Regional deletion and amplification on chromosome 6 in a uveal melanoma case without abnormalities on chromosomes 1p, 3 and 8

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Loss of the long arm and gain of the short arm of chromosome 6 are frequently observed chromosomal aberrations in UM, together with loss of chromosome 1p36, loss of chromosome 3 and gain of chromosome 8. This suggests the presence of one or more oncogenes on 6p and tumor suppressor genes at 6q that are involved in UM development. Both regions, however, have not been well defined yet. Furthermore in other neoplasms gain of 6p and loss of 6q are frequently occurring events. In this case report, we describe the delineation of a partial gain on chromosome 6p and a partial deletion on 6q in a UM with the objective to pinpoint smaller candidate regions on chromosome 6 involved in UM development. Conventional cytogenetics, comparative genomic hybridization (CGH) and fluorescence in-situ hybridization (FISH) were used to delineate regions of loss and gain on chromosome 6 in this UM patient. With conventional cytogenetics a deleted region was found on chromosome 6q that was further delineated to a region ranging from 6q16.1 to 6q22 using CGH and FISH. A region of gain from 6pter to 6p21.2 was also demarcated with CGH and FISH. No other deletions or amplifications on recurrently involved chromosomes were found in this patient. This study indicates the presence of one or more tumor suppressor genes on chromosomal region 6q16.1-6q22 and the presence of one or more oncogenes on chromosomal region 6pter-6p21.2, which are likely to be important in UM and other tumors.     

Chromosomal alterations in small cell lung cancer revealed by multicolour fluorescence in situ hybridization

Small cell lung cancer (SCLC) is a major cause of cancer related morbidity and mortality. Karyotypic studies have revealed numerous chromosomal aberrations in most SCLC however, classical G-banding analysis is unable to fully characterise complex marker chromosomes. Recent developments in molecular cytogenetics now allow accurate identification of the chromosomal components of complicated rearrangements. We have applied the technique of multicolour fluorescence in situ hybridization (M-FISH) in combination with comparative genomic hybridization (CGH) to the analysis of 5 SCLC cell lines and 1 primary tumour specimen to characterise the chromosomal abnormalities. CGH analysis identified many similarities between specimens, with frequent DNA copy number decreases on chromosomes 3p, 5q, 10, 16q, 17p and frequent gains on 3q, 1p, 1q and 14q. In contrast, M-FISH analysis revealed a large number of structural abnormalities, with each specimen demonstrating an individual pattern of chromosomal translocations. Forty different translocations were identified with the vast majority (39) being unbalanced. Chromosome 5 was the most frequently rearranged chromosome (9 translocations) followed by chromosomes 2, 10 and 16 (6 translocations each). Further investigation of these frequently involved chromosomes is warranted to establish whether consistent break points are involved in these translocations, causing dysregulation of specific genes that are crucial for tumour progression and secondly to identify the affected genes.     

Chromosomal analysis of non-small-cell lung cancer by multicolour fluorescent in situ hybridisation

The cytogenetic abnormalities in non-small-cell lung cancer remain elusive due primarily to the difficulty in obtaining metaphase spreads from solid tumours. We have used the molecular cytogenetic techniques of multicolour fluorescent in situ hybridisation (M-FISH) and comparative genomic hybridisation (CGH) to analyse four primary non-small-cell lung cancer samples and two established cell lines (COR-L23 and COR-L105) in order to identify common chromosomal aberrations. CGH revealed regions on 5p, 3q, 8q, 11q, 2q, 12p and 12q to be commonly over-represented and regions on 9p, 3p, 6q, 17p, 22q, 8p, 10p, 10q and 19p to be commonly under-represented. M-FISH revealed numerous complex chromosomal rearrangements. Translocations between chromosomes 5 and 14, 5 and 11 and 1 and 6 were observed in three of the six samples, with a further 14 translocations being observed in two samples each. Loss of the Y chromosome and gains of chromosomes 20 and 5p were also frequent. Chromosomes 4, 5, 8, 11, 12 and 19 were most frequently involved in interchromosomal translocations. Further investigation of the recurrent aberrations will be necessary to identify the specific breakpoints involved and any role they may have in the aetiology, diagnosis and prognosis of non-small-cell lung cancer.     

