DNA Sequence Copy Number Changes Research Articles

Molecular Diagnostics of Gliomas Using Next Generation Sequencing of a Glioma-Tailored Gene Panel.

Current classification of gliomas is based on histological criteria according to the World Health Organization (WHO) classification of tumors of the central nervous system. Over the past years, characteristic genetic profiles have been identified in various glioma types. These can refine tumor diagnostics and provide important prognostic and predictive information. We report on the establishment and validation of gene panel next generation sequencing (NGS) for the molecular diagnostics of gliomas. We designed a glioma-tailored gene panel covering 660 amplicons derived from 20 genes frequently aberrant in different glioma types. Sensitivity and specificity of glioma gene panel NGS for detection of DNA sequence variants and copy number changes were validated by single gene analyses. NGS-based mutation detection was optimized for application on formalin-fixed paraffin-embedded tissue specimens including small stereotactic biopsy samples. NGS data obtained in a retrospective analysis of 121 gliomas allowed for their molecular classification into distinct biological groups, including (i) isocitrate dehydrogenase gene (IDH) 1 or 2 mutant astrocytic gliomas with frequent α-thalassemia/mental retardation syndrome X-linked (ATRX) and tumor protein p53 (TP53) gene mutations, (ii) IDH mutant oligodendroglial tumors with 1p/19q codeletion, telomerase reverse transcriptase (TERT) promoter mutation and frequent Drosophila homolog of capicua (CIC) gene mutation, as well as (iii) IDH wildtype glioblastomas with frequent TERT promoter mutation, phosphatase and tensin homolog (PTEN) mutation and/or epidermal growth factor receptor (EGFR) amplification. Oligoastrocytic gliomas were genetically assigned to either of these groups. Our findings implicate gene panel NGS as a promising diagnostic technique that may facilitate integrated histological and molecular glioma classification.

Comparison of Clinical Subgroup aCGH Profiles through Pseudolikelihood Ratio Tests

Array-based Comparative Genomic Hybridization (aCGH) is a microarray-based technology that assists in identification of DNA sequence copy number changes across the genome. Examination of differences in instability phenotype, or pattern of copy number alterations, between cancer subtypes can aid in classification of cancers and lead to better understanding of the underlying cytogenic mechanism. Instability phenotypes are composed of a variety of copy number alteration features including height or magnitude of copy number alteration level, frequency of transition between copy number states such as gain and loss, and total number of altered clones or probes. That is, instability phenotype is multivariate in nature. Current methods of instability phenotype assessment, however, are limited to univariate measures and are therefore limited in both sensitivity and interpretability. In this paper, a novel method of instability assessment is presented that is based on the Engler et al. (2006) pseudolikelhood approach for aCGH data analysis. Through use of a pseudolikelihood ratio test (PLRT), more sensitive assessment of instability phenotype differences between cancer subtypes is possible. Evaluation of the PLRT method is conducted through analysis of a meningioma data set and through simulation studies. Results are shown to be more accurate and more easily interpretable than current measures of instability assessment.

Genes Proximal and Distal to MYCN Are Highly Expressed in Human Neuroblastoma as Visualized by Comparative Expressed Sequence Hybridization

MYCN amplification is associated with poor prognosis in neuroblastoma disease. To improve our understanding of the influence of the MYCN amplicon and its corresponding expression, we investigated the 2p expression pattern of MYCN amplified (n = 13) and nonamplified (n = 4) cell lines and corresponding primary tumors (n = 3) using the comparative expressed sequence hybridization technique. All but one MYCN amplified cell line displayed overexpression at 2p. Expression peaks were observed frequently at 2pter and less frequently at 2p24 (MYCN locus), 2p23.3-23.2, and/or 2p23.1. Importantly, cell lines and two corresponding primary tumors displayed expression peaks at similar loci. No significant 2p24 expression level was observed for those cell lines displaying a low amplification rate (n = 3) by comparative genomic hybridization. Only the cell lines with an enhanced peak at 2p23.2-23.3 displayed coamplification of the ALK gene (2p23.2), reported to be associated with unfavorable prognosis. Finally, two of four cell lines without MYCN amplification, both derived from patients with poor outcome, also showed an expression peak at 2p23.2. These data indicate that, besides MYCN, other genes proximal and distal to MYCN are highly expressed in neuroblastoma. The prognostic significance of expression peaks at 2p23.2-23.3, independent of MYCN and ALK status, remains to be investigated.

