Array comparative genomic hybridization and cytogenetic analysis in pediatric acute leukemias

Most patients with acute lymphocytic leukemia (all) are reported to have acquired chromosomal abnormalities in their leukemic bone marrow cells. Many established chromosome rearrangements have been described, and their associations with specific clinical, biologic, and prognostic features are well defined. However, approximately 30% of pediatric and 50% of adult patients with all do not have cytogenetic abnormalities of clinical significance. Despite significant improvements in outcome for pediatric all, therapy fails in approximately 25% of patients, and these failures often occur unpredictably in patients with a favorable prognosis and "good" cytogenetics at diagnosis.It is well known that karyotype analysis in hematologic malignancies, although genome-wide, is limited because of altered cell kinetics (mitotic rate), a propensity of leukemic blasts to undergo apoptosis in culture, overgrowth by normal cells, and chromosomes of poor quality in the abnormal clone. Array comparative genomic hybridization (acgh-"microarray") has a greatly increased genomic resolution over classical cytogenetics. Cytogenetic microarray, which uses genomic dna, is a powerful tool in the analysis of unbalanced chromosome rearrangements, such as copy number gains and losses, and it is the method of choice when the mitotic index is low and the quality of metaphases is suboptimal. The copy number profile obtained by microarray is often called a "molecular karyotype."In the present study, microarray was applied to 9 retrospective cases of pediatric all either with initial high-risk features or with at least 1 relapse. The conventional karyotype was compared to the "molecular karyotype" to assess abnormalities as interpreted by classical cytogenetics. Not only were previously undetected chromosome losses and gains identified by microarray, but several karyotypes interpreted by classical cytogenetics were shown to be discordant with the microarray results. The complementary use of microarray and conventional cytogenetics would allow for more sensitive, comprehensive, and accurate analysis of the underlying genetic profile, with concomitant improvement in prognosis and treatment, not only for pediatric all, but for neoplastic disorders in general.     

Interglandular cytogenetic heterogeneity detected by comparative genomic hybridization in pancreatic cancer

The aim of this study is to explore the mechanisms of intratumoral cytogenetic heterogeneity (ICH) in pancreatic cancer. Using comparative genomic hybridization (CGH) analysis, we investigated interglandular variation in 20 primary invasive ductal adenocarcinomas of the pancreas. Three or four adjacent neoplastic glands were individually microdissected from a tumor specimen. Extracted DNA from each gland was amplified by degenerate oligonucleotide primed-PCR, followed by CGH. In addition, DNA index (DI) was measured by laser scanning cytometry in each case. CGH profiles displayed a wide variety of differences between glands within the same tumor in all cases, i.e., interglandular cytogenetic heterogeneity was distinct in pancreatic cancers. In this study, genetic changes detected in all regions of a tumor were classified as "region-independent" alterations, whereas changes seen in at least one, but not all regions were designated as "region-dependent" alterations, which resulted in ICH. The degree of ICH, which was manifested as the ratio of these two types of alterations, correlated closely with DI (Spearman rho = 0.842; P = 0.0002). Therefore, DI might be a surrogate marker for ICH. These results suggest that with tumor progression, ICH and DNA aneuploidy result from the successive appearance of region-dependent alterations attributable to chromosomal instability in tumor cells. Our data support a concept of individual cell heterogeneity in pancreatic cancer.     

Molecular cytogenetic characterization of drug-resistant leukemia cell lines by comparative genomic hybridization and fluorescence in situ hybridization.