采用比较基因组杂交方法分析原发性食管癌染色体异常

背景与目的:有研究表明原发性食管癌常有染色体的改变,包括染色体基因组异常扩增和缺失.比较基因组杂交技术可以显示这些染色体的异常变化.本实验采用比较基因组杂交技术研究和分析原发性食管癌染色体基因组的变化特点及其与预后的关系.方法:采用比较基因组杂交技术检测16例食管癌组织中染色体的异常改变,并分析染色体异常与预后的关系.研究的病例中7例食管癌术后2年内死亡(对照组),9例术后生存3年以上(生存组).结果:食管癌患者中多数染色体基因组发生改变,最常见的染色体基因组高扩增频率发生在1q/p,2q/p,3q,5q/p,8q/p,9q/p,11q/p,17和20q/p染色体区段上,在染色体1q/p,4p,9p,18q和xp中常见染色体基因缺失.染色体7q/p和19扩增频率和染色体4q/p和18q缺失频率,生存组与对照组间存在显著性差异.结论:食管癌患者染色体区段基因易发生异常扩增和缺失,生存组与对照组存在明显差异.     

Identification of novel cryptic translocations involving IGH in B-cell non-Hodgkin's lymphomas

Chromosomal rearrangements involving the immunoglobulin heavy chain gene (IGH) at 14q32 are observed in approximately 50% of patients with B-cell non-Hodgkin's lymphoma (NHL). The 5' end of the IGH gene is located within 8 kb of the telomeric repeats of 14q. Translocations involving the IGH locus and the telomeric band of a partner chromosome are difficult to identify, because most terminal bands of human chromosomes appear pale by conventional G-banding techniques. To determine whether there are cryptic translocations involving the IGH locus, we used dual-color fluorescence in situ hybridization (FISH) of 5' and 3' IGH genomic clones containing the variable sequences, or the J(H) and the 5' constant regions, respectively. We examined cells from 51 patients with B-cell NHL who had a normal karyotype (3 patients), clonal abnormalities not involving 14q32 (35 patients), or alterations of 14q32 other than recurring translocations, i.e., add(14)(q32) (13 patients). FISH detected 17 IGH translocations in 16 of 51 (31%) cases. Of the 13 cases with add(14)(q32), FISH identified the partner chromosome in 9 cases (69%; 3q27, 6 cases; 2p13, 19p13.3, and 18q21.3, 1 case each). Six of thirty-eight (16%) patients without visible alterations of 14q32 and 2 of 13 (15%) patients with an abnormality of one chromosome 14 had masked (5 patients) or cryptic IGH translocations (3 patients), involving 3q27 (3 patients), 5p15.3 (2 patients), 19p13.3 (3 patients), or 14q32 (1 patient; 1 patient had two rearrangements). We identified two novel, recurring, cryptic translocations: t(5;14)(p15.3;q32) (2 patients) and t(14;19)(q32;p13.3) (3 patients). In summary, FISH permitted the detection of cryptic or masked IGH rearrangements in approximately 20% of lymphoma cases without visible rearrangements of 14q32 analyzed retrospectively.     

Molecular Cytogenetic Characterization in Four Pediatric Pheochromocytomas and Paragangliomas

Pheochromocytomas (PCCs) are rare tumors among children and adolescents and therefore are not genetically well characterized. The most frequently observed chromosomal changes in PCC are losses of 1p, 3q and/or 3p, 6q, 17p, 11q, 22q, and gains of 9q and 17q. Aberrations involving chromosome 11 are more common in malignant tumors. Unfortunately information about gene aberrations in childhood PCC’s is limited. We used comparative genomic hybridization (CGH) and array comparative genomic hybridization (aCGH) to screen for copy number changes in four children suffering from pheochromocytoma or paraganglioma. Patients were diagnosed at the age 13 or 14 years. Bilateral pheochromocytoma was associated with von Hippel-Lindau syndrome (VHL). Multiple paraganglioma was associated with a germline mutation in SDHB. We found very good concordance between the results of CGH and aCGH techniques. Losses were observed more frequently than gains. All cases had a loss of chromosome 11 or 11p. Other aberrations were loss of chromosome 3 and 11 in sporadic pheochromocytoma, and loss of 3p and 11p in pheochromocytoma, which carried the VHL mutation. The deletion of chromosome 1p and other changes were observed in paragangliomas. We conclude that both array CGH and CGH analysis identified similar chromosomal regions involved in tumorigenesis of pheochromocytoma and paragangliomas, but we found 3 discrepancies between the methods. We didn’t find any, of the proposed, molecular markers of malignancy in our benign cases and therefore we speculate that molecular cytogenetic examination may be helpful in separating benign and malignant forms in the future.     

