Sakari Knuutila

This review summarizes reports of recurrent DNA sequence copy number losses in human neoplasms detected by comparative genomic hybridization. Recurrent losses that affect each of the chromosome arms in 73 tumor types are tabulated from 169 reports. The tables are available online at http://www.amjpathol.org and http://www. helsinki.fi/ approximately lglvwww/CMG.html. The genes relevant to the lost regions are discussed for each of the chromosomes. The review is supplemented also by a list of known and putative tumor suppressor genes and DNA repair genes (see Table 1, online). Losses are found in all chromosome arms, but they seem to be relatively rare at 1q, 2p, 3q, 5p, 6p, 7p, 7q, 8q, 12p, and 20q. Losses and their minimal common overlapping areas that were present in a great proportion of the 73 tumor entities reported in Table 2 (see online) are (in descending order of frequency): 9p23-p24 (48%), 13q21 (47%), 6q16 (44%), 6q26-q27 (44%), 8p23 (37%), 18q22-q23 (37%), 17p12-p13 (34%), 1p36.1 (34%), 11q23 (33%), 1p22 (32%), 4q32-qter (31%), 14q22-q23 (25%), 10q23 (25%), 10q25-qter (25%),15q21 (23%), 16q22 (23%), 5q21 (23%), 3p12-p14 (22%), 22q12 (22%), Xp21 (21%), Xq21 (21%), and 10p12 (20%). The frequency of losses at chromosomes 7 and 20 was less than 10% in all tumors. The chromosomal regions in which the most frequent losses are found implicate locations of essential tumor suppressor genes and DNA repair genes that may be involved in the pathogenesis of several tumor types.

Endothelial cell surfaces play key roles in several important physiological and pathological processes such as blood clotting, angiogenic responses, and inflammation. Here we describe the cloning and characterization of tie, a novel type of human endothelial cell surface receptor tyrosine kinase. The extracellular domain of the predicted tie protein product has an exceptional multidomain structure consisting of a cluster of three epidermal growth factor homology motifs embedded between two immunoglobulinlike loops, which are followed by three fibronectin type III repeats next to the transmembrane region. Additionally, a cDNA form lacking the first of the three epidermal growth factor homology domains was isolated, suggesting that alternative splicing creates different tie-type receptors. Cells transfected with tie cDNA expression vector produce glycosylated polypeptides of 117 kDa which are reactive to antisera raised against the tie carboxy terminus. The tie gene was located in chromosomal region 1p33 to 1p34. Expression of the tie gene appeared to be restricted in some cell lines; large amounts of tie mRNA were detected in endothelial cell lines and in some myeloid leukemia cell lines with erythroid and megakaryoblastoid characteristics. In addition, mRNA in situ studies further indicated the endothelial expression of the tie gene. The tie receptor tyrosine kinase may have evolved for multiple protein-protein interactions, possibly including cell adhesion to the vascular endothelium.

Abstract Malignant mesothelioma (MM) is an aggressive cancer arising from mesothelial cells, mainly due to former asbestos exposure. Little is known about the microRNA (miRNA) expression of MM. miRNAs are small noncoding RNAs, which play an essential role in the regulation of gene expression. This study was carried out to analyze the miRNA expression profile of 17 MM samples using miRNA microarray. The analysis distinguished the overall miRNA expression profiles of tumor tissue and normal mesothelium. Differentially expressed miRNAs were found in tumor samples compared with normal sample. Twelve of them, let‐7b*, miR‐1228*, miR‐195*, miR‐30b*, miR‐32*, miR‐345, miR‐483‐3p, miR‐584, miR‐595, miR‐615‐3p, and miR‐885‐3p, were highly expressed whereas the remaining nine, let‐7e*, miR‐144*, miR‐203, miR‐340*, miR‐34a*, miR‐423, miR‐582, miR‐7‐1*, and miR‐9, were unexpressed or had severely reduced expression levels. Target genes for these miRNAs include the most frequently affected genes in MM such as CDKN2A , NF2 , JUN , HGF , and PDGFA . Many of the miRNAs were located in chromosomal areas known to be deleted or gained in MM such as 8q24, 1p36, and 14q32. Furthermore, we could identify specific miRNAs for each histopathological subtype of MM. Regarding risk factors such as smoking status and asbestos exposure, significantly differentially expressed miRNAs were identified in smokers versus nonsmokers (miR‐379, miR‐301a, miR‐299‐3p, miR‐455‐3p, and miR‐127‐3p), but not in asbestos‐exposed patients versus nonexposed ones. This could be related to the method of assessment of asbestos exposure as asbestos remains to be the main contributor to the development of MM. © 2009 Wiley‐Liss,Inc.

