15 each) and 2, 4, 6, and 13 (2/15 each) were observed. In ccRCCs, statistical evaluation revealed significant correlations of chromosomal imbalances with tumor stage and grade, i.e., a gain in copy number of chromosome 5 correlated positively with low tumor grade, whereas a gain of chromosomes 10 and 17 correlated positively with high tumor grade. Furthermore, loss of chromosome 4 correlated positively with high tumor stage.     

Sublocalization of c-myb to 6q21----q23 by in situ hybridization and c-myb expression in a human teratocarcinoma with 6q rearrangements

We have sublocalized the human proto-oncogene c-myb by applying two different techniques: in situ hybridization of metaphase spreads and chromosome spot hybridization of flow-sorted chromosomes. For this we used a teratocarcinoma cell line carrying specific chromosome translocations involving the two chromosomes 6 and one chromosome 11. The distribution of the c-myb gene copies on the different translocation chromosomes revealed that c-myb is located in the region 6q21----q23. Because of the close proximity of the c-myb locus to the chromosomal breakpoints in the teratocarcinoma, we investigated whether c-myb was implicated in the development of this tumor. No rearrangement, deletion, or amplification of the gene was detected in the teratocarcinoma cells. Furthermore, the level of c-myb expression was comparable to that of other cell lines of nonhematopoietic origin. These results suggest that c-myb was not affected by the translocation and played no significant role in the development of this teratocarcinoma.     

Protocols for cytogenetic studies of human embryonic stem cells

All cultured cells develop chromosome changes over time, including cultures of human embryonic stem cells (hESC), but only those cells with adaptive chromosomes changes survive. The most frequent chromosome changes in hESC cultures are trisomy 12 and trisomy 17. Cells with these trisomies are indistinguishable from normal cells by appearance and also demonstrate typical markers of pluripotency, making them difficult to identify without cytogenetic analysis. Early detection of these cells is essential since cells with trisomy 12 and 17 can replace the normal cell population in 5-10 passages. Cytogenetic analysis using G-banding is considered to be the gold standard for detecting chromosome abnormalities and, when used in combination with interphase FISH, provides a sensitive method for early detection of cytogenetic aberrations, such as full and partial trisomies of chromosomes 12 and 17. The following discussion describes the cytogenetic methods used in our laboratory to study cultured hESCs, along with recommendations for integrating these methods into a plan for routine cell line quality control.     

Chromosomal rearrangements and their effects in spontaneous immortalization and transformation of rat embryo cells in vitro

G-banding analysis of LRec-1 and LRec-3, spontaneously immortalized cell lines from rat embryo fibroblast, revealed diploid karyotypes with specific clonal structural rearrangements of chromosomes 7 and 19 - del(7)(q11.2q22.1), t(7;19)(q11.1;q12) in malignant stage. Both clonal abnormalities of chromosomes 7 and 19 were also revealed in LRec-1k clone and LRec-1 sf cell line. Previous study of LRec-1 and LRec-3 cells showed the presence of karyotypes with pseudodiploid modal chromosome number, partial trisomy of chromosome 7 and same clonal structural rearrangements of chromosomes 7 and 19 in immortalized stage. In malignant stage, the trisomy 6 and new clonal structural rearrangements of chromosomes 1, 2, 11, 15, 18, 19 and of chromosomes 10, 20 were also found in LRec-1 sf and LRec-1 cells, accordingly. There were no new clonal structural chromosome rearrangements in LRec-1 k and LRec-3 cells. We compared locies of chromosomes involved in rearrangements with mapped genes on these chromosomes according to RATMAP. Supposedly these genes are involved in spontaneous immortalization of rat embryo fibroblast and malignant transformation of LRec-1 and LRec-3 cells and rearrangements of chromosomes 1, 2, 11, 15 and 18 facilitate expression of growth factors of LRec-1 sf cells.     

Analysis of the cytogenetic stability of the human embryonal kidney cell line 293 by cytogenetic and STR profiling approaches

We have characterized the cytogenetic alterations of the human embryonal cell line 293 by spectral karyotyping and G-banding analysis. To investigate its genomic stability, we compared the karyotypes of 293 and its daughter line EcR-293. Genotype profiling through short tandem repeats complemented the analysis. While displaying almost identical STR profiles and thus verifying their origin and their close relation, the two lines were remarkably different in their number of chromosomes and setup of aberrant chromosomes. However, the cell lines retained a stable karyotype in long term culture. The establishment of subclones from EcR-293, expressing inducible lacZ or MEN1 transgenes, only added minor changes to the karyotype. Our study shows that the cytogenetic constitution of a clonal cell line of the 293 origin appears to be sufficiently stable. However, care should be taken when comparing the properties of independent 293 lineages, since clonal variations might be substantial.     

Derivation, culture, and characterization of VUB hESC lines

In this report, we present the derivation and characterization of 15 hESC lines established at the Vrije Universiteit Brussel, Belgium in collaboration with the Universitair Ziekenhuis Brussel, Belgium, using surplus in vitro fertilization embryos and embryos carrying monogenic disorders donated for research. Four lines were derived from blastocyst-stage embryos presumed to be genetically normal, and 11 hESC lines were obtained from embryos shown to carry genetic mutations by preimplantation genetic diagnosis. All the lines express markers of pluripotency as determined by immunocytochemistry and RT-PCR, and formed teratomas when injected into SCID mice. All VUB hESC lines, except for VUB17, are reported in the European hESC registry and are available upon request after signing a Material Transfer Agreement from the VUB (contact person: Prof. Dr. Karen Sermon; Karen.Sermon@uzbrussel.be).     