Nonrandom chromosomal rearrangements in pancreatic carcinomas.

Short-term cultures were initiated from 20 carcinomas of the pancreas, 17 of which could be successfully cytogenetically analyzed. In eight carcinomas, only normal karyotypes were detected, probably representing dividing stromal cells. Three cases had -Y as the sole anomaly, which also may have occurred in cells that do not belong to the tumor parenchyma. Massively rearranged karyotypes with modal chromosome numbers in the triploid (five cases) and diploid-triploid (one case) ranges were found in the remaining six carcinomas. Structural rearrangements, including deletions and unbalanced translocations, of the long arm of chromosome 6, involving bands q13 and q15 twice and q11 and q16 once, occurred in four tumors. All of these aberrations led to loss of chromosome material from 6q, always involving 6q15. Deletions and unbalanced translocations of the short arm of chromosome 1 also were found in four cases, affecting band p32 in three of them. In all four cases, the abnormalities resulted in loss of genetic material distal to 1p32. Chromosome 17 was involved in structural aberrations in three cases, twice as unbalanced translocations leading to loss of 17p material. Deletions of the short arms of chromosomes 3 and 8 were detected in two carcinomas. The most consistent numerical abnormalities were +2, +10, +11, +14, and tetrasomy 20, which were seen in all six cases. The findings suggest that structural rearrangements, or loss, of genes located on 1p, 3p, 6q, 8p, and 17p are of pathogenetic importance in pancreatic carcinogenesis.     

Candidate genes in breast cancer revealed by microarray-based comparative genomic hybridization of archived tissue

Genomic imbalances in 31 formalin-fixed and paraffin-embedded primary tumors of advanced breast cancer were analyzed by microarray-based comparative genomic hybridization (matrix-CGH). A DNA chip was designed comprising 422 mapped genomic sequences including 47 proto-oncogenes, 15 tumor suppressor genes, as well as frequently imbalanced chromosomal regions. Analysis of the data was challenging due to the impaired quality of DNA prepared from paraffin-embedded samples. Nevertheless, using a method for the statistical evaluation of the balanced state for each individual experiment, we were able to reveal imbalances with high significance, which were in good concordance with previous data collected by chromosomal CGH from the same patients. Owing to the improved resolution of matrix-CGH, genomic imbalances could be narrowed down to the level of individual bacterial artificial chromosome and P1-derived artificial chromosome clones. On average 37 gains and 13 losses per tumor cell genome were scored. Gains in more than 30% of the cases were found on 1p, 1q, 6p, 7p, 8q, 9q, 11q, 12q, 17p, 17q, 20q, and 22q, and losses on 6q, 9p, 11q, and 17p. Of the 51 chromosomal regions found amplified by matrix-CGH, only 12 had been identified by chromosomal CGH. Within these 51 amplicons, genome database information defined 112 candidate genes, 44 of which were validated by either PCR amplification of sequence tag sites or DNA sequence analysis.     

Recurrent deletions of chromosomes 11q and 3p in anal canal carcinoma

A cytogenetic study of 8 cases of anal canal cancer, including 1 cloacogenic and 7 squamous-cell carcinomas, was performed. All tumors exhibited chromosomal abnormalities. A rearrangement involving the long arm of chromosome 11 was seen in all instances, and, with the exception of the i (11q) found in one tumor, all the observed rearrangements resulted in a deletion of the distal segment. Rearrangements of chromosome 3, detected in 6 tumors, led to a deletion of the short arm in 5 cases. The association of these 2 deletions may characterize the anal canal carcinoma, the smallest common deleted segments being distal to 11q22 or q23 and 3p22.     