This review summarizes reports of recurrent DNA sequence copy number amplifications in human neoplasms detected by comparative genomic hybridization. Some of the chromosomal areas with recurrent DNA copy number amplifications (amplicons) of 1p22-p31, 1p32-p36, 1q, 2p13-p16, 2p23-p25, 2q31-q33, 3q, 5p, 6p12-pter, 7p12-p13, 7q11.2, 7q21-q22, 8p11-p12, 8q, 11q13-q14, 12p, 12q13-q21, 13q14, 13q22-qter, 14q13-q21, 15q24-qter, 17p11.2-p12, 17q12-q21, 17q22-qter, 18q, 19p13.2-pter, 19cen-q13.3, 20p11.2-p12, 20q, Xp11.2-p21, and Xp11-q13 and genes therein are presented in more detail. The paper with more than 150 references and two tables can be accessed from our web site http://www.helsinki.fi/lglvwww/CMG.html. The data will be updated biannually until the year 2001.

No clear patterns in molecular changes underlying the malignant processes in lung cancer of different histological types have been found so far. To identify critical genes in lung cancer progression we compared the expression profile of cancer related genes in 14 pulmonary adenocarcinoma patients with normal lung tissue by using the cDNA array technique. Principal component analyses (PCA) and permutation test were used to detect the differentially expressed genes. The expression profiles of 10 genes were confirmed by semi-quantitative real-time RT-PCR. In tumour samples, as compared to normal lung tissue, the up-regulated genes included such known tumour markers as CCNB1, PLK, tenascin, KRT8, KRT19 and TOP2A. The down-regulated genes included caveolin 1 and 2, and TIMP3. We also describe, for the first time, down-regulation of the interesting SOCS2 and 3, DOC2 and gravin. We show that silencing of SOCS2 is not caused by methylation of exon 1 of the gene. In conclusion, by using the cDNA array technique we were able to reveal marked differences in the gene expression level between normal lung and tumour tissue and find possible new tumour markers for pulmonary adenocarcinoma.

Uniparental disomy (UPD) results when both copies of a chromosome pair originate from one parent. In humans, this might result in developmental disease or cancer due to either the production of homozygosity (caused by mutated or methylated genes or by microRNA sequences) or an aberrant pattern of imprinting. Constitutional UPD is associated with meiotic errors, resulting in developmental diseases, whereas acquired UPD probably occurs as a result of a mitotic error in somatic cells, which can be an important step in cancer development and progression. This review summarizes the mechanisms underlying UPD and their emerging association with cancer.

Abstract The development of tyrosine kinase inhibitor treatments has made it important to test cancer patients for clinically significant gene mutations that influence the benefit of treatment. Targeted next‐generation sequencing (NGS) provides a promising method for diagnostic purposes by enabling the simultaneous detection of multiple mutations in various genes in a single test. The aim of our study was to screen EGFR , KRAS , and BRAF mutations by targeted NGS and commonly used real‐time polymerase chain reaction (PCR) methods to evaluate the feasibility of targeted NGS for the detection of the mutations. Furthermore, we aimed to identify potential novel mutations by targeted NGS. We analyzed formalin‐fixed, paraffin‐embedded (FFPE) tumor tissue specimens from 81 non‐small cell lung carcinoma patients. We observed a significant concordance (from 96.3 to 100%) of the EGFR , KRAS , and BRAF mutation detection results between targeted NGS and real‐time PCR. Moreover, targeted NGS revealed seven nonsynonymous single‐nucleotide variations and one insertion‐deletion variation in EGFR not detectable by the real‐time PCR methods. The potential clinical significance of these variants requires elucidation in future studies. Our results support the use of targeted NGS in the screening of EGFR , KRAS , and BRAF mutations in FFPE tissue material. © 2012 Wiley Periodicals, Inc.