Significant involvement of chromosome 13q deletions in progression of larynx cancer, detected by comparative genomic hybridization

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumours with various clinical characteristics. These tumours generally exhibit complex karyotypes. Few studies of genomic imbalances have been performed exclusively in subgroups of larynx cancer samples at different stages of the disease. In the present study, chromosomal gains and losses were investigated in 52 larynx tumours, by using comparative genomic hybridization (CGH). The mean number of observed alterations was 37.7 per tumour. The most common sites of losses were 1p, 13q, Xp, and the most common gains were located in 1p, 9q, 16q. The overall number of gains was negatively associated with cancer grading. G1 tumours were also characterized by a higher frequency of deletions in 13q32 and amplifications in 1q23, than tumours in other grades (p < 0.05). The frequency of losses of 13q22 also positively associated with tumour size. There was no association between the frequency of losses in 13q and the presence of lymph node metastases at the time of diagnosis. Another statistically significant association was observed for gains at 1q22-23 and tumour size (p < 0.01). No statistically significant difference in the frequency of most common imbalances was detected between primary tumours with lymph node metastases and those without metastases. In conclusion, we discovered a significant involvement of 13q deletions in the progression of larynx cancer. All the other significant changes observed in the present study were reported previously as being important for HNSCC progression. It seems that multiple genes are disrupted in the process of neoplastic transformation in the larynx, and the networks of events remain to be elucidated.     

Inverted duplication pattern in anaphase bridges confirms the breakage-fusion-bridge (BFB) cycle model for 11q13 amplification

Conventional cytogenetic analyses and comparative genomic hybridization have revealed a complex and even chaotic nature of chromosomal aberrations in pleural malignant mesothelioma (MM). We set out to describe the complex gene copy number changes and screen for novel genetic aberrations using a high-density oligonucleotide microarray platform for comparative genomic hybridization (aCGH) of a series of 26 well-characterized MM tumor samples. The number of copy number changes varied from zero to 40 per sample. Gene copy number losses predominated over gains, and the most frequent region of loss was 9p21.3 (17/26 cases), the locus of CDKN2A and CDKN2B, both known to be commonly lost in MM. The most recurrent minimal regions of losses were 1p31.1--> p13.2, 3p22.1-->p14.2, 6q22.1, 9p21.3, 13cen-->q14.12, 14q22.1-->qter, and 22qcen-->q12.3. Previously unreported gains included 9p13.3, 7p22.3-->p22.2, 12q13.3, and 17q21.32-->qter. The results suggest that gene copy number losses are a major mechanism of MM carcinogenesis and reveal a recurrent pattern of copy number changes in MM.     

Identification of DNA copy-number aberrations by array-comparative genomic hybridization in patients with schizophrenia

Chromosomal abnormalities are implicated as important markers for the pathogenesis in patients with schizophrenia. In this study, with using bacterial artificial chromosome (BAC) array-based comparative genomic hybridization (CGH), we analyzed DNA copy-number changes among 30 patients with schizophrenia. The most frequent changes were partial gain of Xq23 (52%) and loss of 3q13.12 (32%). Other frequent gains were found in: 1p, 6q, 10p, 11p, 11q, 14p, and 15q regions, and frequent losses were found in: 2p, 9q, 10q, 14q, 20q, and 22q regions. The set of abnormal regions was confirmed by real-time PCR (9q12, 9q34.2, 11p15.4, 14q32.33, 15q15.1, 22q11.21, and Xq23). All real-time PCR results were consistent with the array-CGH results. Therefore, it is suggested that array-CGH and real-time PCR analysis could be used as powerful tools in screening for schizophrenia-related genes. Our results might be useful for further exploration of candidate genomic regions in the pathogenesis of schizophrenia.     

Analyses by comparative genomic hybridization of genes relating with cisplatin-resistance in ovarian cancer

Cisplatin has played a key-role in the management of ovarian cancer patients. Since the mechanisms of cisplatin-resistance have been reported to be multifactorial, it is quite difficult to predict effectiveness of cisplatin-based chemotherapy. In the present study, we have screened abnormal chromosomal regions in cisplatin-resistant and paclitaxel-resistant human ovarian cancer cell lines using comparative genomic hybridization (CGH). Increased copy number at 6q21-25 and decreased copy number at 7q21-36 and 10q12-15 were observed in the cisplatin-resistant cell line. Increased copy number at 7q11.2-21 was observed in paclitaxel-resistant cell lines. Messenger RNA of MDR1 located on chromosomal region of 7q11.2-21 was overexpressed in the paclitaxel-resistant cell lines and recognized as a potential mechanism of acquired paclitaxel-resistance. In CGH analyses of 28 primary epithelial ovarian cancer patients, gains of 1q21-22 (p = 0.0183) and 13q12-14 (p = 0.0407) were observed in significantly high abundance in the cisplatin-resistant tumor group, compared with the cisplatin-sensitive tumor group. These genetic alterations were suggested to be potential indicators for drug resistance.