Hodgkin's lymphoma cell lines are characterized by frequent aberrations on chromosomes 2p and 9p including REL and JAK2

Four Hodgkin's lymphoma cell lines (KM-H2, HDLM-2, L428, L1236) were analyzed for cytogenetic aberrations, applying multiplex fluorescence in situ hybridization, chromosome banding and comparative genomic hybridization. Each line was characterized by a highly heterogeneous pattern of karyotypic changes with a large spectrum of different translocated chromosomes (range 22-57). A recurrent finding in all cell lines was the presence of chromosomal rearrangements of the short arm of chromosome 2 involving the REL oncogene locus. Furthermore, multiple translocated copies of telomeric chromosomal segments were frequently detected. This resulted in a copy number increase of putative oncogenes, e.g., JAK2 (9p24) in 3 cell lines, FGFR3 (4p16) and CCND2 (12p13) in 2 cell lines as well as MYC (8q24) in 1 cell line. Our data confirm previous cytogenetic results from primary Hodgkin's tumors suggesting an important pathogenic role of REL and JAK2 in this disease. In addition, they provide evidence for a novel cytogenetic pathomechanism leading to increased copy numbers of putative oncogenes from terminal chromosomal regions, most probably in the course of chromosomal stabilization by telomeric capture.     

Genomic imbalances and patterns of karyotypic variability in mantle-cell lymphoma cell lines

Mantle-cell lymphoma (MCL) is genetically characterized by 11q13 chromosomal translocations involving the CCND1 gene. We have characterized five MCL cell lines, JVM-2, GRANTA-519, REC-1, JEKO-1, and NCEB-1, combining metaphase and array comparative genomic hybridization, multicolor-FISH, and molecular analysis. Our results revealed common gained regions at 2p14, 9q31.2-qter, 11q13.1-q21, 13q14-q21.2, 13q34-qter and 18q21.1-q22.1, and losses at 1p21.2-p31.1, 2p11.2, 8p21.2-pter, 9p21.3-pter, 11q23.3-qter, 17p11.2-pter, and 17q21.2-q22.2. All cell lines except JVM-2, displayed moderate or high numerical chromosome instability. In addition, an ongoing level of chromosome rearrangements was observed in REC-1. Surprisingly, NCEB-1 carried several stable mouse chromosomes and showed expression of both human and murine bcl-2 protein. Our findings indicate that these cell lines represent three patterns of chromosome evolution in MCL and may be useful to understand the pathogenesis of this neoplasm.     

Genotypic analysis of esophageal squamous cell carcinoma by molecular cytogenetics and real-time quantitative polymerase chain reaction

We performed an integrated cytogenetic study using a combination of comparative genomic hybridization (CGH), spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) to analyze chromosomal aberrations associated with 8 human esophageal squamous cell carcinoma (EC-SCC) cell lines, and used real-time quantitative PCR (Q-PCR) to study the copy number changes of two candidate genes of chromosome 3q, PIK3CA and TP63, in 20 primary tumors of EC-SCC. The pooled CGH results revealed frequent gain abnormalities on chromosome arms 1p, 1q, 3q, 5p, 6p, 7p, 7q, 8q, 9q, 11q, 12p, 14q, 15q, 16p, 16q, 17q, 18p, 19q, 20q, 22q, and Xq, while frequent losses were found on 3p, 4, 5q, 6q, 7q, 9p, and 18q. SKY detected 195 translocations, 13 deletions and 2 duplications. Among the 374 breakpoints, most clustered at the centromeric regions, such as 8q10, 13q10, 7q10, 9q10, 14q10, 15q10, 16q10, 21q10, and 22q10, but also at other regions, including 3q (3q21, 3q22, 3q25), 7p (7p22, 7p14, 7p12), 7q (7q21, 7q31, 7q32), 8q (8q21.1, 8q23), 11q (11q21, 11q24), 13q (13q14) and 18q (18q21). There was a good correlation between the number of aberrations identified by CGH and SKY (r=0.667; p=0.035). Combined CGH and SKY analyses indicated that chromosomes 3, 7, 9, 11, 14, 16, 18, 19, 20, and 22 harbored higher frequency of chromosomal aberrations than expected. FISH using BAC clones containing oncogene PIK3CA and TP63 found that both genes were amplified in 6 and 5 cell lines, respectively. Q-PCR analysis of primary tumors revealed amplification of PIK3CA and TP63 in 100% and 80% of the cases. Average copy number of PIK3CA per haploid genome was greater than that of TP63 (6.27 vs 2.73), and the difference showed statistical significance (p<0.001). Combination of CGH, SKY and FISH could reveal detailed chromosomal changes associated with esophageal cancer cells, and Q-PCR could assess the change of the candidate genes in clinical samples in a high throughput way.     