Lung cancer has the highest mortality rate of all of the cancers in the world and asbestos-related lung cancer is one of the leading occupational cancers. The identification of asbestos-related molecular changes has long been a topic of increasing research interest. The aim of this study was to identify novel asbestos-related molecular correlates by integrating miRNA expression profiling with previously obtained profiling data (aCGH and mRNA expression) from the same patient material. miRNA profiling was performed on 26 tumor and corresponding normal lung tissue samples from highly asbestos-exposed and non-exposed patients, and on eight control lung tissue samples. Data analyses on miRNA expression, and integration of miRNA and previously obtained mRNA data were performed using Chipster. A separate analysis was used to integrate miRNA and previously obtained aCGH data. Both known and new lung cancer-associated miRNAs and target genes with inverse correlation were discovered. Furthermore, DNA copy number alterations (e.g., gain at 12p13.31) were correlated with the deregulated miRNAs. Specifically, thirteen novel asbestos-related miRNAs (over-expressed: miR-148b, miR-374a, miR-24-1*, Let-7d, Let-7e, miR-199b-5p, miR-331-3p, and miR-96 and under-expressed: miR-939, miR-671-5p, miR-605, miR-1224-5p and miR-202) and inversely correlated target genes (e.g., GADD45A, LTBP1, FOSB, NCALD, CACNA2D2, MTSS1, EPB41L3) were identified. In addition, over-expression of the well known squamous cell carcinoma-associated miR-205 was linked to down-regulation of the DOK4 gene. The miRNAs/genes presented here may represent interesting targets for further investigation and could eventually have potential diagnostic implications.

We studied DNA copy number changes in diffuse large B-cell lymphoma using comparative genomic hybridization analysis on 20 primary tumors and on 12 recurrent tumors excised after chemotherapy or radiotherapy. Twenty-nine (91%) of the cases showed abnormal copy number karyotypes. Chromosomal regions at X (41%), 1q (38%), 7 (31%), 3 (24%), 6p (21%), 11 (21%), 12 (21%), and 18 (21%) were most frequently gained, and the most common losses involved 6q (38%), X (21%), 1p (14%), and 8p (10%). High-level amplifications were observed at 6p23-ter, 10p12–14, 17p1l.2, 18q21-ter, and Xq22-ter, all but 18q appearing only in the recurrent tumors. Gains (median, 2; range, 0 to 10) were more frequent than losses (median, 1; range, 0 to 7; P = .0004). The median number of aberrations found in the recurrent tumors (6.5) was greater than that in the primary tumors (2; P = .01). The copy number changes found in the recurrent tumors were more random than those found in the primary tumors, which were mainly located in the most frequently affected regions. Our findings are in line with those observed using conventional cytogenetic analysis, but especially novel high-level amplifications were detected. Southern blot analysis showed BCL2 amplification, but not translocation t(14;18)(q32;q21), in cases in which a gain at 18q was detected by comparative genomic hybridization, which strongly suggests that, in addition to translocation, gene amplification is another mechanism for the overexpression of the BCL2 protein.

Histologic response to chemotherapy is currently the best prognostic parameter in high-grade osteosarcoma but it can be evaluated only after several weeks of chemotherapy. Thus a prognostic parameter known at the time of diagnosis would be of great clinical benefit. In the present study, we present the results of 31 primary high-grade osteosarcomas analyzed by comparative genomic hybridization (CGH). CGH allows for genome-wide screening of a tumor by detecting alterations in DNA sequence copy number. The most frequent aberrations were copy number increases at 1q21 in 58% of the tumors and at 8q (8q21.3-q22 in 52% and 8cen-q13 in 45%), followed by copy number increases at 14q24-qter (35%) and Xp11.2-p21 (35%). The most common losses were detected at 6q16 (32%) and 6q21-q22 (32%). Patients with a copy number increase at 8q21.3-q22 and/or at 8cen-q13 had a statistically significant poor distant disease-free survival (p = 0.003) and showed a trend toward short overall survival (p = 0.04). Patients with a copy number increase at 1q21 showed a trend toward short overall survival (p = 0.04). Thus, specific genetic aberrations detected at the time of the diagnosis could be used in prognostic evaluation of high-grade osteosarcoma. Int. J. Cancer (Pred. Oncol.) 84:114–121, 1999. © 1999 Wiley-Liss, Inc.

We used comparative genomic hybridization (CGH) to screen for DNA copy number changes in 34 specimens from 27 cases of mantle cell lymphoma (MCL). The most common gains were detected at 3q (52%), 8q (30%), and 15q (26%), whereas the most frequent losses involved 13q (41%), 1p (33%), 6q (30%), 9p (30%), and 11q (30%). The gain of 3q, with a minimal common region at 3q26.1-27, appeared in more than half of the lymphomas, suggesting the location of an important oncogene here. A common deleted region at 11q22 was found in one-third of the patients, which suggests that this region may harbor a tumor suppressor gene important in the tumorigenesis of MCL. The mean number of changes was higher in more aggressive blastoid variants of MCL than in lymphomas with typical morphology. Our results show that the chromosomal regions affected in MCL are highly consistent and are different from those seen in other types of non-Hodgkin's lymphoma.

