Comparative Genomic Hybridization Studies Research Articles

9p21 and 13q14 dosages in ependymomas. A clinicopathologic study of 101 cases

Ependymomas are glial neoplasms whose clinical behavior is difficult to predict based on histology alone. Recently, a comparative genomic hybridization study identified frequent chromosome 9p and 13q losses in anaplastic ependymomas, suggesting that p16 and RB alterations may be involved in tumor progression. In order to test this hypothesis further, 101 myxopapillary, conventional, and anaplastic ependymomas (51 spinal and 50 intracranial tumors) were tested for RB and p16 deletions using fluorescence in situ hybridization. Clinical follow-up, ranging from 2 to 198 months (median 46 months), was obtained in 90 cases (91%). RB and p16 deletions were seen in 22 of 92 (24%) and 22 of 89 (25%) informative cases, respectively. Polysomies were more frequent in the grade I and II spinal tumors, consistent with prior reports of increased aneuploidy in such cases. No significant genetic associations were seen with tumor grade, recurrence, or death, suggesting that 9p and 13q deletions do not play a prominent role in the malignant progression of ependymomas, as has been implicated in other glioma subtypes.

Comprehensive genome-wide comparison of DNA and RNA level scan using microarray technology for identification of candidate cancer-related genes in the HL-60 cell line

Genome-wide scans for DNA and RNA changes in the HL-60 cell line relative to normal leukocytes were conducted. Microarray-based comparative genome hybridization (CGH) studies were performed with the Spectral Genomics Human Bacterial Artificial Chromosome (BAC) 3MB system. Transcriptional measurements of approximately 12,500 human genes were monitored using Affymetrix U95A GeneChips. In HL-60, genomic DNA amplification of the 8q24 locus, trisomy 18, and deletions at loci 5q11.2∼q31, 6q12, 9p21.3∼p22, 10p12∼p15, 14q22∼q31, 17p12∼p13.3, and monosomy X were detected. After obtaining locus information about the RNA transcripts from the Affymetrix database, 4368 genes were stratified both according to status of RNA expression and the DNA copy number of their designated loci. The expression level of 2326 (53.25%) of 4368 transcripts is concordant with DNA copy number. Examples of specific, highly expressed, cancer-associated genes in amplified loci include SERPINB10, MYC, TYMS, HEC, and EPB41L3, while CD14, GZMK, TCF7, FOS, MLH3, CTNNA1, IRF1, VIM, CRK, MAP3K1, STAM, MAX, SFRG5, ENC1, PURA, MNT, RASA1, GLRX, UBE2B, NR3C1, PTENP1, BS69, COPEB, SKIP, PIM2, and MIC2 represent cancer-associated genes in deleted loci with decreased expression. The complementary usage of genome-wide DNA and RNA scans should enhance the identification of candidate genes in the neoplastic process.

P.2.102 Aripiprazole treatment for psychosis in patients with Alzheimer's disease

Adrenocortical tumors (ACT) are rare neoplasms of the adrenal glands accounting for 0.2% of all pediatric cancers. However, the incidence of ACT in South Brazilian children is 10 to 15 times greater than the worldwide incidence. Comparative genomic hybridization studies have revealed the presence of a high degree of chromosomal instability in ACT. We evaluated 16 ACT, 8 of them carcinomas and 8 adenomas. The presence of changes in DNA copy numbers was determined by comparative genomic hybridization, and the findings were validated by real-time polymerase chain reaction on the basis of IGF-II gene expression. The adenomas showed a mean of 19.7 imbalances per case, with the most frequent gain and loss being 4p15.1–p15.3 and 20p11.2–p13.2, respectively. The carcinomas presented with a mean of 35.5 imbalances per case, with the more frequent gain being 2q14.1–q24.3 and the more frequent losses being 3q21–q26.2, 20q12–qter, and 22q11.2–q13.3. The most frequent imbalances in both adenomas and carcinomas were gains of 1p21–p31.2, 2p12–p21 and loss of 20p11.2–p12. The expression of IGF-II mRNA (11p15.5) was higher in samples that presented with a gain of this region. It has been established that great genomic instability exists in pediatric ACT.