三种食管鳞癌细胞系的染色体异常研究

目的:分析食管鳞癌(ESCC)细胞系的染色体异常,为将来寻找食管癌相关基因提供线索。方法:采用比较基因组杂交法(CGH)分析3种ESCC细胞系的染色体DNA拷贝数改变情况。结果:9q(3/3)、3q(2/3)、5q(2/3)、5p(2/3)、8q(2/3)、12p(2/3)和20q(2/3)为常见的染色体增加区。4q(2/3)和6q(2/3)是常见的染色体丢失区。结论:这些常见的染色体异常有助于寻找和定位食管癌相关基因。     

Chromosome 1 abnormalities in cervical carcinoma

Abnormalities of chromosome 1 have been reported in a number of solid tumors and hematologic malignancies, indicating that this is a frequent event in neoplasia. Here we report our observations on aberrations of chromosome 1 in malignancies of the uterine cervix. Tumor material obtained from 148 patients with invasive carcinoma of the cervix and two cases of carcinoma in situ (CIS) was analyzed on direct preparations by G-banding. The results showed abnormalities of chromosome 1 to be one of the most common karyotypic changes, with 95% of the patients showing rearrangements of this chromosome. These changes were never seen as the sole abnormality but were always found in association with other chromosomal aberrations. Numerical rearrangements were present in 54% of the cases, with losses of unaltered chromosome 1 predominating. Consistent marker chromosomes included deletions of chromosome 1 at bands q32, p34, q42, p32, and p22, isochromosomes of both the "p" and "q" arms and translocations, particularly on the long arm. Specific regions on both arms of chromosome 1 (1p11-p13 and 1q21-q32) were preferentially overrepresented in changes involving this chromosome. Certain breakpoints were nonrandomly involved in the structural changes, particularly band 1q32 breaks occurring at this site in 88 instances. The presence of chromosome 1 aberrations in the two cases of CIS suggests that rearrangements of this chromosome are not always a secondary change contributing to the progression of the cancer, but also may represent an early cytogenetic event as in neuroblastoma, some leukemias, and myeloproliferative disorders.     

Translocation t(2;3)(p15-23;q26-27) in myeloid malignancies: report of 21 new cases, clinical, cytogenetic and molecular genetic features.

Chromosomal rearrangements involving 3q26 either due to inversion or translocation with various partner chromosomes are a recurrent finding in malignant myeloid disorders. Typically, these chromosome aberrations contribute to ectopic expression of or to the formation of fusion genes involving the EVI1 proto-oncogene. Chromosomal translocations involving the short arm of chromosome 2 (p15-p23) and the distal part of the long arm of chromosome 3 (q26-q27) are a rare but recurrent finding in patients with myeloid malignancies, and are assumed to be part of this spectrum of disorders. Thus far, however, these translocations have been poorly studied. Here, we present 21 new cases with myelodysplasia, acute myeloid leukemia or CML in blast crisis, which upon karyotyping showed the presence of a t(2;3). Furthermore, an extensive literature review disclosed 29 additional cases. Morphological, clinical and cytogenetic assessment revealed the typical hallmarks of 3q26/EVI1 rearrangements, that is, trilineage dysplasia and dysmegakaryopoiesis, poor prognosis and additional monosomy 7. Molecular cytogenetic analysis and PCR in selected samples indicated that in most cases the translocation indeed targets the EVI1 locus. Mapping of the chromosome 2 breakpoints confirmed the initially suspected cytogenetic breakpoint heterogeneity at the 2p arm.     

