Gains of 8q23-qter and 20q and loss of 11q22-qter in esophageal squamous cell carcinoma associated with lymph node metastasis

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is associated with poor prognosis and lymph node metastasis is one of the critical prognostic factors. Although it is important to elucidate the genetic aberrations underlying its lymph node metastasis, to the authors' knowledge little is known regarding alterations in the primary ESCC that are linked with ESCC metastasis to the lymph nodes. METHODS: To elucidate genetic aberrations involved in the lymph node metastasis of ESCC, comparative genomic hybridization analysis was applied to 36 ESCC specimens, from 12 cases with no lymph node metastasis and 24 cases with lymph node metastasis. RESULTS: Copy number gains frequently were detected at 3q (75%), 8q23-qter (50%), 11q13 (44%), 5p14-pter (25%), 20q (25%), 7q (22%), 2p (19%), 12p (17%), and 20p (17%) and losses were detected at 18q (58%), 3p (50%), 9p (44%), 5q14-23 (39%), 4q (33%), 13q (22%), and 11q22-qter (19%). DNA amplifications were detected at four loci: 11q13, 2q12, 7q21, and 20q11.2 It is interesting to note that the gains of 8q23-qter (P < 0.0005) and 20q (P < 0.02) and loss of 11q22-qter (P < 0.05) were observed in tumors metastatic to the lymph nodes. The gains of 3q and 11q13 and losses of 18q, 3p, 9p, 5q14-23, and 4q were detected in both early and advanced stage ESCCs. CONCLUSIONS: These observations suggest that gains of 8q23-qter and 20q and loss of 11q22-qter allow the prediction of lymph node metastasis, and that gains of 3q and 11q13 and losses of 18q, 3p, 9p, 5q14-23, and 4q are associated with the development of ESCC.     

Detection of chromosomal alterations in bladder transitional cell carcinomas from Northern China by comparative genomic hybridization

To identify chromosome alterations in Chinese bladder cancer, forty-six transitional cell carcinomas of the bladder were analyzed by comparative genomic hybridization. Frequent gains of DNA copy number were observed on 1p (13/46), 1q (13/46), 5p (8/46), 6p (9/46), 7p (7/46), 8q (12/46), 11q (8/46), 17q (11/46), 19q (7/46), 20q (8/46) and Yq (8/46), with minimal overlapping regions at 1p32-pter (10/46), 1q21-q24 (12/46), 5p (8/46), 6p22-p23 (7/46), 7p11.2-p14 (7/46), 8q22-q24 (12/46), 11q13-q14 (8/46), 17q22-qter (11/46), 19q11-13.2 (7/46), 20q11-q13.2 (8/46) and Yq11 (8/46). Losses were predominantly found on 2q (16/46), 5q (8/46), 8p (7/46), 9p (8/46), 9q (13/46), 11p (7/46), 13q (7/46), 17p (12/46), 18q (7/46), Xp (18/46) and Xq (19/46), with smallest overlapping regions at 2q32-qter (16/46), 5q12-q31 (8/46), 8p12-pter (7/46), 9p21-pter (10/46), 9q (13/46), 11p (7/46), 13q13-q22 (7/46), 17p (12/46), 18q21-qter (7/46), Xp (18/46) and Xq (19/46). There were significantly higher frequencies of gains of 1q21-q24 and 17q22-qter in moderately differentiated tumors as compared with those in well-differentiated tumors, indicating a possible association of these two abnormalities with the dedifferentiation of tumor cells. Gains of 1p32-pter, 5p, 6p22-p23, 11q13-q14, 17q22-qter and losses of 2q32-qter, 9q, 17p were more frequent in pT1 as compared with those in pTa carcinomas. Gains at 1q21-q24, 7p11.2-p14, 8q22-q24, 19q, 20q11-q13.2 and losses at 5q12-q31, 8p12-pter, 9p21-pter, 11p, 13q13-q22 and 18q21-qter were unique to pT1 and higher stage tumors, suggesting that genes responsible for the invasion and progression of bladder cancer might be located at these chromosomal regions. In multiple tumors from the same patients, consistent alterations such as gains of 8q, 11q13-q14, 12q13-q15, 13q12, 20q and losses of 2q32-qter, 8p, 9, 11p, 11q21-qter, 13q13-qter, X were detected. These abnormalities were possibly earlier events, which might play a critical role during the genesis of the tumors. Further detailed studies to the recurrent aberration regions may lead to the identification of oncogenes and tumor suppressor genes involved in the development and progression of Chinese bladder cancer.     