Malignant fibrous histiocytoma of bone: Analysis of genomic imbalances by comparative genomic hybridisation and C-MYC expression by immunohistochemistry

Malignant fibrous histiocytoma (MFH) of bone is a rare, highly malignant tumour. As very little is known about its genetic alterations, 26 bone MFHs were analysed by comparative genomic hybridisation (CGH). Twenty-three tumours (89%) had DNA sequence copy number changes (mean, 7.2 changes per sample). Gains were more frequent than losses (gains:losses = 2.5:1). Minimal common regions for the most frequent gains were 8q21.3-qter (35%), 9q32-qter (35%), 7q22-q31 (35%), 1q21-q23 (31%), 7p12-pter (31%), 7cen-q11.2 (31%) and 15q21 (31%). Minimal common regions for the most frequent losses were 13q21-q22 (42%) and 18q12-q22 (27%). High-level amplifications were detected in 8 out of the 26 tumours (31%). The only recurrent amplifications, 1q21-q23 and 8q21.2-q22, were present in two samples (8%). As copy number increase at 8q24 (the locus of C- MYC) was frequent, the expression of C- MYC was studied by immunohistochemistry. Increased levels of c-myc protein were detected in 7 out of 21 tumours studied (33%). 81% of the samples studied both by CGH and immunohistochemistry showed concordant results. Furthermore, the findings of the present study were compared to previous publications on osteosarcoma, soft tissue MFH and fibrosarcoma of bone. Clear differences were detected in CGH aberration patterns, further supporting the concept of bone MFH as an individual bone tumour entity. Finally, the findings of the present study reflect well the high malignancy and aggressive nature of bone MFH.

Gains of 13q are correlated with a poor prognosis in liposarcoma

Liposarcomas are a phenotypical heterogenous group of tumors divided into four main subtypes: well-differentiated, dedifferentiated, myxoid/round cell, and pleomorphic. The aim of this study was to compare DNA sequence copy number changes of these subtypes as investigated by comparative genomic hybridization in 36 patients. Comparative genomic hybridization revealed genomic imbalances in tumors of 27 patients (mean 5.6 imbalances per tumor). The most frequent gains were within single regions of 1q, 12q, and 13q. We found a significant correlation of poor overall survival and gain of 13q21 (P=0.0221), 13q22 (P=0.0341), 13q31 (P=0.0410), and 13q32 (P=0.0074). The univariate Cox regression analysis revealed an increased risk of tumor-related death for patients whose liposarcomas possess with gains of 13q21 and 13q32 simultaneously (P=0.010; RR=7.1; 95% CI 1.6–31.7). Furthermore, 12 high-level amplifications were found in tumors of seven patients. In four cases 12q14–q15 and in two cases 13q32–q33 were amplified. We identified in different liposarcoma subtypes characteristic genomic changes: Gains and high-level amplifications of 12q occurred in all 11 investigated well-differentiated liposarcomas, and these changes were often present simultaneously with gains of 1q (mean 5.5 changes). In the two dedifferentiated liposarcomas, gains of 1q in both liposarcomas, and a high-level amplification of 13q were striking. Only eight of the 17 patients with myxoid/round cell liposarcomas showed changes in DNA copy number (mean 3.4 imbalances). In four of these eight cases gains of 13q occurred. The six pleomorphic liposarcomas possessed the most frequent genomic imbalances (mean number 16.3) of all liposarcoma subtypes investigated. These imbalances were in almost all chromosomal regions detected predominantly as over-representations of chromosomes 1, 5p, 13q, and 22q. Summarizing, all subtypes but well-differentiated liposarcomas showed gains of 13q, which were associated with a poor prognosis.