Frequent allelic loss at 10q23 but low incidence of PTEN mutations in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare, highly metastatic skin tumor of neuroectodermal origin. The disease shares clinical and histopathological features with small cell lung carcinoma (SCLC). The genetic mechanisms underlying the development and tumor progression of MCC are poorly understood. We recently showed by comparative genomic hybridization (CGH) that the pattern of chromosomal abnormalities in MCC resembles that of SCLC. One of the most frequently observed losses involved the entire chromosome 10 or partial loss of the chromosome 10 long arm (33% of examined MCC cases). The PTEN tumor-suppressor gene has been mapped to 10q23.3 and was shown to be mutated in a variety of human cancers including SCLC. Germline PTEN mutations have been observed in familial predisposing cancer syndromes including Cowden disease. Interestingly, an association between Cowden syndrome and Merkel cell carcinoma has been reported. To study the possible role of PTEN in MCC oncogenesis, loss of heterozygosity (LOH) analysis for the 10q23 region was performed on 26 MCC tumor samples from 23 MCC patients. The PTEN locus was deleted in 9 of 21 (43%) informative MCC tumor samples [7 of 18 (39%) MCC patients]. Despite this high frequency of LOH at 10q23, mutation and homozygous deletion screening of the PTEN gene revealed only one tumor with a nonsense mutation and a second with a homozygous deletion of exon 9. These data suggest that either alternative mechanisms lead to inactivation of the PTEN gene or that other tumor-suppressor genes at chromosome 10 are implicated in the development of MCC.     

Investigations for fine mapping of amplifications in chromosome 3q26.3-28 frequently occurring in squamous cell carcinomas of the head and neck

OBJECTIVE: Overrepresentations of chromosomal material on the long arm of chromosome 3 frequently occur in squamous cell carcinoma of the head and neck. This experimental study was conducted for further fine mapping of these overrepresentations by interphase fluorescence in situ hybridization (FISH) of tumor cells in cell lines. METHODS: Seven cell lines derived from squamous cell carcinomas of the head and neck were investigated by comparative genomic hybridization to analyze unbalanced chromosomal aberrations. Overrepresentations of chromosomal material on the telomeric part of the long arm of chromosome 3 were further analyzed by interphase FISH using YAC contig clones. RESULTS: Chromosomal aberrations which frequently occurred were overrepresentations on 5p (n = 4), 7p (n = 5), 11q13 (n = 3), 15q (n = 5), 17q (n = 3), 19q (n = 2), 20q (n = 2) and 22q (n = 3). Reoccurring losses of chromosomal material were found in 3p (n = 3), 7q (n = 2), 18q (n = 3) and 19p (n = 2). Gains of chromosomal material on chromosome 3q were found in 4 out of 7 cell lines, with a high copy number of amplifications occurring in the chromosomal region of 3q26.3-28. Further experiments revealed a physical mapping of this amplification to a narrow band of 13.8 Mbp on chromosome 3q, whose amplification borders were represented by the YAC clones 754_f_3 centomeric and 955_b_2 telomeric. CONCLUSIONS: By FISH, the amplification of chromosomal material on 3q could be fine mapped on a narrow band on 3q26.3-27. This aberration can be considered as a breakpoint in tumorigenesis. Putative candidate oncogenes and tumor suppressor genes located in this region might be a target for mutations leading to tumor progression.     

人肝细胞癌4号染色体长臂缺失的细胞遗传学及分子遗传学研究

肝癌的发生是一个多基因、多途径、多阶段的复杂过程,其中染色体的缺失及对应区域的抑癌基因失活是HCC发生发展的重要生物学过程,4q在HCC中经常发生缺失,提示在4q存在肝癌相关的特异性抑癌基因.本文对HCC中有关4q缺失的研究进行了综述,涉及细胞遗传学及分子遗传学水平,包括利用荧光原位杂交、比较基因组技术及限制性片段多态性、微卫星、单核苷酸多态性的杂合性缺失和芯片等技术所做的研究;归纳了4q在不同国家与地区缺失的热点区域,以及与HBV感染、HCC分化程度及肿瘤大小等临床参数的关系;并且列举了4q上与HCC相关的可能的抑癌基因.