Resistance to chemotherapeutic drugs is one of the major difficulties encountered during cancer chemotherapy. To detect genomic aberrations underlying the acquired drug resistance, we examined three cultured human myelomonocytic leukemia cell sublines each resistant to adriamycin (ADR), 1-beta-1-D-arabinofuranosylcytosine (ara-C), or vincristine (VCR), using comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), RT-PCR, and western blot techniques. Chromosomes 7, 10 and 16 most conspicuously showed frequent aberrations among the resistant sublines as compared to the parental KY-821 cell line. In ADR-resistant cells, gains at 7q21, 16p12, 16p13.1-13.3, 16q11.1-q12.1, and losses at 7p22-pter, 7q36-qter, 10p12, 10p11.2-pter, 10q21-q25, 10q26-qter were notable. In ara-C-resistant cells, no remarkable gain or loss on chromosome 7, but losses at 10p14-pter, 10q26-qter and 16p11.2-p11.3 were observed. In VCR-resistant cells, gain at 7q21 and losses at 10p11-p13, 10p15 and 16p11.2-p13.3 were found. FISH identified amplified signals for the MDR-1 gene located at 7q21.1 in ADR- and VCR- but not ara-C-resistant cells, and for the MRP-1 gene located at 16p13.1 in ADR-resistant cells. These findings were validated at the mRNA and protein levels. Overlapping of the amplified MRP-1 gene with MDR-1 gene may play a critical part in the acquisition of resistance to ADR. Resistance to ara-C excluded MDR-1 gene involvement and highlighted other key genes such as MXR gene. Several other genes putatively involved in the development of drug resistance might lie in other aberrated chromosomal regions.     

Analysis of cytogenetic aberrations in sporadic vestibular schwannoma by comparative genomic hybridization

Vestibular schwannomas (VS) are benign tumors of the nervous system that are usually sporadic but also occur in the inherited disorder neurofibromatosis type 2 (NF2). The NF2 gene is a tumor suppressor gene located on chromosome 22. Loss of the NF2 protein product, Merlin, is universal in both sporadic and NF2-related schwannomas and the loss or mutation of the gene is the only established causative event underlying schwannoma formation. Comparative genomic hybridization (CGH) was used to screen 20 sporadic VS to identify additional chromosomal regions that may harbor genes involved in VS-tumorigenesis. The most common change were losses on chromosome 22q. Additionally, losses were observed on chromosome 9p indicating a possible participation of the CDKN2A tumor suppressor gene in the genesis of VS. Gains were observed on 17q, 19p and 19q, which have been reported before in malignant peripheral nerve sheath tumors that are associated with neurofibromatosis type 1. Importantly, high level amplifications have been observed on 16p and 16q as well as on 9q, suggesting the possible involvement of several oncogenes in the tumorigenesis of VS. Our data suggest the involvement of various oncogenes and tumor suppressor genes might play a role in the genesis of the vestibular schwannomas apart from the inactivation of the NF2 gene.     

Malignant placental site trophoblastic tumor: a cytogenetic study using comparative genomic hybridization and chromosome in situ hybridization

BACKGROUND: Placental site trophoblastic tumor (PSTT) is a rare form of gestational trophoblastic neoplasm composed predominantly of intermediate trophoblast. Most showed benign behavior whereas 10-15% of PSTTs were clinically malignant with later recurrence and metastasis. Currently, there are no reliable means to predict clinical outcome, and cytogenetic information is scanty. METHODS: The clinicopathologic features of two cases of malignant PSTT were analyzed. Cytogenetic analysis was performed by comparative genomic hybridization (CGH) and chromosome in situ hybridization (CISH) using frozen tissue and paraffin embedded sections, respectively. RESULTS: Both patients were 32 years old at time of diagnosis. One patient with PSTT presented with menorrhagia, and the other presented with symptoms of missed abortion. Elevated serum human chorionic gonadotropin (HCG) was detected in both patients. Histologic examination showed the typical features of PSTT with high mitotic count (> 5/10 high-power fields). Ovarian and lung metastasis occurred in both patients. Immunohistochemical staining revealed an equal distribution of HCG and human placental lactogen. Cytogenetic studies by CISH showed that karyotypes of these two malignant PSTTs were diploid. Analysis of the tumor tissue by CGH did not show any changes in DNA copy numbers. CONCLUSIONS: The authors' study indicated that the two metastasizing PSTTs had balanced diploid karyotype. The malignant behavior of PSTTs may be not related to the DNA copy number changes. Such cytogenetic study may be useful in distinguishing metastatic PSTT from choriocarcinoma.     