Comparative Genomic Hybridization Analysis of Astrocytomas: Prognostic and Diagnostic Implications

Astrocytoma is comprised of a group of common intracranial neoplasms that are classified into four grades based on the World Health Organization histological criteria and patient survival. To date, histological grade, patient age, and clinical performance, as reflected in the Karnofsky score, are the most reliable prognostic predictors. Recently, there has been a significant effort to identify additional prognostic markers using objective molecular genetic techniques. We believe that the identification of such markers will characterize new chromosomal loci important in astrocytoma progression and aid clinical diagnosis and prognosis. To this end, our laboratory used comparative genomic hybridization to identify DNA sequence copy number changes in 102 astrocytomas. Novel losses of 19p loci were detected in low-grade pilocytic astrocytomas and losses of loci on 9p, 10, and 22 along with gains on 7, 19, and 20 were detected in a significant proportion of high-grade astrocytomas. The Cox proportional hazards statistical modeling showed that the presence of +7q and -10q comparative genomic hybridization alterations significantly increased a patient's risk of dying, independent of histological grade. This investigation demonstrates the efficacy of comparative genomic hybridization for identifying tumor suppressor and oncogene loci in different astrocytic grades. The cumulative effect of these loci is an important consideration in their diagnostic and prognostic implications.

Array-based comparative genomic hybridization for the detection of DNA sequence copy number changes in Barrett's adenocarcinoma.

Array-based comparative genomic hybridization (aCGH) allows the identification of DNA sequence copy number changes at high resolution by co-hybridizing differentially labelled test and control DNAs to a micro-array of genomic clones. The present study has analysed a series of 23 formalin-fixed, paraffin wax-embedded tissue samples of Barrett's adenocarcinoma (BCA, n = 18) and non-neoplastic squamous oesophageal (n = 2) and gastric cardia mucosa (n = 3) by aCGH. The micro-arrays used contained 287 genomic targets covering oncogenes, tumour suppressor genes, and DNA sequences localized within chromosomal regions previously reported to be altered in BCA. DNA sequence copy number changes for a panel of approximately 50 genes were identified, most of which have not been previously described in BCA. DNA sequence copy number gains (mean 41 +/- 25/BCA) were more frequent than DNA sequence copy number losses (mean 20 +/- 15/BCA). The highest frequencies for DNA sequence copy number gains were detected for SNRPN (61%); GNLY (44%); NME1 (44%); DDX15, ABCB1 (MDR), ATM, LAMA3, MYBL2, ZNF217, and TNFRSF6B (39% each); and MSH2, TERC, SERPINE1, AFM137XA11, IGF1R, and PTPN1 (33% each). DNA sequence copy number losses were identified for PDGFB (44%); D17S125 (39%); AKT3 (28%); and RASSFI, FHIT, CDKN2A (p16), and SAS (CDK4) (28% each). In all non-neoplastic tissue samples of squamous oesophageal and gastric cardia mucosa, the measured mean ratios were 1.00 (squamous oesophageal mucosa) or 1.01 (gastric mucosa), indicating that no DNA sequence copy number chances were present. For validation, the DNA sequence copy number changes of selected clones (SNRPN, CMYC, HER2, ZNF217) detected by aCGH were confirmed by fluorescence in situ hybridization (FISH). These data show the sensitivity of aCGH for the identification of DNA sequence copy number changes at high resolution in BCA. The newly identified genes may include so far unknown biomarkers in BCA and are therefore a starting point for further studies elucidating their possible role in Barrett's carcinogenesis.