11q13 amplification status and human papillomavirus in relation to p16 expression defines two distinct etiologies of head and neck tumours

Two distinct etiologies of head and neck squamous cell carcinoma (HNSCC) have been proposed, DNA damage owing to tobacco and alcohol exposure and human papillomavirus (HPV) oncogene-mediated transformation. Common genetic alterations in HNSCC include TP53 mutations, 11q13 amplification (amp) and CDKN2A/p16 mutations or promoter methlyation. However, in HPV+ HNSCC it is frequent to observe wild-type TP53 and expression of p16. The relationship of this unusual pattern with 11q13 amp has not been tested. In a retrospective study on 125 HNSCC patients, only 17% (five out of 30) of HPV+ vs 44% (39 out of 89) of HPV - tumours expressed 11q13 amp (adjusted odds ratio (OR)=0.2, 95% confidence interval (CI)=0.1-0.6). A subpopulation of tumours (n=69) were classified according to the three molecular markers, TP53, p16 and 11q13 amp. In addition to wild-type TP53, and p16 expression, HPV+ tumours were more likely not to be amplified at 11q13 (OR=6.5, 95% CI=1.8-23.9). As HPV+ HNSCC lack the genetic alterations which are common in other tumours, we hypothesise that HPV infection may represent an early event in the HNSCC carcinogenic process, thus suggesting a distinct molecular pathway.     

Patterns of chromosomal imbalances in invasive breast cancer

Invasive breast carcinomas are characterized by a complex pattern of chromosomal alterations. We applied comparative genomic hybridization (CGH) to analyze 105 primary breast carcinomas using histograms to indicate the incidence of DNA imbalances of tumor subgroups and difference histograms to compare invasive ductal carcinomas (IDC) with lobular carcinomas (ILC), well and poorly differentiated carcinomas (G1/G3) and estrogen receptor-positive and -negative tumors (ER(+)/ER(-)). Only single imbalances showed a higher incidence in ILC compared with IDC, i.e., gains on chromosomes 4 and 5q13-q23 as well as deletions on chromosomes 6q, 11q14-qter, 12p12-pter, 16q, 17p, 18q, 19, and 22q. Of these, particularly gains of 4 and losses at 16q21-q23, and 18q12-q21 were statistically significant. For most loci, IDC showed more alterations providing a genetic correlate to the fact that ductal carcinoma overall is associated with a worse prognosis than ILC. Of these, many imbalances showing statistical significance were also observed in G3 and ER(-) tumors, i.e., deletions at 2q35-q37, 3p12-p14, 4p15-p16, 5q, 7p15, 8p22-p23, 10q, 11p, 14q21-q31, 15q, and gains at 2p, 3q21-qter, 6p, 8q21-qter, 10p, 18p11-q11, and 20q, suggesting that they contribute to a more aggressive tumor phenotype. By contrast, gains on chromosome 5q13-q23 as well as deletions at 6q, 16q and 22q were more prevalent in G1 and ER(+) tumors. The ratio profiles of all cases as well as histograms are accessible at our CGH online tumor database at http://amba.charite.de/cgh. Our results highlight distinct chromosomal subregions for cancer-associated genes. In addition, these imbalances may serve as markers for a genetic classification of invasive breast 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.     

Establishment of a new human pleomorphic malignant fibrous histiocytoma cell line,FU-MFH-2: molecular cytogenetic characterization by multicolor fluorescence in situ hybridization and comparative genomic hybridization

Background

Pleomorphic malignant fibrous histiocytoma (MFH) is one of the most frequent malignant soft tissue tumors in adults. Despite the considerable amount of research on MFH cell lines, their characterization at a molecular cytogenetic level has not been extensively analyzed.