Combined classical and molecular cytogenetic analysis of cancer

While chromosome-banding analysis has set the standard for karyotyping from 1970 onwards, fluorescent in situ hybridisation (FISH) has more recently been used to complement the study of chromosomal rearrangements. Especially useful has been the appearance of FISH methodologies with screening abilities, namely comparative genome hybridisation (CGH), multicolour-FISH (m-FISH), and cross-species colour banding (RxFISH). These FISH-based screening techniques are reviewed here together with methodologies using chromosome- or locus-specific probes. Emphasis is put on the strengths and limitations of these FISH techniques to complement standard chromosome banding analysis. Judicious choice from the molecular cytogenetic techniques now available has significantly improved our ability to characterise the genomic rearrangements of cancer cells.     

应用比较基因组杂交技术分析肺癌染色体异常

背景与目的:肺癌的发生是一个多基因、多阶段、多步骤的复杂生物学过程.研究肿瘤发生发展过程中的基因异常改变是肿瘤发生机制研究的一个热点.本研究应用比较基因组杂交技术分析肺癌的染色体异常,探讨染色体异常与肺癌不同病理类型和临床特征之间的关系.方法:收集胸外科2005年10月-2006年9月手术切除的新鲜肺癌标本30例,其中小细胞肺癌、腺癌和鳞癌各10例,应用比较基因组杂交技术(CGH)分析患者肺癌组织中全基因组的变化.结果:30例肺癌标本中CGH都发现有染色体异常改变,不同病理类型肺癌在1p11-p22、5p11-p14、16p11-p12、19q13、19p13、20p12和21q21等区域均有高频的扩增,在5q、6p24-pter、9p31-qter、13q21-qter和14q21-qter等区域均有高频缺失.同时不同病理类型肺癌的染色体异常表现也有一定区别.结论:遗传物质的异常改变在肺癌细胞中普遍存在,遗传物质异常是肺癌发生和发展的基础.不同病理类型肺癌在一些染色体区域的异常有所不同,这可以为三者的鉴别诊断提供一种遗传学标志.随着恶性肿瘤病程的进展,染色体畸变的复杂性也明显的提高.不同的致癌因素(如吸烟)可导致不同的染色体异常.     

Cytogenetic analysis of infantile neuroblastomas by comparative genomic hybridization

Neuroblastomas are heterogeneous tumors. Their clinical behavior varies from spontaneous regression to malignant progression. To investigate the cytogenetic heterogeneity of infantile neuroblastomas, we employed comparative genomic hybridization (CGH). To characterize chromosomal imbalances in 35 infantile neuroblastomas, we performed CGH and compared our results with those of other clinical and biological studies. The most frequent genetic imbalances were found in chromosome 17 (43%), including whole chromosome 17 gains in eight patients (23%) and 17q gains in seven patients (20%). A 1p loss and a 2p gain were detected in six patients each (17%). Losses of 11q and 14q were detected in two patients (6%) and one (3%) patient, respectively. The number of gains of 17q were significantly higher in DNA diploid tumors than in aneuploid tumors (P=0.006). Conversely, whole chromosome 17 gains were not found in DNA diploid tumors and/or MYCN amplification. Interestingly, nine of 17 tumors that were histologically evaluated showed a spontaneous regression and did not demonstrate any partial chromosomal abnormalities (i.e. 17q gain, 1p loss, 2p gain, 11q loss and 14q loss). These results suggest that a gene on chromosome 17q is associated with neuroblastoma progression. Finally, our observations indicate that the chromosomal imbalances observed in infantile neuroblastomas are different from those observed in older patients.     

Progression analysis of lung squamous cell carcinomas by comparative genomic hybridization.