Genetic analysis of fibrosarcoma of bone, a rare tumour entity closely related to osteosarcoma and malignant fibrous histiocytoma of bone

Fibrosarcoma (FS) of bone is an extremely rare and genetically uncharacterised malignant tumour arising in the skeleton. On the basis of clinicopathologic features it appears to be closely related to either fibroblastic osteosarcoma (OS) or malignant fibrous histiocytoma (MFH) of bone. In this study, 27 decalcified, paraffin-embedded FS of bone were collected for genetic and immunohistochemical characterisation. Good quality DNA, suitable for genetic analyses, was isolated from nine cases (7 primary tumours, 1 local recurrence, and 1 lung metastasis), which were analysed by comparative genomic hybridisation (CGH) on chromosomes and DNA microarrays. DNA sequence copy number changes were found in five out of seven primary tumours (72%), as well as in both, the local recurrence and the metastatic lesion, by CGH on chromosomes. The most frequent aberration was gain of the chromosomal region 22q, which was present in four out of the five primary tumours with genetic changes, in the local recurrence and, as the sole genetic aberration, in the lung metastasis. DNA microarray analysis showed that gain of the platelet-derived growth factor beta (PDGF-B) gene (located at 22q12.3-q13.1) was the most frequent gene imbalance, which was present in three out of the five analysed tumours. In these three cases, real-time PCR revealed a 2.1- to 2.7-fold increase of PDGF-B gene copy numbers. By immunohistochemistry, a positive reaction for B-chain-containing PDGF proteins was revealed in all the cases showing gain of 22q. A more extensive immunohistochemical analysis identified the presence of PDGF-B proteins in 8/20 primary FS of bone (40%), 3/3 lung metastases and in 1/2 local recurrences. A simultaneous positive reaction for PDGF-B proteins and PDGF receptors was found in two third of PDGF-B-positive cases (8/12). Taken together, the genetic and immunohistochemical data indicate that over-representation of the chromosomal region 22q, including particularly the PDGF-B gene, may be important for the pathogenesis of FS of bone. Our results also demonstrate that CGH on chromosomes and DNA microarrays are suitable for the genetic characterisation of decalcified, paraffin-embedded tumour tissue samples and may facilitate, combined with other techniques, the rapid acquisition of data providing insight into the molecular genetic and biologic basis of rare bone sarcomas. Moreover, these findings suggest the possible presence of an autocrine loop in FS of bone, which might be taken into account for planning innovative therapeutic strategies for patients unresponsive to conventional treatments.

DNA copy number changes detected by comparative genomic hybridization and their association with clinicopathologic parameters in breast tumors

The purpose of this study was to use comparative genomic hybridization (CGH) to screen breast tumors for copy number changes: 22 ductal, 9 lobular, 7 mixed, 2 micropapillary carcinomas, and 2 ductal carcinoma in situ were studied and various regional genomic imbalances were detected. The majority of the aberrations identified in this study were in line with previous CGH findings. The most frequent DNA sequence copy number changes were 1q, 8q, and 20q gains. The frequency of 16q losses was significantly higher in lobular carcinomas. The nodal involvement was 10 times higher in cases showing losses of 13q than in cases having normal peak profile at this region. Estrogen receptor positivity was significantly higher in cases displaying 20q gains and 16q losses. Unambiguous high-level DNA amplifications have also been detected. These mapped to 4q31, 6q21∼q22, 8q21∼q24, 8p11.2∼p12, 11q13, 15q24∼qter, 20q13.1∼qter, and 20q12∼qter chromosomal locations. Our results highlight several chromosomal regions that may be important in the molecular genetics of distinct clinicopathologic breast cancer subgroups.

Establishment of a novel human dedifferentiated liposarcoma cell line, FU-DDLS-1: conventional and molecular cytogenetic characterization.