Methods and results

We established a new permanent human cell line, FU-MFH-2, from a metastatic pleomorphic MFH of a 72-year-old Japanese man, and applied multicolor fluorescence in situ hybridization (M-FISH), Urovysion™ FISH, and comparative genomic hybridization (CGH) for the characterization of chromosomal aberrations. FU-MFH-2 cells were spindle or polygonal in shape with oval nuclei, and were successfully maintained in vitro for over 80 passages. The histological features of heterotransplanted tumors in severe combined immunodeficiency mice were essentially the same as those of the original tumor. Cytogenetic and M-FISH analyses displayed a hypotriploid karyotype with numerous structural aberrations. Urovysion™ FISH revealed a homozygous deletion of the p16^INK4A locus on chromosome band 9p21. CGH analysis showed a high-level amplification of 9q31-q34, gains of 1p12-p34.3, 2p21, 2q11.2-q21, 3p, 4p, 6q22-qter, 8p11.2, 8q11.2-q21.1, 9q21-qter, 11q13, 12q24, 15q21-qter, 16p13, 17, 20, and X, and losses of 1q43-qter, 4q32-qter, 5q14-q23, 7q32-qter, 8p21-pter, 8q23, 9p21-pter, 10p11.2-p13, and 10q11.2-q22.

Conclusion

The FU-MFH-2 cell line will be a particularly useful model for studying molecular pathogenesis of human pleomorphic MFH.     

Chromosomal imbalances in malignant peripheral nerve sheath tumor detected by metaphase and microarray comparative genomic hybridization

Malignant peripheral nerve sheath tumors (MPNSTs) are highly malignant tumors affecting adolescents and adults. There have been a few reports on chromosomal aberrations of MPNSTs; however, the tumor-specific alteration remains unknown. We characterized the genomic alterations in 8 MPNSTs and 8 schwannomas by metaphase comparative genomic hybridization (CGH). In 5 of 8 MPNSTs, microarray CGH was added for more detailed analyses. Frequent gains were identified on 3q13-26, 5p13-14, and 12q11-23 and frequent losses were at 1p31, 10p, 11q24-qter, 16, and 17. Microarray CGH revealed frequent gains of EGFR, DAB2, MSH2, KCNK12, DDX15, CDK6, and LAMA3, and losses of CDH1, GLTSCR2, EGR1, CTSB, GATA3, and SULT2A1. These genes seem to be responsible for developing MPNSTs. The concordance rate between metaphase CGH and microarray CGH was 66%. Metaphase CGH was useful for identifying chromosomal alterations before applying microarray CGH.     

Comparative genomic hybridization in primary sinonasal adenocarcinomas

BACKGROUND: Little is known about the genetic alterations that occur in sinonasal adenocarcinomas. The goal of the current study was to detect recurrent chromosomal gains and losses in a series of 21 primary sinonasal adenocarcinomas using comparative genomic hybridization (CGH). METHODS: The authors examined ethmoid sinus adenocarcinoma samples from 21 patients. All 21 adenocarcinomas were associated with work-related exposure to wood dust. CGH was used to detect chromosomal abnormalities, and the results of CGH analysis were evaluated for correlations with clinicopathologic characteristics. RESULTS: Chromosomal gains and losses were detected in all 21 adenocarcinomas. Gains were detected at high frequencies at 7q11-21 (n = 15 [71%]), 18p11 (n = 14 [66%]), 8q11-22 (n = 13 [62%]), 5p11-13 (n = 12 [57%]), 12q11-13 and 19p (n = 11 [52%]), 20q (n = 10 [47%]), X and 5p (n = 9 [43%]), and 3q26-27 (n = 8 [38%]); and losses were detected at 8p22-23 (n = 18 [86%]), 18q22-23 (n = 17 [80%]), 17p13 (n = 12 [57%]), and 5q31-qter (n = 11 [52%]). Aside from low-level gains, 43 high-level amplifications were observed in the current series of 21 tumors, most commonly at Xq13 (n = 7 [33%]). CONCLUSIONS: CGH revealed that ethmoid sinus adenocarcinomas carry a large number of chromosomal losses and gains, including high-level amplifications. To the authors' knowledge, the current study represents the first attempt to investigate sinonasal adenocarcinomas on a genetic level by using CGH. The pattern of chromosomal abnormalities in these tumors was different from the pattern in other tumors within the same anatomic region (e.g., squamous cell carcinomas and salivary gland tumors); this finding may be explained by differences in etiology. Nonetheless, sinonasal adenocarcinomas appear to be genetically similar to adenocarcinomas of the stomach and colon, which also have an etiology that differs from that of sinonasal adenocarcinomas. Further study is necessary to better understand the molecular genetic basis underlying the development of sinonasal adenocarcinomas. In the near future, this type of understanding may present new possibilities for prevention and treatment of malignant disease.     