OBJECTIVE: The genetic mechanisms underlying the development and progression of lung squamous cell carcinoma (SCC), the major subtype of non-small cell lung cancer, are still unknown. To better understand this disease, we studied the association between genetic alterations and the progression of lung SCC. METHODS: Chromosomal aberrations in 39 samples of lung SCC, including 21 nonmetastatic and 18 metastatic carcinomas, were characterized by comparative genomic hybridization and statistically correlated to clinical staging and metastatic ability. RESULTS: The average gains and losses per patient were significantly higher in the advanced-stage lung SCC and metastatic SCC group compared to the early-stage lung SCC and nonmetastatic SCC group. Gains of 2p, 20p and losses of 2q, 4q, 5q, 9q, 13p, 18q correlated with advanced-stage lung SCC. Losses on 2q, 4q, 6p, 16p, 16q, 18q, 20q, 21q and gains on 2p, 7p, 7q, 20p were more frequent in the metastatic SCC group, which was significantly different from the nonmetastatic SCC group. Gains on 2p, 20p and losses on 2q, 4q, 18q were not only associated with an advanced clinical stage but also with metastases of lung SCC. CONCLUSIONS: The results suggest that several chromosomal aberrations (e.g. gains on 2p, 20p and losses on 2q, 4q, 18q) may contribute to the progression of lung SCC.     

Whole-genome analysis of human astrocytic tumors by comparative genomic hybridization

Frozen sections of 35 astrocytic tumors of various histologic malignancies were analyzed by comparative genomic hybridization in an attempt to characterize the profile of genetic aberrations. Over 94% of the samples revealed DNA copy number aberrations, which increased with higher histological malignancy grades, and also involvement of more than one chromosome was seen in 85% of instances. The aberrations observed were mainly deletions and most frequently incorporated chromosomes 1p, 10, 19q, and 22q. On the other hand, gains or amplifications were detected only in glioblastomas. Additionally, such gains or amplifications were present in all tumor samples where the initial histopathological diagnosis was glioblastoma and immunohistochemical study disclosed p53 tumor suppressor protein negative and epidermal growth factor receptor positive immunoreactivity; such glioblastomas possessing p53 tumor suppressor protein positive and epidermal growth factor receptor negative immunoreactivity seldom displayed any gain. Thus, glioblastomas exhibiting two different profiles of genetic aberrations were recognized—one with and the other without any gains/amplifications. We speculate that the former variety is de novo glioblastoma.     

Combined cDNA array comparative genomic hybridization and serial analysis of gene expression analysis of breast tumor progression

To identify genetic changes involved in the progression of breast carcinoma, we did cDNA array comparative genomic hybridization (CGH) on a panel of breast tumors, including 10 ductal carcinoma in situ (DCIS), 18 invasive breast carcinomas, and two lymph node metastases. We identified 49 minimal commonly amplified regions (MCRs) that included known (1q, 8q24, 11q13, 17q21-q23, and 20q13) and several uncharacterized (12p13 and 16p13) regional copy number gains. With the exception of the 17q21 (ERBB2) amplicon, the overall frequency of copy number alterations was higher in invasive tumors than that in DCIS, with several of them present only in invasive cancer. Amplification of candidate loci was confirmed by quantitative PCR in breast carcinomas and cell lines. To identify putative targets of amplicons, we developed a method combining array CGH and serial analysis of gene expression (SAGE) data to correlate copy number and expression levels for each gene within MCRs. Using this approach, we were able to distinguish a few candidate targets from a set of coamplified genes. Analysis of the 12p13-p12 amplicon identified four putative targets: TEL/ETV6, H2AFJ, EPS8, and KRAS2. The amplification of all four candidates was confirmed by quantitative PCR and fluorescence in situ hybridization, but only H2AFJ and EPS8 were overexpressed in breast tumors with 12p13 amplification compared with a panel of normal mammary epithelial cells. These results show the power of combined array CGH and SAGE analysis for the identification of candidate amplicon targets and identify H2AFJ and EPS8 as novel putative oncogenes in breast cancer.     

应用比较基因组杂交技术研究外阴鳞癌组织的遗传变异

背景与目的:外阴鳞状细胞癌约占女性外阴恶性肿瘤的80%～90%.但有关其遗传学研究却不多,本研究采用比较基因组杂交(comparative genomichybridization,CGH)技术分析外阴鳞癌组织的遗传变异,了解其遗传改变的特征.以期发现外阴鳞癌相关基因.方法:应用CGH技术检测21例外阴鳞癌基因组的不平衡性即其DNA的扩增或丢失.结果:外阴鳞癌常见的扩增染色体是3q,8q,12q.常见的丢失染色体是4p和3p.结论:外阴鳞癌细胞中存在多条染色体拷贝数的改变,由此引起相应癌基因的扩增和抑癌基因的丢失可能参与了外阴鳞癌的发生、发展.     