A number of human cell lines derived from well-differentiated, myxoid/round cell, or pleomorphic liposarcoma have been described. To our knowledge, however, no human cell line established from dedifferentiated liposarcoma has been reported. In this study, we established a new human cell line, FU-DDLS-1, which originated from a dedifferentiated liposarcoma arising in the retroperitoneum of a 61-year-old man. This cell line was characterized by immunocytochemistry, conventional banding analysis, fluorescence in situ hybridization with chromosome painting probe, and comparative genomic hybridization (CGH). FU-DDLS-1 cells were spindle or polygonal shaped and possessed oval nuclei and slender cytoplasmic processes. The cultured cells were successfully maintained in vitro for over 90 passages over more than 30 months. The histologic features of heterotransplanted tumors in severe combined immunodeficiency mice were essentially the same as those of the original nonlipogenic sarcoma resembling a malignant fibrous histiocytoma. Both in vitro and in vivo, the cells exhibited immunopositive reaction for mdm2 and p53 proteins. Cytogenetically, FU-DDLS-1 displayed a hypertetraploid karyotype with giant marker chromosomes composed partly of chromosome 12 material. In addition, CGH analysis demonstrated that DNA sequence copy number changes including a gain of 12q12-q21 detected in FU-DDLS-1 were essentially the same as those in the original sarcoma. The FU-DDLS-1 cell line, which exhibits the unique conventional and molecular cytogenetic characteristics of dedifferentiated liposarcoma, should be a particularly useful model for studying the molecular pathogenesis of human dedifferentiated liposarcoma.

Prognostic factors influencing surgical management and outcome of gastrointestinal stromal tumours

The purpose of this study was to review surgical experience with gastrointestinal stromal tumours (GISTs) at a single tertiary university hospital, and to identify morphological and genetic prognostic markers of tumour progression. Forty-eight GISTs from 39 patients were reviewed retrospectively. The prognostic significance of DNA copy number changes, measured by comparative genomic hybridization (CGH), and morphological markers in low-risk and high-risk tumours were investigated. Significantly more patients died from disease after incomplete tumour resection than after complete primary resection (P = 0.020). Tumour size of 5 cm or greater, mitotic count of 2 or more, and proliferative activity greater than 10 per cent were significantly associated with a shorter recurrence-free survival (P = 0.020, P = 0.001 and P = 0.002 respectively). Patients with low-risk tumours had a significantly better outcome than those with high-risk GISTs, both in terms of overall and recurrence-free survival (P < or = 0.001). CGH performed on 16 tumours revealed fewer DNA sequence copy number changes in low-risk than in high-risk GISTs. Non-progressive GISTs contained significantly fewer genetic alterations than recurrent or metastatic tumours (P < 0.001). Only tumours with more than five changes showed disease progression. Complete surgical resection is the most important means of cure for GISTs. DNA copy number changes are related to the behaviour of these tumours and may serve as additional prognostic markers.

Independent Genetic Events Associated with the Development of Multiple Parathyroid Tumors in Patients with Primary Hyperparathyroidism

Multiple parathyroid tumors, as opposed to hyperplasia, have been reported in a subset of patients with sporadic primary hyperparathyroidism (PHPT). It is not clear whether these multiple tumors are representative of a neoplastic process or whether they merely represent hyperplasia that has affected the parathyroid glands differentially and resulted in asynchronous growth. The molecular genetic techniques of comparative genomic hybridization (CGH), loss of heterozygosity (LOH), and MEN1 mutation analysis were performed on a series of five patients with multiglandular PHPT, each of which had two parathyroid tumors removed. Analysis of these multiple parathyroid tumors from patients with PHPT revealed that independent genetic events were associated with the development of a subset of these tumors. The DNA sequence copy number changes, identified by CGH analyses, either involved different chromosomal regions in the paired glands of a patient (two patients), or those regions implicated in one gland were not changed in a second gland from the same patient (two patients). Each of the three patients exhibiting LOH demonstrated different changes between the paired glands. Where LOH was detected in one gland from a patient, the other gland from the same patient either exhibited no allelic loss or the loss detected was in another region. Each of the three tumors exhibiting LOH at 11q13 was found to contain a somatic MEN1 mutation in the remaining allele, however these mutations were not present in the germline or in the paired gland from the same patient. Although it is possible that a separate series of genetic changes has arisen randomly in two separate glands within the same individual, it seems more likely that the development of these multiple tumors has arisen because of the involvement of other unknown factors. These factors may be genetic [such as the involvement of one or more germline mutations in an unknown low-penetrance gene(s), germline mosaicism or alterations in calcium-sensing receptor gene(s)], epigenetic, physiological, or environmental.