Chromosomal alterations during lymphatic and liver metastasis formation of colorectal cancer

Comparative genomic hybridization (CGH) was used to screen colorectal carcinomas for chromosomal aberrations that are associated with metastatic phenotype. In total, 63 tumor specimens from 40 patients were investigated, comprising 30 primary tumors, 22 systemic metastases (12 liver, 6 brain, and 4 abdominal wall metastases) and 11 lymph node tumors. Using statistical analysis and histograms to evaluate the chromosomal imbalances, overrepresentations were detected most frequently at 20q11.2-20q13.2, 7q11.1-7q12, 13q11.2-13q14, 16p12, 19p13, 9q34, and 19q13.1-19q13.2. Deletions were prominent at 18q12-18q23, 4q27-4q28, 4p14, 5q21, 1p21-1p22, 21q21, 6q16-6q21, 3p12, 8p22-8p23, 9p21, 11q22, and 14q13-14q21. Hematogenous metastases showed more alterations than lymph node tumors, particularly more deletions at 1p, 3, 4, 5q, 10q, 14, and 21q21 and gains at 1q, 7p, 12qter, 13, 16, and 22q. Comparing liver metastases with their corresponding primary tumors, particularly deletions at 2q, 5q, 8p, 9p, 10q, and 21q21 and gains at 1q, 11, 12qter, 17q12-q21, 19, and 22q were more often observed. The analysis suggested that the different pathways of tumor dissemination are reflected by a nonrandom accumulation of chromosomal alterations with specific changes being responsible for the different characteristics of the metastatic phenotype.     

Chromosomal basis of adenocarcinoma of the prostate.

Prostate cancer is the most frequent malignancy and the second leading cause of cancer deaths among males in the Western world. The clinical course of the disease is highly complex, and genetic factors underlying tumorigenesis are poorly understood. The challenge that lies ahead is to identify the important gene(s) that causes adenocarcinoma of the prostate. Chromosomal findings by cytogenetic and molecular methods, including Southern blotting, microsatellite analysis, fluorescence in situ hybridization, and comparative genomic hybridization, revealed a high frequency of chromosomal aberrations of heterogeneous nature, including: -1, +1, -1q, +4, -6q, -7, +7, -8, -8p, -8q, +i(8q), -9, -9p, -10, +10, +11, -12, -13q, -16, -16q, +16, -17, +17, +17q, -18, +18, -18q, +19p, +20q, +X, -Xq, -Y, and +Y. Specific chromosomal regions of alterations were 1q24-25, 2cen-q31, 5cen-q23.3, 6q14-23.2, 7q22-q31, 8p12-21, 8p22, 8q24-qter, 10q22.1, 10q23-25, 11p11.2, 16q24, 17p13.1, 18q12.2, and Xq11-12. Recently, a predisposing gene for early onset has been localized on 1q42.2-43. The losses of heterozygosity at specific chromosomal loci from chromosomes 5q, 6q, 7q, 8p, 8q, 10q, 13q, 16q, 17p, 17q, and 18q are generally correlated with poor prognosis in advanced tumor stage. In addition, an abnormal function of known tumor suppressor genes from these regions have been observed in prostate cancer. Although, the amplification of the androgen receptor gene at Xq11-13 and HER-2/neu gene at 17q11.2-q12 are novel findings, no single gene has been implicated in harboring prostate cancer. Frequent inactivation of PTEN/MMAC1 tumor suppressor gene at 10q23, MXI-1 at 10q25, KAI-1 at 11p11.2, Rb at 13q14.2, and p53 at 17p13.1 and deregulation of c-myc oncogene at 8q24 have recently been the subject of intense scrutiny and debate.     