比较基因组杂交技术检测肝细胞癌染色体异常及其临床意义

目的探讨肝细胞癌染色体异常及其临床意义。方法运用比较基因组杂交技术检测25例肝细胞癌染色体DNA异常情况,并与临床指标作相关分析。结果25例肝癌均存在不同程度的染色体DNA拷贝数的扩增或缺失,较为常见的染色体DNA异常是＋1q（72％）、＋1p（64％）、＋2q（、48％）、＋2p（48％）、＋5q（48％）、＋Xq（48％）、＋7q（44％）、-4q（48％）、-16p（48％）、-8p（40％）、-17p（36％）;相关分析显示＋17p、＋18p、-8p、-13q、-11q、-8q染色体异常事件与临床指标部分相关。结论肝细胞癌存在明显的染色体异常,部分染色体异常事件是非随机性的,可能与肝癌的发生、发展有关,并与肿瘤的生物学行为和预后相关。     

比较基因组杂交技术检测肝细胞癌染色体异常及其临床意义

目的探讨肝细胞癌染色体异常及其临床意义。方法运用比较基因组杂交技术检测25例肝细胞癌染色体DNA异常情况,并与临床指标作相关分析。结果 25例肝癌均存在不同程度的染色体DNA拷贝数的扩增或缺失,较为常见的染色体DNA异常是+1q(72%)、+1p(64%)、+2q(48%)、+2p(48%)、+5q(48%)、+Xq(48%)、+7q(44%)、-4q(48%)、-16p(48%)、-8p(40%)、-17p(36%);相关分析显示+17p、+18p、-8p、-13q、-11q、-8q染色体异常事件与临床指标部分相关。结论肝细胞癌存在明显的染色体异常,部分染色体异常事件是非随机性的,可能与肝癌的发生、发展有关,并与肿瘤的生物学行为和预后相关。     

High-resolution analysis of genomic copy number alterations in bladder cancer by microarray-based comparative genomic hybridization

We have screened 22 bladder tumour-derived cell lines and one normal urothelium-derived cell line for genome-wide copy number changes using array comparative genomic hybridization (CGH). Comparison of array CGH with existing multiplex-fluorescence in situ hybridization (M-FISH) results revealed excellent concordance. Regions of gain and loss were defined more accurately by array CGH, and several small regions of deletion were detected that were not identified by M-FISH. Numerous genetic changes were identified, many of which were compatible with previous results from conventional CGH and loss of heterozygosity analyses on bladder tumours. The most frequent changes involved complete or partial loss of 4q (83%) and gain of 20q (78%). Other frequent losses were of 18q (65%), 8p (65%), 2q (61%), 6q (61%), 3p (56%), 13q (56%), 4p (52%), 6p (52%), 10p (52%), 10q (52%) and 5p (43%). We have refined the localization of a region of deletion at 8p21.2-p21.3 to an interval of approximately 1 Mb. Five homozygous deletions of tumour suppressor genes were confirmed, and several potentially novel homozygous deletions were identified. In all, 15 high-level amplifications were detected, with a previously reported amplification at 6p22.3 being the most frequent. Real-time PCR analysis revealed a novel candidate gene with consistent overexpression in all cell lines with the 6p22.3 amplicon.     

比较基因组杂交在肿瘤研究中的应用

比较基因组杂交(CGH)是一种能对不同基因组间染色体上DNA序列拷贝数进行检测并定位的分子细胞遗传学技术.现综述比较基因组杂交的基本原理、方法、应用、优缺点及近期进展.     

比较基因组杂交在肿瘤研究中的应用

比较基因组杂交（CGH）是一种能对不同基因组间染色体上DNA序列拷贝数进行检测并定位的分子细胞遗传学技术.现综述比较基因组杂交的基本原理、方法、应用、优缺点及近期进展.     