DNA copy number gains and amplifications at 17q are frequent in gastriccancer, yet systematic analyses of the 17q amplicon have not been performed. In this study, we carried out a comprehensive analysis of copy number and expression levels of 636 chromosome 17-specific genes in gastric cancer by using a custom-made chromosome 17-specific cDNA microarray. Analysis of DNA copy number changes by comparative genomic hybridization on cDNA microarray revealed increased copy numbers of 11 known genes (ERBB2, TOP2A, GRB7, ACLY, PIP5K2B, MPRL45, MKP-L, LHX1, MLN51, MLN64, and RPL27) and seven expressed sequence tags (ESTs) that mapped to 17q12-q21 region. To investigate the genes transcribed at the 17q, we performed gene expression analyses on an identical cDNA microarray. Our expression analysis showed overexpression of 8 genes (ERBB2, TOP2A, GRB2, AOC3, AP2B1, KRT14, JUP, and ITGA3) and two ESTs. Of the commonly amplified transcripts, an uncharacterized EST AA552509 and the TOP2A gene were most frequently overexpressed in 82% of the samples. Additional studies will be initiated to understand the possible biological and clinical significance of these genes in gastric cancer development and progression.

We performed a comparative genomic hybridization study on 25 samples of adenocarcinoma and 19 samples of squamous cell carcinoma of the lung to detect recurrent changes in the genetic material. DNA copy number changes were found in 16 squamous cell carcinoma samples and 17 adenocarcinoma samples. The most common changes were gains of DNA sequences in 3q (43%), 1q (34%), 8q (32%), 5p, (30%), 7p (25%), and 12p (25%). Of the squamous cell carcinoma samples with DNA copy number changes, 94% (15/16) had a gain in 3q (minimal common region of overlap q24-qter), whereas only 24% (4/17) of the adenocarcinoma samples with DNA copy number changes showed a gain in 3q (q22-qter) (P< 0.001). Six high-level amplifications in 3q (q26.2-q26.3) were detected in the squamous cell carcinoma samples but none were observed in the adenocarcinoma samples. Our results suggest that amplification of genes in 3q may be important in the tumorigenesis of squamous cell carcinoma but not necessarily of adenocarcinoma. Genes Chromosomes Cancer 22:79–82, 1998. © 1998 Wiley-Liss, Inc.

Abstract Little is known about the genomic alterations underlying osteosarcoma. We performed a genomewide high‐resolution gene copy number analysis of 22 osteosarcoma samples using comparative genomic hybridization on a cDNA microarray that contained cDNA clones of about 13,000 genes. Nineteen of the 22 cases had amplifications that on average spanned more than 1 Mb and contained more than 10 genes. Numerous regions of gain and loss were identified, and their boundaries were defined at high resolution. Novel amplicons were found at 14q11, 17q25, and 22q11–q13. Earlier‐known large amplified regions were detected at 12q11–q15, 8q24, 6p12–p13, and 17p11–p13 in 8, 6, 5, and 4 of the 22 samples, respectively. Amplification of 12q was observed more frequently (36% of the cases) than previously reported. Previously known small amplicons at 1p34–p36, 1q21, 19q13, and 21q22 were seen in at least three cases. Our results implicate TOM1L2 and CYP27B1 as having roles as novel targets for the 17p and 12q amplicons, respectively. Details ( www.helsinki.fi/cmg ) of the amplified genes in each amplicon provide valuable raw data for further in silico studies. © 2004 Wiley‐Liss, Inc.

CD99 is a unique 32-kDa cell surface molecule with broad cellular expression but still poorly understood biological functions. In cancer cells, CD99 is highly expressed in virtually all Ewing's sarcoma (ES). Engagement of CD99 induces fast homotypic aggregation of ES cells and caspase-independent apoptosis. In this study, we analysed signal transduction after CD99 engagement on ES cells. Findings obtained with selective inhibitors indicated that only actin cytoskeleton integrity was essential for cell-cell adhesion and apoptosis of ES cells. Indeed, CD99 stimulation induced actin repolymerization, further supporting the role of cytoskeleton in CD99 signaling. Gene expression profiling of ES cells after CD99 engagement showed modulation in the expression of 32 genes. Among the pool of upregulated genes reported to be involved in cell adhesion, we chose to analyse the role of zyxin, a cytoplasmic adherens junction protein found to play a role in the regulation of the actin cytoskeleton. Overexpression of zyxin after CD99 ligation was confirmed by real-time PCR and Western blot. Treatment of ES cells with zyxin antisense oligonucleotides inhibited CD99-induced cell aggregation and apoptosis, suggesting a functional role for this protein. Therefore, our findings indicate that CD99 functions occur through reorganization of cytoskeleton and identify actin and zyxin as the early signaling events driven by CD99 engagement.