Chromosomal aberrations detected by comparative genomic hybridization predict outcome in patients with colorectal carcinoma

To obtain comprehensive information regarding the correlation between genomic changes and clinicopathological parameters such as disease stage, metastases, and survival, we investigated genomic changes by comparative genomic hybridization (CGH) in 73 patients with colorectal cancer (CRC), and assessed the associations of such charges with clinicopathological parameters. Gains of 8q21-22, 13q21-31 and 20q12-qter and loss of 17p12-pter were detected in >50% of stage I tumors. Gain of 8q23-qter and losses of 8p12-pter and 18q12-qter were observed more frequently in stage III/IV tumors than in stage I tumors (all P<0.05). Loss of 8p12-pter and gain of 8q23-qter were linked to nodal metastasis (all P<0.05). Loss of 18q12-qter and gain of 8q23-qter were associated with distant organ metastasis at diagnosis and/or recurrence after surgery (all P<0.05). Moreover, losses of 8p12-pter and 18q12-qter and gains of 8q23 and 8q24-qter were associated significantly with unfavorable prognosis (all P<0.05). Furthermore, combined examination of the above four changes can provide a more accurate assessment for patient's prognosis. Specifically, 11 of 19 patients with these four changes died, but only 1 of 21 cases without these four changes died during the follow-up period (P<0.0001). Multivariate analysis revealed that loss of 18q12-qter is an independent prognostic marker (P=0.031). Our findings indicate that genetic aberrations detected by CGH may predict outcome in patients with CRC.     

Comparative genomic hybridization of esophageal squamous cell carcinoma: correlations between chromosomal aberrations and disease progression/prognosis.

BACKGROUND: Esophageal carcinoma is a major cause of cancer-related deaths among males in Taiwan. However, to date, the genetic alterations that accompany this lethal disease are not understood. METHODS: Chromosomal aberrations of 46 samples of esophageal squamous cell carcinoma (EC-SCC) were analyzed by comparative genomic hybridization (CGH), and their correlations with pathologic staging and prognosis were analyzed statistically. RESULTS: In total, 321 gains and 252 losses were found in 46 tumor samples; thus, the average gains and losses per patient were 6.98 and 5.47, respectively. Frequent gain abnormalities were found on chromosome arms 1q, 2q, 3q, 5p, 7p, 7q, 8q, 11q, 12p, 12q, 14q, 17q, 20q, and Xq. Frequent deletions were found on chromosome arms 1p, 3p, 4p, 5q, 8p, 9p, 9q, 11q, 13q, 16p, 17p, 18q, 19p, and 19q. It was found that deletions of 4p and 13q12-q14 and gain of 5p were significantly correlated with pathologic staging. Losses of 8p22-pter and 9p also were found more frequently in patients with advanced disease. Gain of 8q24-qter was seen more frequently in patients with Grade 3 tumors. A univariate analysis found that pathologic staging; gains of 5p and 7q; and deletions of 4p, 9p, and 11q were significant prognostic factors. However, pathologic staging became the only significant factor in a multivariate analysis. CONCLUSIONS: CGH not only revealed novel chromosomal aberrations in EC-SCC, but also found possible genotypic changes associated with disease progression. Despite all of the possible associations of chromosomal aberrations with disease progression, the most important prognostic factor for patients with EC-SCC was pathologic staging.     

High-resolution genome-wide allelotype analysis identifies loss of chromosome 14q as a recurrent genetic alteration in astrocytic tumours

Diffusely infiltrative astrocytic tumours are the most common neoplasms in the human brain. To localise putative tumour suppressor loci that are involved in low-grade astrocytomas, we performed high-resolution genome-wide allelotype analysis on 17 fibrillary astrocytomas. Non-random allelic losses were identified on chromosomal arms 10p (29%), 10q (29%), 14q (35%), 17p (53%), and 19q (29%), with their respective common regions of deletions delineated at 10p14-15.1, 10q25.1-qter, 14q212.2-qer, 17p11.2-pter and 19q12-13.4. These results suggest that alterations of these chromosomal regions play important roles in the development of astrocytoma. We also allelotyped 21 de novo glioblastoma multiforme with an aim to unveil genetic changes that are common to both types of astrocytic tumours. Non-random allelic losses were identified on 9p (67%), 10p (62%), 10q (76%), 13q (60%), 14q (50%), and 17p (65%). Allelic losses of 10p, 10q, 14q and 17p were common genetic alterations detectable in both fibrillary astrocytomas and glioblastoma multiforme. In addition, two common regions of deletions on chromosome 14 were mapped to 14q22.3-32.1 and 14q32.1-qter, suggesting the presence of two putative tumour suppressor genes. In conclusion, our comprehensive allelotype analysis has unveiled several critical tumour suppressor loci that are involved in the development of fibrillary astrocytomas and glioblastoma multiforme. Although these two types of brain tumours are believed to evolve from different genetic pathways, they do share some common genetic changes. Our results indicate that deletions of chromosome 14q is a recurrent genetic event in the development of astrocytoma and highlight the subchromosomal regions on this chromosome that are likely to contain putative tumour suppressor genes involved in the oncogenesis of astrocytic tumours.     