Chromosomal changes in incidental prostatic carcinomas detected by comparative genomic hybridization.

The genetic changes underlying the development and progression of prostate cancer are poorly understood. To identify chromosomal regions in incidental prostatic carcinoma (T1a and T1b) was the primary aim of this study. We used comparative genomic hybridization (CGH) to search for DNA sequence copy number changes on a series of 48 T1 prostate cancer diagnosed by transurethral resection (TURP) and by adenomectomy. Incidental prostatic carcinomas have not been studied by CGH previously. CGH analysis indicated that 14 cases (29.2%) of incidental prostatic carcinoma showed chromosome alterations. The most frequent alterations were chromosomal losses of 8p (10.4%), 13q (6.3%), 5q (4.2%) and 18q (4.2%), and gains of 17p (10.4%), 17q (10.4%), 9q (6.3%) and 7q (4.2%). Minimal overlapping chromosomal regions of loss, indicative for the presence of tumor suppressor genes (TSGs), were mapped to 8p22 and 13q14.1-q21.3, and minimal overlapping regions of gain, indicative for the presence of oncogenes, were found at 9q34.2-qter, 17p12 and 17q24-qter. The statistical analysis displayed a significant association between chromosomal aberration detected by CGH and high Gleason score (P < 0.005) as well as between tumor categories T1a and T1b and chromosomal imbalance (P = 0.041). Studies directed at incidental prostatic carcinomas allow discovery of chromosomal changes in small and highly malignant tumors. Our results suggest that loss or gain of DNA in these regions are important in prostate cancer. This is the first study, which documents the spectrum of chromosomal changes in incidental prostatic carcinomas.

Genetic changes in stage pT2N0 prostate cancer studied by comparative genomic hybridization.

To identify chromosomal regions important for progression in clinically organ-confined prostate cancer, as the genetic changes underlying the development and progression of prostate cancer are poorly understood. Comparative genomic hybridization (CGH) was used to search for DNA sequence copy-number changes in a series of 50 primary organ-confined prostate adenocarcinomas (pT2N0) removed by radical prostatectomy. CGH analysis indicated that 23 (46%) of the primary prostate adenocarcinomas showed chromosome alterations. The percentage of tumours with losses (38%) was higher than with gains (28%). Losses of 13q (24%), 8p (18%), 6q (10%), 16q (8%), 18q (6%) and 5q (6%) and gains of 17q (12%), 20q (12%), 9q (10%), 17p (8%) and 8q (6%) were the most frequent alterations. Amplifications were found at 8q24-qter. Minimal overlapping regions of loss, indicative of the presence of tumour-suppressor genes, were mapped to 13q21.1-q21.3 and 8p21.2, and minimal overlapping regions of gain, indicative of the presence of oncogenes, were found at 9q34.4-qter, 17q25-qter and 20q13.3-qter. There was a significant association between Gleason score and losses and gains (P = 0.003), and an association between chromosomal imbalance and high histological grade (P = 0.008). These results suggest that losses or gains of DNA in these regions are important for prostate cancer progression, and document the spectrum of chromosomal alterations in stage pT2N0 of clinically organ-confined prostate cancer.

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.

Genome-wide screening of laser capture microdissected gastric signet-ring cell carcinomas.