Malignant mesothelioma (MM) results from the accumulation of a number of acquired genetic events, especially deletions, which lead to the inactivation of multiple onco-suppressor genes in a multistep cascade mechanism. Past asbestos exposure represents the major risk factor for MM, and the link between asbestos fibers and MM has been largely proved by several epidemiologic and experimental studies. Asbestos fibers induce DNA and chromosomal damage. Most MM cases have shown multiple chromosomal abnormalities. Chromosomal losses are more common than gains. The most common cytogenetic abnormality in MM is a deletion in 9p21, the locus of CDKN2A, a tumor suppressor gene (TSG). The deletion of CDKN2A is a negative prognostic factor in MM. Loss of TSG CDKN2A/p14ARF is also common in MM and mutations in NF2 occur in approximately half of the cases. Despite the ban on asbestos use in Western countries, the incidence of MM is increasing, and asbestos is still used in developing countries. This epidemiologic situation calls for further research. Ongoing studies are already applying high-throughput genomic profiling methods in MM. Genetic alterations observed in MM may be useful in differential diagnosis between lung cancer and MM, as diagnostic markers or therapeutic targets, and as indicators of premalignancy for primary prevention and health surveillance.

Abstract Recent studies have shown the important role of microRNAs (miRNAs) in a variety of biological processes, and in its ability to distinguish tumors according to their prognostic and predictive properties. To identify miRNA signatures associated with colorectal carcinoma (CRC) and with KRAS status, we studied, using Agilent's miRNA microarrays, miRNA expression in primary tumors from 55 metastatic CRC patients, including 15 with mutant and 40 with wild‐type KRAS. Comparing these with normal colon tissue, we identified 49 miRNAs—including 19 novel miRNAs—significantly deregulated in tumor tissue. The presence of the KRAS mutation was associated with up‐regulation of miR‐127‐3p , miR‐92a , and miR‐486‐3p and down‐regulation of miR‐378 . Increased expression of miR‐127‐3p and miR‐92a in KRAS mutant tumors was significantly confirmed by quantitative reverse transcriptase polymerase chain reaction (qRT‐PCR) ( P &lt; 0.05). We identified some predicted target genes of differentially expressed miRNAs between mutated and wild‐type KRAS , such as RSG3 and TOB1 , which are involved in apoptosis and proliferation. Target prediction and pathway analysis suggest a possible role for deregulated miRNAs in nicotinamide adenine dinucleotide phosphate (NADPH) regeneration and G protein‐coupled receptor signaling pathways. © 2011 Wiley Periodicals, Inc.

The effect of short-term culture on the type and frequency of chromosomal aberrations found in bone marrow cells from patients with hematologic disease was evaluated.The classic direct method (1 -2 hr incubation) was compared with methods involving culture for 1 -4 days with or without methotrexate.Data on 32 samples from 21 's Hospital, and the Third

Despite extensive characterization of the role of the EWS-ETS fusions, little is known about secondary genetic alterations and their clinical contribution to Ewing sarcoma (ES). It has been demonstrated that the molecular structure of EWS-ETS lacks prognostic value. Moreover, CDKN2A deletion and TP53 mutation, despite carrying a poor prognosis, are infrequent. In this scenario identifying secondary genetic alterations with a significant prevalence could contribute to understand the molecular mechanisms underlying the most aggressive forms of ES.We screened a 67 ES tumor set for copy number alterations by array comparative genomic hybridization. 1q gain (1qG), detected in 31% of tumor samples, was found markedly associated with relapse and poor overall and disease-free survival and demonstrated a prognostic value independent of classical clinical parameters. Reanalysis of an expression dataset belonging to an independent tumor set (n=37) not only validated this finding but also led us to identify a transcriptomic profile of severe cell cycle deregulation in 1qG ES tumors. Consistently, a higher proliferation rate was detected in this tumor subset by Ki-67 immunohistochemistry. CDT2, a 1q-located candidate gene encoding a protein involved in ubiquitin ligase activity and significantly overexpressed in 1qG ES tumors, was validated in vitro and in vivo proving its major contribution to this molecular and clinical phenotype. This integrative genomic study of 105 ES tumors in overall renders the potential value of 1qG and CDT2 overexpression as prognostic biomarkers and also affords a rationale for the application of already available new therapeutic compounds selectively targeting the protein-ubiquitin machinery.