Microsatellite alterations in hepatocellular carcinoma and intrahepatic cholangiocarcinoma

A series of 20 hepatocellular carcinomas and 8 intrahepatic cholangiocarcinomas was screened from the Korean population for microsatellite alterations, including a loss of heterozygosity and replication errors using nine microsatellite markers containing several genes. The microsatellite results and our previous comparative genomic hybridization results of two tumors were compared at each locus, and the correlations between these and clinicopathologic variables were examined. The most characteristic findings were found at 13q. Replication errors were prevalent at D13S160 (13q21.2∼q31) and D13S292(13q12). The incidence of loss of heterozygosity, however, was higher at D13S153 (13q14.1∼q14.3) and D13S265(13q31∼q32). In contrast, there were higher deletion frequencies observed in hepatocellular carcinoma (HCC) and higher amplification frequencies observed in intrahepatic cholangiocarcinoma at 13q in our previous comparative genomic hybridization (CGH) study. Higher frequencies of replication errors were observed at D16S408 (13q12∼q21) and D16S504(13q23∼q24) in the HCC. This study found that significant differences in the patterns of genetic instability of microsatellites were dependent on the chromosomal loci. It is believed that certain genes at altered CGH regions, which are relevant to the development and/or progression of these cancers, are activated by different mutation mechanisms.

Recurrent genetic alterations in 26 colorectal carcinomas and 21 adenomas from Chinese patients

Colorectal cancer (CRC) is one of the most common malignancies worldwide. The incidence of CRC in the Chinese population has increased dramatically during the last two decades; however, nonrandom chromosomal alterations in Chinese patients have not been described. In the present study, comparative genomic hybridization (CGH) was applied to detect recurrent chromosome alterations in 26 primary colorectal carcinomas and 21 colorectal adenomas from Chinese patients. In CRC, several recurrent chromosomal changes were found, including gains of 8q (14/26 cases, 54%), 20q (54%), 3q (50%), 13q (50%), 5p (46%), 7p (42%), 7q (42%), and 12p (38%) and losses of 18q (65%) and 17p (42%). From comparison with previous CGH studies, the frequent gains of 3q and 12p might be distinctive occurrences in Chinese patients. The distribution of frequently found chromosomal alterations in different locations was studied. The gain of 20q was more frequently found in colon cancer ( P<0.01) and the gain of 12p was more frequently found in rectal cancer. Chromosomal alterations were found in 19/21 of adenomas; the most frequent chromosomal alteration was the loss of 18q (9/21 cases, 43%). These recurrent alterations provide several starting points for the isolation of candidate oncogenes and tumor suppressor genes.

Elevated expression levels of NCOA3, TOP1, and TFAP2C in breast tumors as predictors of poor prognosis.

Amplification of DNA in certain chromosomal regions plays a crucial role in the development and progression of human malignancies, specifically when protooncogenic target genes within those amplicons are overexpressed. Comparative genomic hybridization studies have revealed frequent amplification at 20q in primary breast tumors. The aim of the current study was to identify specific genes in the 20q amplicon that were likely to have clinical significance. The authors examined 38 primary breast tumors by using a quantitative real-time reverse transcription-polymerase chain reaction assay to determine expression levels of 18 potential targets for amplification events involving 20q. Potential correlations between elevated expression of the genes in question and clinicopathologic parameters or clinical outcomes were analyzed. Elevated expression of NABC1 was significantly associated with positive estrogen (P < 0.001) and progesterone (P = 0.027) receptors in breast tumors, and high expression of PTK6 was significantly correlated with positive estrogen receptor status (P = 0.022) and postmenopausal status (P = 0.008). Patients whose tumors showed elevated expression of NCOA3 (AIB1) had significantly shorter disease-free (P = 0.017) and overall (P = 0.0021) survival times after surgery than did other patients with breast tumors. Reduced disease-free survival, but not reduced overall survival, was associated with high expression of TOP1 (P = 0.035) and TFAP2C (P = 0.035). TOP1, TFAP2C, and (particularly) NCOA3 may be prognostic indicators for patients with breast tumors.