Malignant and benign ganglioglioma: a pathological and molecular study

Gangliogliomas are generally benign tumors, composed of transformed neuronal and glial elements, with rare malignant progression of the glial component. The current study of a rare case of a woman harboring a ganglioglioma with areas of malignant transformation addresses two fundamental questions: (1) Are the ganglioglioma and its malignant component clonal in origin? (2) What are the genetic alterations associated with the initiation and subsequent malignant progression of ganglioglioma? By using the human androgen receptor gene (HUMARA) assay, we found the ganglioglioma and the malignant component to be clonal in origin, suggestive of initial transformation of a single neuroglial precursor cell with subsequent malignant progression. Conventional and array comparative genomic hybridization (approximately 2.5-Mb resolution) analyses found chromosomal losses to be predominant in the benign areas of the ganglioglioma, with gains more prevalent in the malignant component. Regions of chromosomal loss, postulated to harbor genes involved in the initiation of ganglioglioma, included 1p35-36, 2p16-15, 3q13.1-13.3, 3q24-25.3, 6p21.3-21.2, 6q24-25.2, 9p12, Xp11.3-11.22, and Xq22.1-22.3. Direct analysis demonstrated loss of p19 expression and p53 mutation in the malignant areas, highly suggestive of these alterations being involved in the malignant progression of the ganglioglioma. Additional chromosomal alterations specific to the malignancy involved gains on 1p35-34.2, 2q24.1-32.3, 3q13.1-13.3, 6q13-16.2, 7q11.2-31.3, 8q21.1-23, 11q12-31, and 12q13.2-21.3. This molecular-pathological study has provided insight into the pathogenesis of gangliogliomas and associated rare malignant progression. Deciphering the specific genes residing in these chromosomal regions may further our understanding of not only these rare tumors but also the more common gliomas.     

Clinicopathologic significance and prognostic value of chromosomal imbalances in diffuse large B-cell lymphomas.

PURPOSE: To determine the clinicopathologic significance and prognostic value of chromosomal imbalances in diffuse large B-cell lymphomas (DLBCL). PATIENTS AND METHODS: We have examined 64 tumors at diagnosis using comparative genomic hybridization and real-time quantitative polymerase chain reaction (PCR), single-stranded conformational polymorphism, and DNA sequencing for the analysis of several potential target genes. RESULTS: The most recurrent alterations were gains of 18q (20%), Xq (15%), 2p, 7q, and 12p (14%), and losses of 6q and 17p (14%). Frequent high-level DNA amplifications were detected at 2p13-p16 and 18q21 loci. Real-time quantitative PCR detected REL and BCL11A gene amplifications in the nine patients with gains at 2p13-p16 and only in one additional patient with normal chromosome 2. Similarly, the BCL-2 gene was amplified in the 12 tumors with gains of 18q21 but in none of 39 patients with normal 18q profile. p53 gene inactivation was detected in nine of 58 (16%) tumors and was commonly associated with 17p losses. Tumors with 18q gains were significantly associated with a high number of chromosomal imbalances, primary nodal presentation, high serum lactate dehydrogenase levels, high International Prognostic Index, shorter cause-specific survival, and a high risk of relapse. Losses of 17p and p53 gene alterations were associated with an absence of complete response achievement. CONCLUSION: These results suggest that DLBCLs have a characteristic pattern of genomic alterations; 18q gains or amplifications and 17p losses are associated with particular clinicopathological features and aggressive clinical behavior. Additional studies are needed to confirm these observations in larger series of patients.     