Array comparative genomic hybridization analysis of genomic alterations in breast cancer subtypes

In this study, we performed high-resolution array comparative genomic hybridization with an array of 4153 bacterial artificial chromosome clones to assess copy number changes in 44 archival breast cancers. The tumors were flow sorted to exclude non-tumor DNA and increase our ability to detect gene copy number changes. In these tumors, losses were more frequent than gains, and gains in 1q and loss in 16q were the most frequent alterations. We compared gene copy number changes in the tumors based on histologic subtype and estrogen receptor (ER) status, i.e., ER-negative infiltrating ductal carcinoma, ER-positive infiltrating ductal carcinoma, and ER-positive infiltrating lobular carcinoma. We observed a consistent association between loss in regions of 5q and ER-negative infiltrating ductal carcinoma, as well as more frequent loss in 4p16, 8p23, 8p21, 10q25, and 17p11.2 in ER-negative infiltrating ductal carcinoma compared with ER-positive infiltrating ductal carcinoma (adjusted P values < or = 0.05). We also observed high-level amplifications in ER-negative infiltrating ductal carcinoma in regions of 8q24 and 17q12 encompassing the c-myc and c-erbB-2 genes and apparent homozygous deletions in 3p21, 5q33, 8p23, 8p21, 9q34, 16q24, and 19q13. ER-positive infiltrating ductal carcinoma showed a higher frequency of gain in 16p13 and loss in 16q21 than ER-negative infiltrating ductal carcinoma. Correlation analysis highlighted regions of change commonly seen together in ER-negative infiltrating ductal carcinoma. ER-positive infiltrating lobular carcinoma differed from ER-positive infiltrating ductal carcinoma in the frequency of gain in 1q and loss in 11q and showed high-level amplifications in 1q32, 8p23, 11q13, and 11q14. These results indicate that array comparative genomic hybridization can identify significant differences in the genomic alterations between subtypes of breast cancer.     

Analysis of pilocytic astrocytoma by comparative genomic hybridization

Very little is known about genetic abnormalities involved in the development of pilocytic astrocytoma, the most frequently occurring brain tumour of childhood. We have analysed 48 pilocytic astrocytoma specimens using comparative genomic hybridization. Only five of 41 tumours from children showed abnormalities detectable by comparative genomic hybridization, and in each case this represented gain of a single chromosome. Interestingly, two of seven tumours from adults showed abnormalities, which were multiple and relatively complex. Six of the seven tumours showing abnormalities were from female patients (two adults and four children). The most frequently detectable abnormality was gain of 9q34.1-qter, which was present in three cases (two adult and one paediatric).     

Genetic analysis of sorted Hodgkin and Reed-Sternberg cells using comparative genomic hybridization.

Hodgkin and Reed-Sternberg (H and RS) cells are generally considered to be the neoplastic cells of Hodgkin's disease (HD); however, such cells are found only in a minority of HD lesions. Very few data have so far been published on the cytogenetic abnormalities in HD. An analysis of unknown genetic aberrations has only recently become possible through the use of comparative genomic hybridization (CGH), which is based on the competitive binding of tumor and control DNA to metaphase chromosomes. In order to exclude the reaction of non-tumor cells, we used 100 sorted H-RS cells as the tumor DNA, then 100 sorted reactive T cells or B cells as the control DNA. We obtained the amplified DNA, using degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). In addition, to confirm whether or not the lymphocytes in the background were reactive, we performed CGH with 100 sorted B cells and 100 sorted T cells. CGH was thus performed on 9 HDs, including 6 cases of mixed-cellularity (MC) sub-type and 3 cases of nodular-sclerosis (NS) sub-type. CGH of the B and T cells showed no genetic changes in any cases. In contrast, CGH of H-RS cells revealed both gains and losses of DNA in all 9 cases, and multiple changes were also observed. In situ hybridization showed an Epstein-Barr-virus infection in 5 cases of MC; however, no definite relationship was observed between the EBV infection and genetic changes. The most commonly observed genetic aberrations were a loss on 16q11/21 in 6 cases, a gain on 1p13 in 5 cases, and a gain on 7q35/36 in 5 cases. The large number of chromosomal alterations in HD suggests, therefore, that an increased degree of genetic instability play a role in the formation of H-RS cells.