Gastric signet-ring cell carcinoma comprises a distinct category of gastric cancers and has been reported to have poor prognosis. In an attempt to define genetic changes involved in the pathogenesis of this lesion in an in vivo state, we isolated signet-ring cell carcinoma cells from freshly fixed smears of tumor tissues of 7 primary gastric signet-ring cell carcinomas by laser capture microdissection and applied comparative genomic hybridization (CGH) to screen for DNA sequence copy number changes. Frequent chromosomal gains were detected on 2q, 5p, 7q, 14q and 20q, each in 6/7 cases, on 9q, 12q, 17q, and 19q, each in 5/7 cases, and on 18p in 4/7 cases. Frequent losses were observed on 6p and 17p, each in 5/7 cases, on 6q, and 21p, each in 4/7 cases, and on 3p, 8p and 8q, each in 3/7 cases. Losses on 6p have rarely been observed in conventional types of gastric carcinomas reported in the literature. These data provide the first evidence for the occurrence of specific genomic aberrations in gastric signet-ring cell carcinomas. Our observation of frequent losses on 6p chromosomal arm may provide novel abnormalities of potential significance in gastric signet-ring cell carcinomas, suggesting the involvement of genes residing in this region in the genesis of the disease.

Chromosomal changes pattern and gene amplification in T cell non-Hodgkin's lymphomas.

T cell non-Hodgkin's lymphomas are a heterogeneous group of lymphomas with poor prognosis, and whose genetic alterations are not well understood. Comparative genomic hybridization (CGH) is a technique that allows the identification of DNA imbalances without cytogenetic studies. We have studied 37 samples from 29 T cell non-Hodgkin's lymphomas (25 peripheral and four lymphoblastic lymphomas) by CGH in order to detect DNA sequence copy number changes of putative importance in the biology and prognosis of these neoplasms. We detected abnormal CGH profiles in 16/27 (59%) of samples at diagnosis, a ratio that increased to 66% (23/37) when we included the relapsed samples. The most common recurrent changes were gains related to the X chromosome, either the whole chromosome or partially the Xq26-27 bands (19%). Other recurrent changes included gains of bands 9q34, gains of chromosomes 17, 19, and 20, and complete or partial deletions of chromosome 13 (10%). Cancer-related genes located at Xq26-28 region were analyzed by Southern blot and fluorescence in situ hybridization (FISH). Low level amplification of some of these genes was detected by this technique confirming the results obtained by CGH in this region. The detection of abnormal CGH profiles in these T cell lymphomas could have clinical implications. Patients with abnormal CGH profiles showed significant associations with advanced stage of disease, overexpression of P53, and higher proliferative index.

Synovial sarcoma. A Scandinavian Sarcoma Group project.

Synovial sarcoma accounts for 5-10% of all soft tissue sarcomas. More than 90% are found in the extremities or trunk wall. Characteristic for synovial sarcoma is the translocation t(X;18) (p11.2;q11.2). Cloning of the breakpoints of this translocation revealed fusion of two novel genes, SYT and SSX. The SYT gene, located on chromosome 18, is fused with one of three closely related genes; SSX1, SSX2 or SSX4 located on the X chromosome. The long term survival rates have continuously improved and have at best been reported to around 50%. However, since almost no population based studies on synovial sarcoma have been reported, these improvements may be due to differences in patient selection due to a changes in referral practice. This project was based on a consecutive series of synovial sarcoma patients from the Scandinavian Sarcoma Group Register acquired during a 9-year period. Only surgically treated patients without metastases at diagnosis were included in the prognostic analyses. The tumors were defined clinically, histopathologically, molecular and cytogenetically and these features were related to clinical course. 34 of 104 patients developed metastases. The overall 5 and 7 years survival rates were 0.76 (95% CI 0.66-0.83) and 0.69 (0.58-0.78), respectively. Large tumor size and amputation were significantly associated with impaired metastasis free survival. In addition, patients with local recurrence had a higher risk for metastases following the local event. All were high grade lesions, 74 Grade III and 30 IV. Kaplan-Meier estimates of metastasis-free survival at 5 years were 83% (95% CI 72-92%) for patients with Grade III tumors versus 31% (95% CI 13-51%) for Grade IV. Histologic grading conveyed more prognostic information than any single histologic factor. Immunostaining with anti-Ki-67 antibodies (MIB1) and p53 based on formalin-fixed paraffin-embedded material from 86 patients revealed that MIB-1 > or = 10% was associated with poorer metastasis-free survival but p53 was not. Type of fusion transcripts (SYT-SSX1 or SYT-SSX2) and Ki-67 were assessed in fresh frozen tissue from 33 patients. The 5-year metastasis-free survival for patients with SYT-SSX1 was 42% versus 89% for those with SYT-SSX2. The hazard ratio for metastasis associated with the SYT-SSX1 fusion transcripts was 7 (95% CI 1.5-36, log-rank p = 0.004). There was a significant association between SYT-SSX1 and high tumor proliferation rate. Comparative Genomic Hybridization revealed DNA sequence copy number changes in 35 of 69 tumor specimens. The frequency of aberrations/tumor were higher in monophasic tumors than in biphasic. Gains of chromosome 8 were associated with large tumors (> 5 cm). There was no obvious association between secondary aberrations and clinical outcome. Large tumor size, local recurrence, histologic Grade IV, MIB1 index > or = 10 and possibly SYT-SSX1 fusion transcript were associated with impaired clinical outcome.