Identification of Four Distinct Regions of Allelic Imbalances on Chromosome 1 by the Combined Comparative Genomic Hybridization and Microsatellite Analysis on Hepatocellular Carcinoma

Frequent chromosome 1 abnormalities detected in human hepatocellular carcinoma have been implicated in early genetic events of liver carcinogenesis. Recurrent loss of 1p with a common deleted region 1p36–p34 has been reported from microsatellite analysis, whereas common gain of the whole chromosome q-arm was described from several comparative genomic hybridization studies. The relationships between copy number changes and allelic status however remains unclear. In this study, we have conducted a simultaneous comparative genomic hybridization and microsatellite analysis study on chromosome 1 in 31 hepatocellular carcinoma cases. Microsatellite analysis revealed frequent loss of heterozygosity on 1p at loci D1S468 (74%), D1S450 (67%), D1S2667 (65%), D1S2697 (75%), D1S199 (52%), and D1S234 (67%) corresponded to the distal 1p36 region and coincided with 12 cases (86%) that presented losses on 1p by comparative genomic hybridization analysis. Although comparative genomic hybridization indicated a common deleted region of 1p36–p35 in the current series, microsatellite analysis has refined the smallest overlapping region (SOR) to 1p36.13–p36.22. Gain of 1q as revealed by comparative genomic hybridization suggested low and high-level gains, and cases that displayed an amplicon below the heterochromatic region 1q21–q25. Common allelic imbalances of polymorphic markers D1S2635 (64%), D1S484 (67%), D1S2878 (65%), D1S196 (70%), D1S249 (64%) D1S2785 (75%), D1S2842 (73%) and D1S2836 (74%) that corresponded to the regions 1q23.1–q24.2, 1q32.1 and 1q43–q44 were detected. Three distinct regions of allelic imbalances were thus suggested on recurring 1q gain found in hepatocellular carcinoma. Furthermore, microsatellite analysis has enabled a mapping of common overrepresented regions and suggested SOR on 1q23.1–q23.3 (D1S2635-D1S2878), 1q25.1–q31.1 (D1S452-D1S238), and 1q43 (D1S2785-D1S2842). Our current study has refined chromosome 1 aberrations in hepatocellular carcinoma to four regions of allelic imbalances. The SORs delineated should provide basis for further molecular investigation in hepatocarcinogenesis on genes residing on these chromosomal regions.

Mutational analysis of Smad3, a candidate tumor suppressor implicated in TGF-beta and menin pathways, in parathyroid adenomas and enteropancreatic endocrine tumors.

Based upon molecular allelotyping and comparative genomic hybridization studies, chromosome 15q is the likely location of a tumor suppressor gene important in the pathogeneses of sporadic enteropancreatic endocrine tumors and parathyroid adenomas. Interest has focused on Smad3 as a candidate endocrine tumor suppressor gene because 1) it is localized to 15q and 2) it encodes a TGF beta signaling molecule that has been identified as a binding partner of the multiple endocrine neoplasm type 1 gene product menin, itself involved in enteropancreatic and parathyroid neoplasia. To determine whether Smad3 plays a primary role in development of these tumors, 20 enteropancreatic tumors and 67 parathyroid adenomas were investigated for loss of heterozygosity at DNA markers surrounding Smad3. Twenty percent of enteropancreatic tumors and 24% of parathyroid adenomas showed loss. All 9 coding exons and intron-exon boundaries of the Smad3 gene were then sequenced in genomic DNA from all 20 enteropancreatic and 25 parathyroid tumors, including every case with loss of heterozygosity. No acquired clonal mutations, insertions, or microdeletions in Smad3 were detected in any tumors. Because inactivating somatic mutation is the hallmark of an authentic tumor suppressor, Smad3 is unlikely to function as a classical tumor suppressor gene in the pathogenesis of sporadic parathyroid or enteropancreatic endocrine tumors.

Rearrangements involving the 13q chromosome arm committed to the progression of laryngeal squamous cell carcinoma

Our recent comparative genomic hybridization (CGH) study has shown that losses of the long arm of chromosome 13 were the most common aberrations in primary larynx tumors and their corresponding metastases. In the present study, 20 pairs of primary larynx tumors and their metastases were analyzed by interphase fluorescence in situ hybridization (FISH) with three different 13q-specific probes ( RB1, D13S25, and 13qtel). Our experiments were generally consistent with the CGH results, with some differences indicating cell population heterogeneity in the analyzed cohort of tumors. The results provided further evidence for the putative role of the RB1 gene alterations in the metastatic process, although a contribution by other gene(s) during metastasis cannot be ruled out.