Anti-EGFR monoclonal antibodies (anti-EGFRmAb) serve in the treatment of metastatic colorectal cancer (mCRC), but patients with a mutation in KRAS/BRAF and nearly one-half of those without the mutation fail to respond. We performed microRNA (miRNA) analysis to find miRNAs predicting anti-EGFRmAb efficacy. Of the 99 mCRC patients, we studied differential miRNA expression by microarrays from primary tumors of 33 patients who had wild-type KRAS/BRAF and third- to sixth-line anti-EGFRmAb treatment, with/without irinotecan. We tested the association of each miRNA with overall survival (OS) by the Cox proportional hazards regression model. Significant miR-31* up-regulation and miR-592 down-regulation appeared in progressive disease versus disease control. miR-31* expression and down-regulation of its target genes SLC26A3 and ATN1 were verified by quantitative reverse transcriptase polymerase chain reaction. Clustering of patients based on miRNA expression revealed a significant difference in OS between patient clusters. Members of the let-7 family showed significant up-regulation in the patient cluster with poor OS. Additionally, miR-140-5p up-regulation and miR-1224-5p down-regulation were significantly associated with poor OS in both cluster analysis and the Cox proportional hazards regression model. In mCRC patients with wild-type KRAS/BRAF, miRNA profiling can efficiently predict the benefits of anti-EGFRmAb treatment. Larger series of patients are necessary for application of these miRNAs as predictive/prognostic markers. Anti-EGFR monoclonal antibodies (anti-EGFRmAb) serve in the treatment of metastatic colorectal cancer (mCRC), but patients with a mutation in KRAS/BRAF and nearly one-half of those without the mutation fail to respond. We performed microRNA (miRNA) analysis to find miRNAs predicting anti-EGFRmAb efficacy. Of the 99 mCRC patients, we studied differential miRNA expression by microarrays from primary tumors of 33 patients who had wild-type KRAS/BRAF and third- to sixth-line anti-EGFRmAb treatment, with/without irinotecan. We tested the association of each miRNA with overall survival (OS) by the Cox proportional hazards regression model. Significant miR-31* up-regulation and miR-592 down-regulation appeared in progressive disease versus disease control. miR-31* expression and down-regulation of its target genes SLC26A3 and ATN1 were verified by quantitative reverse transcriptase polymerase chain reaction. Clustering of patients based on miRNA expression revealed a significant difference in OS between patient clusters. Members of the let-7 family showed significant up-regulation in the patient cluster with poor OS. Additionally, miR-140-5p up-regulation and miR-1224-5p down-regulation were significantly associated with poor OS in both cluster analysis and the Cox proportional hazards regression model. In mCRC patients with wild-type KRAS/BRAF, miRNA profiling can efficiently predict the benefits of anti-EGFRmAb treatment. Larger series of patients are necessary for application of these miRNAs as predictive/prognostic markers.

To identify genetic changes related to tumor progression and find out diagnostic and prognostic genetic markers in gastrointestinal stromal tumors (GISTs), 95 tumor samples (24 benign GISTs, 36 malignant primary GISTs, and 35 GIST-metastases) from 60 patients were studied using comparative genomic hybridization. DNA copy number changes were detected in all samples. Benign GISTs had a mean of 2.6 aberrations/ sample (losses:gains, 5:1) and significantly fewer DNA copy number changes and fewer gains than malignant primary and metastatic GISTs (P < 0.01). High-level amplifications were not seen in benign GISTs. Malignant primary GISTs had a mean of 7.5 aberrations/tumor (losses: gains, 1.6:1), whereas the mean number of aberrations/metastatic GIST was 9 (losses:gains, 1.8:1). Frequent changes observed in all GIST groups included losses in chromosome arms 1p (51%), 14q (74%), and 22q (53%). Gains and high-level amplifications at 8q and 17q were significantly more frequent in metastatic GISTs (57 and 43%) than in benign GISTs (8 and 0%; P < 0.001) and malignant primary GISTs (33 and 25%; P < 0.05). Gains and high-level amplifications at 20q were only seen in malignant primary and metastatic GISTs (P < 0.01), and gains at 5p were not detected in benign GISTs (P < 0.01). Losses in chromosome arm 9p were never seen in benign tumors (P < 0.001), and they were more frequent in metastatic GISTs than in malignant primary GISTs (63 and 36%; P < 0.05). Losses in 13q were less frequent in benign GISTs than in malignant primary (P < 0.05) and metastatic (P < 0.01) GISTs. Our results show that several DNA copy number changes are related to the behavior of GISTs and can be used as prognostic markers for tumor progression.