Discovery of over-expressed genes and genetic alterations in breast cancer cells using a combination of suppression subtractive hybridization,multiplex FISH and comparative genomic hybridization

To identify genes that are involved in breast cancer, suppression subtractive hybridization (SSH) was utilized to construct a breast cancer subtracted library. Differential screening of the library isolated 28 genes which by Northern analysis were highly expressed in the breast cancer cell line MDA-MB-231 compared to the normal breast cell line MCF12A. Sequence analysis revealed that 15 clones coded for previously described genes such as SNAP43, Cyr61, Thymosin beta4, tra1, elongation factor 1alpha, BSF-2/IL6, BiP, and GDP/GTP exchange protein. The remaining 13 clones did not match sequences in GenBank/EMBL database, indicating that they may be novel genes. SNAP43, a subunit of the TBP-TAF complex, was expressed 20-fold higher in MDA-MB-231 compared to MCF12A and several breast cancer cell lines, implying that SNAP43 may be involved in tumorigenesis of a specific subset of breast cancers. Amplification of SNAP43 was not found by Southern analysis. However, genetic alterations of MDA-MB-231 included a deletion of chromosome 14 with a reciprocal translocation t(6;14) and two additional translocations [t(12;14) and t(14;15)] as determined by fluorescent in situ hybridization (FISH) with YAC 823G8 located at chromosome 14q23 which contained SNAP43. Because of the numerous alterations observed by FISH in MDA-MB-231, we further explored the genetic abnormalities in this breast cancer cell line using multiplex FISH (M-FISH) and comparative genomic hybridization (CGH). These cells were replete with numerous complex structural rearrangements and had DNA copy-number imbalances involving multiple chromosomes including gains on chromosomes 2p, 2q31-q32, 3p14-pter, 5q, 6p, 7q36-qter, 11, 14q21-q24, 17p11.2-pter, 17q21-qter, 19, 20, Xp11-q13 and losses on chromosomes 4pter-q32, 8p, 9p21-p24, 10q26-qter, 16p13-pter, 18q12-qter, 22, Xp11.3-p22.1, Xq13-qter. In summary, SSH revealed a number of genes that were either novel or previously not associated with breast cancer. In addition, we found that breast cancer cells abounded with abnormalities as observed by M-FISH and CGH. Together, these results may facilitate defining the genetic alterations associated with breast cancer progression.     

Comparative genomic hybridization of postirradiation sarcomas.

BACKGROUND: Radiotherapy is a known risk factor for sarcoma development. Postirradiation sarcomas arise within the radiation field after a latency period of several years and usually are highly malignant. Very little is yet known about their genetic changes. METHODS: Twenty-seven postirradiation sarcomas were analyzed by comparative genomic hybridization, which allows genome-wide screening of DNA sequence copy number changes. RESULTS: Copy-number aberrations were detected in 20 (74%) tumors. The mean number of aberrations per tumor was 5.3 with gains outnumbering losses. The most frequent gains affected the minimal common regions of 7q11.2-q21 and 7q22 in 30% and 7p15-pter in 26%. Gain of 8q23-qter was detected in 22%. The most frequent losses affected 11q23-qter and 13q22-q32 in 22%. In osteosarcomas, the most frequent aberration was loss of 1p21-p31, in malignant fibrous histiocytomas (MFH) gain of 7cen-q22, and in fibrosarcomas gain of 7q22. The findings in postirradiation osteosarcomas and MFHs were compared with findings in sporadic osteosarcomas and MFHs, reported previously by the authors. In sporadic osteosarcomas, gains outnumbered losses, but, in postirradiation osteosarcomas, losses were more frequent than gains. Loss at 1p was rare in sporadic osteosarcoma (3%) but frequent (57%) in postirradiation osteosarcomas. Gains at 7q were frequent both in postirradiation and sporadic MFH. CONCLUSIONS: According to previous studies on different types of sporadic sarcomas, gains at 7q or 8q are associated with poor prognosis or large tumor size. Thus, the frequent gains at 7q and 8q might have been responsible in part for the poor prognosis of postirradiation sarcomas. Also, however, some of their clinical features, i.e., high malignancy grade, late diagnosis, and central location, are associated with a poor prognosis.     

Copy number alterations in urothelial carcinomas: their clinicopathological significance and correlation with DNA methylation alterations