A comparative genomic hybridization study in a 46,XX male

Objective: To identify Y chromosome material in an azoospermic male with an XX karyotype. Design: Case report. Setting: Faculty of medicine and Centro de Patologia Celular (CPC) medical center. Patient(s): A 33-year-old man with infertility. Intervention(s): G-banding, fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), and comparative genomic hybridization (CGH). Main Outcome Measure(s): FISH for X and Y chromosomes, PCR for the SRYgene and amelogenin gene in the Xp (AMGX) and (AMGY), and losses or gains with CGH. Result(s): FISH analysis using X and Y chromosome-specific probes showed an X chromosome containing Y chromosome sequences on the top of the short arm; this Y chromosome region was not visible by conventional cytogenetic analysis. PCR amplification of DNA showed the presence of the sex-determining region of the Y chromosome (SRY) and the amelogenin gene in the pseudoautosomal boundary of the X chromosome (AMGX). CGH confirmed the presence of the chromosome region Yp11.2-pter and detected the presence of the two otherwise normal X chromosomes. Conclusion(s): The two Xpter (XPAR1) pseudoautosomal regions present in this XX male suggest the need to reevaluate XX males using CGH and PCR to characterize the clinical variability in XX males due to genes other than those located on the Y chromosome.

Cytogenetic and molecular analyses of multiple endocrine neoplasias of the MEN1 syndrome

A pancreas carcinoid, an adrenocortical adenoma, three parathyroid adenomas and a parathyroid hyperplasia of 5 MEN1 patients were used for loss of heterozygosity (LOH) and comparative genomic hybridization (CGH) studies. The MEN1 gene is located in the region 11q13, approximately 30 kb distal to PGYM. Four tumors showed LOH on chromosome 11q13 (D11S11335, PGYM, D11S1883, FGF3, D11S937) however LOH was also found beyond 11q13. The pancreas carcinoid and adrenocortical adenoma, both from the same patient, showed LOH at marker 11q23.3 and 11q25. In the three parathyroid adenomas LOH was detected in five different markers: 11q21, 11q22.3, 11q23.2, 11q23.3 and 11q25. No LOH was found in parathyroid hyperplasia. CGH analysis showed in case of the pancreas carcinoid losses on chromosomes 1p, 2q, 3, 6, 9p, 11 and 12p. Gains were found at 4, 5, 7, 8, 13, 14, 15q, 18, 19. The parathyroid adenoma of the third patient showed losses only on chromosome 11 in the region 11p12-p15 and 11q12-q23. Our data indicate that other genes are involved in the tumorigenesis of the MEN1 syndrome. Especially the numerous allelic losses between markers 11q23 and 11q25 (D11S938 and D11S910) are a hint for further tumor suppressor genes on chromosome 11.

Compilation of published comparative genomic hybridization studies

The power of comparative genomic hybridization (CGH) has been clearly proven since the first paper appeared in 1992 as a tool to characterize chromosomal imbalances in neoplasias. This review summarizes the chromosomal imbalances detected by CGH in solid tumors and in hemopathies. In May of 2001, we took a census of 430 articles providing information on 11,984 cases of human solid tumors or hematologic malignancies. Comparative generic hybridization has detected a number of recurrent regions of amplification or deletion that allows for identification of new chromosomal loci (oncogenes, tumor suppressor genes, or other genes) involved in the development, progression, and clonal evolution of tumors. When CGH data from different studies are combined, a pattern of nonrandom genetic aberrations appears. As expected, some of these gains and losses are common to different types of pathologies, while others are more tumor-specific.

DNA Copy Number Changes in Lung Adenocarcinoma in Younger Patients

We performed a comparative genomic hybridization study on 25 lung adenocarcinoma samples from younger patients (<41 y of age) and compared the results with a previous comparative genomic hybridization analysis of lung adenocarcinoma samples from older patients (50–81 y of age). Twenty of the 25 tumor samples from younger patients had DNA copy number changes. Gains, losses, and high-level amplifications were seen more frequently in the specimens from the younger group. The most striking difference between the two groups was the high frequency of gains and/or high-level amplifications in the long arm of chromosome 20 in the samples from the younger patients (14/25, 56%) compared with that in the samples from the older patients (2/24, 8%, P < .001). Gains in the long arm of chromosome 22 and of the chromosomal band 11q13 were also detected significantly more often in the younger group. No correlation was found between DNA copy number changes and clinical parameters. Our results suggest that amplification of genes in the long arm of chromosome 20 may be important in the tumorigenesis of lung adenocarcinoma in young adults. Several candidate genes have already been described in the long arm of chromosome 20, particularly in breast cancer.

Genome-wide detection of chromosomal imbalances in tumors using BAC microarrays.