The genetic changes leading to thyroid cancer are poorly characterized. We studied DNA copy number changes by comparative genomic hybridization (CGH) in 69 primary thyroid carcinomas. In papillary carcinoma, DNA copy number changes were rare (3 of 26, 12%). The changes were all gains, and they were associated with old age (P = 0.01) and the presence of cervical lymph node metastases at presentation (P = 0.08). DNA copy number changes were much more frequent in follicular carcinoma (16 of 20, 80%) than in papillary carcinoma (P < 0.0001), and follicular carcinomas had more often deletions (13/20 versus 0/26, P < 0.0001). Loss of chromosome 22 was common in follicular carcinoma (n = 7, 35%), it was more often seen in widely invasive than in minimally invasive follicular carcinoma (54% versus 0%, P = 0.04), and it was associated with old age at presentation (P = 0.01). In three of the four patients with follicular carcinoma who died of cancer, the tumor had loss of chromosome 22. DNA copy number changes were found in 5 (50%) of the 10 medullary carcinomas studied. Four of these five carcinomas had deletions, and in two of them there was deletion of chromosome 22. Eleven (85%) of the thirteen anaplastic carcinomas investigated had DNA copy number changes, of which five had deletions, and one had deletion of chromosome 22. The most common gains in anaplastic carcinoma were in chromosomes 7p (p22-pter, 31%), 8q (q22-qter, 23%), and 9q (q34-qter, 23%). We conclude that DNA copy number changes are frequent in follicular, medullary, and anaplastic thyroid carcinoma but rare in papillary carcinoma when studied by CGH. Loss of chromosome 22 is particularly common in follicular carcinoma, and it is associated with the widely invasive type. The genetic changes leading to thyroid cancer are poorly characterized. We studied DNA copy number changes by comparative genomic hybridization (CGH) in 69 primary thyroid carcinomas. In papillary carcinoma, DNA copy number changes were rare (3 of 26, 12%). The changes were all gains, and they were associated with old age (P = 0.01) and the presence of cervical lymph node metastases at presentation (P = 0.08). DNA copy number changes were much more frequent in follicular carcinoma (16 of 20, 80%) than in papillary carcinoma (P < 0.0001), and follicular carcinomas had more often deletions (13/20 versus 0/26, P < 0.0001). Loss of chromosome 22 was common in follicular carcinoma (n = 7, 35%), it was more often seen in widely invasive than in minimally invasive follicular carcinoma (54% versus 0%, P = 0.04), and it was associated with old age at presentation (P = 0.01). In three of the four patients with follicular carcinoma who died of cancer, the tumor had loss of chromosome 22. DNA copy number changes were found in 5 (50%) of the 10 medullary carcinomas studied. Four of these five carcinomas had deletions, and in two of them there was deletion of chromosome 22. Eleven (85%) of the thirteen anaplastic carcinomas investigated had DNA copy number changes, of which five had deletions, and one had deletion of chromosome 22. The most common gains in anaplastic carcinoma were in chromosomes 7p (p22-pter, 31%), 8q (q22-qter, 23%), and 9q (q34-qter, 23%). We conclude that DNA copy number changes are frequent in follicular, medullary, and anaplastic thyroid carcinoma but rare in papillary carcinoma when studied by CGH. Loss of chromosome 22 is particularly common in follicular carcinoma, and it is associated with the widely invasive type. The great majority of all thyroid cancers are either papillary, follicular, or anaplastic carcinomas, which are thought to be derived from follicular cells. Only 5% to 10% are medullary carcinomas, which originate from the C-cells.1Franssila K Thyroid.in: Lechago J Gould VE Bloodworth's Endocrine Pathology. ed 3. Williams & Wilkins, Baltimore1997: 171-247Google Scholar This histopathological classification of thyroid carcinomas into four major subtypes has been considered as established, and each of the four entities have typical clinical features. Papillary carcinoma frequently gives rise to cervical lymph node metastases, but distant metastases are rare. This pattern is reversed in follicular carcinoma, which often has distant bone metastases, but cervical lymph node metastases are rare. Papillary and follicular carcinoma are frequently found in the same thyroid as anaplastic carcinoma, suggesting that some anaplastic carcinomas originate from pre-existing differentiated carcinoma.2Demeter JG De Jong SA Lawrence AM Paloyn E Anaplastic thyroid carcinoma: risk factors and outcome.Surgery. 