The aim of this study was to clarify the genetic backgrounds underlying the clinicopathological characteristics of urothelial carcinomas (UCs). Array comparative genomic hybridization analysis using a 244K oligonucleotide array was performed on 49 samples of UC tissue. Losses of 2q33.3-q37.3, 4p15.2-q13.1 and 5q13.3-q35.3 and gains of 7p11.2-q11.23 and 20q13.12-q13.2 were correlated with higher histological grade, and gain of 7p21.2-p21.12 was correlated with deeper invasion. Losses of 6q14.1-q27 and 17p13.3-q11.1 and gains of 19q13.12-q13.2 and 20q13.12-q13.33 were correlated with lymph vessel involvement. Loss of 16p12.2-p12.1 and gain of 3q26.32-q29 were correlated with vascular involvement. Losses of 5q14.1-q23.1, 6q14.1-q27, 8p22-p21.3, 11q13.5-q14.1 and 15q11.2-q22.2 and gains of 7p11.2-q11.22 and 19q13.12-q13.2 were correlated with the development of aggressive non-papillary UCs. Losses of 1p32.2-p31.3, 10q11.23-q21.1 and 15q21.3 were correlated with tumor recurrence. Unsupervised hierarchical clustering analysis based on copy number alterations clustered UCs into three subclasses: copy number alterations associated with genome-wide DNA hypomethylation, regional DNA hypermethylation on C-type CpG islands and genome-wide DNA hypo- and hypermethylation were accumulated in clusters A, B(1) and B(2), respectively. Tumor-related genes that may encode therapeutic targets and/or indicators useful for the diagnosis and prognostication of UCs should be explored in the above regions. Both genetic and epigenetic events appear to accumulate during urothelial carcinogenesis, reflecting the clinicopathological diversity of UCs.     

Cytogenetic findings in malignant peripheral nerve sheath tumors

Clonal chromosome aberrations were detected in 8 short-term cultured malignant peripheral nerve sheath tumors (MPNST). Seven had a near-triploid chromosome number and I was in the hyperhaploid-hypodiploid range. No recurrent structural rearrangements were found; the bands most frequently involved (3 tumors) were 7p11, 12p13 and 14q11. The most common numerical changes were loss of a sex chromosome (all tumors) and loss of at least 1 copy of chromosomes 8, 16 and 22 (4 tumors). Pooling our data with those on the 20 previously published MPNST with abnormal karyotypes, we found that the chromosome number has often been in the triploid range (12 tumors), with stem line variation between 34 and 270. All chromosome arms, except 22p and the Y chromosome, were involved in recombinations. The most frequently rearranged bands were 7p22 (6 tumors) and 1p21, 7p11 and 14q11 (5 tumors each). Most numerical and unbalanced structural aberrations have led to loss of genetic material, in particular from Xq26-qter (13 tumors); 11q22-qter and 13p (12 tumors); 9p22-pter, 11p13-pter, 17p and 17q11-21 (11 tumors); 1p22-32 and 1p34-pter (10 tumors) and 6q25-qter and chromosome 16 (9 tumors). © 1995 Wiley-Liss, Inc.     

Chromosomal alterations in lung adenocarcinoma from smokers and nonsmokers

The etiology of lung tumors arising in nonsmokers remains unclear. Although mutations in the K-ras and p53 genes have been reported to be significantly higher in smoking-related lung carcinomas, in the present study we performed a more comprehensive analysis in search of additional genetic changes between lung adenocarcinoma from tobacco- and non-tobacco-exposed patients. We selected a matched cohort of 18 lifetime nonsmoking and 27 smoking patients diagnosed with primary adenocarcinoma of the lung and searched for chromosomal alterations in each tumor by testing normal and tumor tissue with 54 highly polymorphic microsatellite markers located on 28 different chromosomal arms. Allelic losses or gains at chromosomal arms 3p (37 versus 6%), 6q (46 versus 12%), 9p (65 versus 22%), 16p (28 versus 0%), 17p (45 versus 11%), and 19p (58 versus 16%) were present significantly more often in adenocarcinomas from smokers than from nonsmokers. Chromosomal arms showing allelic imbalance in lung tumors from nonsmokers were rare but occurred more often at 19q (22%), 12p (22%), and 9p (22%). The FAL (fractional allelic loss or gain) is defined as the percentage of chromosomal arm losses/gains among the total informative chromosomal arms. Tumors from smokers harbored higher levels of FAL (13 (48%) of 27 showed FAL > or = 0.3) compared with the lung tumors from the nonsmoker patients (2 (11%) of 18 showed FAL > or = 0.3; P = 0.02; odds ratio, 0.13; 95% confidence interval, 0.01-0.79). Our data demonstrate that widespread chromosomal abnormalities are frequent in lung adenocarcinoma from smokers, whereas these abnormalities are infrequent in such tumors arising in nonsmokers. These observations support the notion that lung cancers in nonsmokers arise through genetic alterations distinct from the common events observed in tumors from smokers.