Chromosomal imbalances such as deletions and amplifications are common rearrangements in most tumors. Specific rearrangements are consistently associated with specific tumor types or stages, implicating the role of the genes in a region of chromosomal imbalance in tumor initiation and progression. The development of comparative genomic hybridization (CGH) has obviated the need to obtain metaphase spreads from tumors, so that the chromosomal imbalances in many solid tumors may be revealed using an extracted genomic DNA sample. However, the resolution of the cytogenetic method remains and the extreme technical difficulty of CGH has restricted its use. Conceptually, DNA microarray-based CGH is an obvious solution to all of the limitations of conventional CGH. Although arrays have been used for CGH studies, their success has been limited by poor specific signal-to-noise ratios. Here we demonstrate a microarray-based CGH method that allows reliable detection of chromosomal deletions and amplifications with high resolution. Our microarray system is fundamentally different from most current microarray technologies in that activated DNA is printed on natural glass surfaces while other systems almost exclusively focus on activating the surfaces, a strategy that invariably introduces hybridization backgrounds. The concept of using pre-modification may be generally applied for making arrays of other biological materials, as modifying the substrates will be more controllable in solution than on surfaces.

A CGH study of 27 patients with CHARGE association.

CHARGE association is a non-random occurrence of congenital malformations including coloboma, heart disease, choanal atresia, retarded growth and/or retarded development, genital hypoplasia, ear anomalies and/or deafness. The cause of this association remains unknown. Various genetic mechanisms have been proposed, including a contiguous gene syndrome but, so far, no recurrent locus has been identified. To address this question, we decided to perform a comparative genomic hybridization (CGH) study on a cohort of 27 patients with CHARGE association and a normal standard karyotype. We found two chromosomal anomalies: a der(9)t(9;13) derived from a paternal translocation and a der(6)t(4;6) of unknown origin. This suggests that chromosome imbalances may well mimic CHARGE association. Therefore patients with CHARGE association must be carefully tested with classical and molecular cytogenetic techniques to detect a potential chromosome imbalance. It is expected that more stringent diagnostic criteria of CHARGE association could define a more homogeneous group of patients where a single genetic cause might be identified.

Comparative genomic hybridization study of genetic changes associated with vindesine resistance in esophageal carcinoma

The acquisition of drug-resistance is a major problem for cancer patients undergoing chemotherapy. To clarify genetic alterations in cancer cells that develop drug-resistance, comparative genomic hybridization (CGH) was applied to esophageal squamous cell carcinoma cell lines (SH-1V1, SH-1V2, SH-1V4 and SH-1V8) and chemoresistance-related genes in altered chromosomal regions were evaluated. These cell lines were derived from the parental SH-1 cell line, after multiple steps of selection by an increasing exposure to vindesine. SH-1V8 cells were strongly resistant to vindesine. DNA copy number at 16p which includes the MRP (multidrug resistance related protein) gene was markedly increased in all cell lines examined. Increased DNA copy numbers were found at the regions of 5q31-32, 10q11.1-23, and 14q32-qter in SH-1V8 cells that acquired resistance to other drugs as well. Both SH-1V4 and SH-1V8 showed increased DNA copy numbers at 7q11.1-22, 16q12.1-qter, 19p13.2-13.3, 19q11-13.2 and 20q13.1-qter. The chromosomal region of 7q11.1-22 including MDR-1 (multidrug resistance-1) gene was highly amplified in SH-1V4 and SH-1V8. Amplification of the MRP region suggests the prerequisite of developing resistance to vindesine, and further amplification of MDR-1 may play a critical role in acquiring drug-resistance. Several unknown genes related to the induction of chemoresistance might be concealed in other altered chromosomal regions.

Overrepresentation of 17q22∼qter and 22q13 in dermatofibrosarcoma protuberans but not in dermatofibroma: a comparative genomic hybridization study

Histopathological differentiation between dermatofibrosarcoma protuberans (DFSP) and dermatofibroma (DF) is often difficult, because both neoplasms share some clinical features and the presence of a storiform pattern. In the present study, we investigated the usefulness of comparative genomic hybridization (CGH) in the diagnosis of these entities by examining 12 DFSP and 12 DF cases. The most frequent DNA sequence copy number changes detected in 10 (83%) of 12 DFSP cases (mean, 1.9 aberrations/tumor; range, 0–3) consisted of gains of 17q22∼qter (10 tumors), 22q13 (nine tumors), and 8q24.1∼qter (three tumors). High-level amplification, which was detected in three tumors, was seen only in chromosome 17, with 17q23∼q25 as the minimal common region. Loss of DNA sequences was not found in DFSP cases. In contrast, two (17%) of the 12 DF cases (mean, 0.5 aberrations/tumor; range, 0–4) showed DNA sequence copy number changes, although recurrent gains and losses and high-level amplifications were not observed. Gains were more common than losses in DF. Overrepresentation of 17q and 22q sequences was a common finding in DFSP but not in DF. Thus, CGH seems to be useful for distinguishing DFSP from DF in most cases.