Molecular Cytogenetic Analysis of Uterine Leiomyoma and Leiomyosarcoma by Comparative Genomic Hybridization

Uterine leiomyomata are among the most common of human neoplasms and are associated with abnormal uterine bleeding, infertility, and abdominal pain. Uterine leiomyosarcomata are presumed to be the malignant counterpart to uterine leiomyomata and are very rare. Transformation of uterine leiomyoma (ULM) into uterine leiomyosarcoma (ULMS) is yet to be conclusively confirmed, and each type of tumor may represent a distinct genetic entity. We used comparative genomic hybridization (CGH) to evaluate DNA sequence copy-number changes in 12 specimens of ULM and 8 of ULMS. CGH analysis of ULM demonstrated chromosomal imbalances in 8 of 12 (66.7%) specimens. The most frequent ULM gains were observed at 9q34 (a novel finding) and on chromosome 19. Other ULM imbalances included gains and losses of chromosome 1p, losses on 7q, and gains on 12q. All ULMS specimens demonstrated chromosomal aberrations. Chromosome 1 imbalances were very prominent. The most frequent losses were detected on 14q and 22q. Losses on 14q are rarely seen in other types of leiomyo–sarcoma and may be a distinctive feature of ULMS. Gains on chromosomes 8, 17, and X were observed in half the cases and were accompanied by high-level amplification. Other chromosome arms overrepresented included 12q and 19p. The absence of specific anomalies common to all ULM and ULMS argues against their being benign–malignant counterparts.

Detection of chromosomal imbalances in central neurocytomas by using comparative genomic hybridization

Central neurocytomas are rare neuronal tumors commonly found in the intraventricular regions. Little is known about the tumorigenesis of these neoplasms. The aim of this study was to provide an overview of genetic imbalances in central neurocytomas. In this study, comparative genomic hybridization was used to identify DNA sequence copy number changes (losses and gains) in a series of 10 central neurocytomas. Tumor DNA and normal reference DNA were differentially labeled and allowed to cohybridize to normal metaphase chromosomes. After hybridization and fluorescent staining of the bound DNA, regions of gain or of loss of DNA sequences were detected as changes in the tumor/normal fluorescence intensity ratio along the target metaphase chromosomes. A gain of DNA sequence was detected in chromosomes 2p, 10q, and 18q. A protooncogene, Bcl2, which maps to 18q21, was evaluated by immunohistochemical analysis to determine its role in the formation of central neurocytomas. In this study the authors identified recurrent genetic changes on chromosomes 2p, 10q, and 18q in central neurocytomas and highlighted chromosomal regions for additional mapping and cloning of candidate genes that are important in the development of central neurocytomas.