Routine acid decalcification of bone marrow samples can preserve DNA for FISH and CGH studies in metastatic prostate cancer.

Production of paraffin-section material from tissue samples that contain bone requires decalcification. Techniques such as acidic decalcification or EDTA chelation are suitable methods. Acid decalcification is generally quicker than EDTA chelation but studies have suggested that it may result in hydrolysis of DNA. Here we show that limited acid decalcification (less than 24 hr) in 5% formic acid can preserve DNA sufficient for fluorescent in situ hybridization (FISH) or comparative genomic hybridization (CGH) and that prolonged 10% formic acid decalcification results in failure of FISH and only limited retrieval of DNA for CGH studies.

Novel targets for the 18p11.3 amplification frequently observed in esophageal squamous cell carcinomas.

Amplification of DNA in certain chromosomal regions, with consequent over-expression of specific genes within these amplicons, plays a crucial role in the development and progression of human cancer. Since our previous comparative genomic hybridization (CGH) study revealed frequent amplifications at 18p in esophageal squamous cell carcinomas (ESC) cell lines, we focused on the identification of genetic target(s) within the 18p amplicon. In four cell lines having remarkable copy-number amplification with homogeneously staining region (HSR) pattern by fluorescence in situ hybridization (FISH), the smallest common region of overlapping covered approximately 3.5 Mb at 18p11.3. We screened 29 ESC cell lines to discern amplifications and expression levels of 14 known genes and 21 uncharacterized transcripts within the amplicon. Only four known genes, YES1, TYMS, HEC and TGIF showed amplification and consequent over-expression. These genes were amplified in several of primary ESCs. Moreover, resistance to transforming growth factor beta (TGFbeta)-induced growth inhibition was enhanced in four cell lines with amplification and expression of TGIF, which encodes the repressor for TGFbeta-activated transcription, appears to be involved in the progression of ESC. Taken together, these results suggest that YES1, TYMS, HEC and TGIF are likely to be candidate targets for 18p11.3 amplification and be associated with esophageal tumorigenesis.

Chromosomal and Comparative Genomic Analyses of HHV-8-Negative Primary Effusion Lymphoma in Five HIV-negative Japanese Patients

A rare subset of HIV lymphoma, known as primary effusion lymphoma (PEL), is a high-grade tumour carrying human herpes virus 8 (HHV-8). Very little is known about genomic aberration in PEL, and only a few HIV-negative PEL have been reported. Here we report the results of chromosomal analysis and comparative genomic hybridisation (CGH) conducted to detect regions of gain and loss, in five HIV-negative Japanese cases of HHV-8-negative PEL. All patients except one (35-year-old female) were elderly men and the morphologic examination showed large cell type. PEL expressed B-cell-associated and activation-associated antigens, and exhibited clonal immunoglobulin genes. No HHV-8 was detected in all four examined cases, but Epstein-Barr virus (EBV) was detected in one case. Genomic abnormalities and aberrations were identified in all HHV-8/HIV-negative PEL. CGH studies showed gain in 19 of 24 chromosomes. Gains of 3q13-27, 8q24, 10q21-23 and Yq were detected in two of the five cases, but other gains were noted in each case. Chromosomal analysis revealed complex abnormalities both in numbers and structures. Burkitt lymphoma-associated t(8;22) was detected in one case, but +8 chromosome and c-myc amplification were detected in the other three cases by Southern blot and/or fluorecence in situ hybridization (FISH). Abnormality of chromosome 8, which associates with c-myc, was detected in four of the five HHV-8/HIV-negative PEL. However, the other common genomic abnormalities of HHV-8/HIV-negative PEL were not detected in our study, but the complex abnormalities seemed to be true rather than the usual large B-cell lymphoma. Our results suggest that multi-step genomic abnormalities might be associated in HHV-8/HIV-negative PEL tumorigenesis.