1991; 110: 956-963PubMed Google Scholar, 3Nel CJ van Heerden JA Goellner JR Gharib H McConahey WM Taylor WF Grant CS Anaplastic carcinoma of the thyroid: a clinicopathologic study of 82 cases.Mayo Clin Proc. 1985; 60: 51-58Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar The molecular genetic events in the evolution of different types of thyroid carcinomas are poorly characterized. However, the central role of mutations in the RET proto-oncogene, located at 10q11.2, in the genesis of hereditary medullary thyroid carcinoma is now well recognized, and used in screening of this disorder. More than 95% of patients with MEN 2A have missense germ line mutations that involve either exon 10 or 11 of this receptor tyrosine kinase,4Mulligan LM Kwok JB Healey CS Elsdon MJ Eng C Gardner E Love DR Mole SE Moore JK Papi L Ponder MA Telenius H Tunnacliffe A Ponder BAJ Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A.Nature. 1993; 363: 458-460Crossref PubMed Scopus (1794) Google Scholar and all patients with MEN 2B have the same missense mutation in RET exon 16 at amino acid position 918 within what is predicted to be the tyrosine kinase catalytic domain.5Carlson KM Dou S Chi D Scavarda N Toshima K Jackson CE Wells Jr, SA Goodfellow PJ Donis-Keller H Single missense mutation in the tyrosine kinase catalytic domain of the RET protooncogene is associated with multiple endocrine neoplasia type 2B.Proc Natl Acad Sci USA. 1994; 91: 1579-1583Crossref PubMed Scopus (579) Google Scholar Moreover, sporadic medullary carcinomas have somatic mutations in RET codon 918 in 33% to 67% of cases,6Blaugrund JE Johns Jr, MM Eby YJ Ball DW Baylin SB Hruban RH Sidransky D RET proto-oncogene mutations in inherited and sporadic medullary thyroid cancer.Hum Mol Genet. 1994; 3: 1895-1897Crossref PubMed Scopus (86) Google Scholar and familial non-MEN medullary carcinomas display also frequently RET mutations.7Mulligan LM Ponder BA Genetic basis of endocrine disease multiple endocrine neoplasia type 2.J Clin Endocrinol Metab. 1995; 80: 1989-1995Crossref PubMed Scopus (137) Google Scholar A hypodiploid chromosomal number in the range of 34 to 44 has been found in primary medullary carcinoma tissue.8Wurster-Hill DH Noll WW Bircher LY Pettengill OS Grizzle WA Cytogenetics of medullary carcinoma of the thyroid.Cancer Genet Cytogenet. 1986; 20: 247-253Abstract Full Text PDF PubMed Scopus (25) Google Scholar, 9Tanaka K Baylin SB Nelkin BD Testa JR Cytogenetic studies of a human medullary thyroid carcinoma cell line.Cancer Genet Cytogenet. 1987; 25: 27-35Abstract Full Text PDF PubMed Scopus (22) Google Scholar Medullary carcinoma has been found to be associated with a constitutional minute deletion in the short arm of chromosome 20 (del(20)(p12.2)).10Babu VR Van Dyke DL Flejter WL Jackson CE Chromosome 20 deletion in multiple endocrine neoplasia type 2: expanded double-blind studies.Am J Med Genet. 1987; 27: 739-748Crossref PubMed Scopus (7) Google Scholar In cytogenetic studies, only ∼30 cases with clonal chromosomal abnormalities have been described in papillary carcinoma.11Bondeson L Bengtsson A Bondeson A-G Dahlenfors R Grimelius L Wedell B Mark J Chromosome studies in thyroid neoplasia.Cancer. 1989; 64: 680-685Crossref PubMed Scopus (83) Google Scholar, 12Jenkins RB Hay ID Herath JF Schultz CG Spurbeck JL Grant CS Goellner JR Dewald GW Frequent occurrence of cytogenetic abnormalities in sporadic nonmedullary thyroid carcinoma.Cancer. 1990; 66: 1213-1220Crossref PubMed Scopus (87) Google Scholar, 13Teyssier J-R Liautaud-Roger F Ferre D Patey M Dufer J Chromosomal changes in thyroid tumours: relation with DNA content, karyotypic features, and clinical data.Cancer Genet Cytogenet. 1990; 50: 249-263Abstract Full Text PDF PubMed Scopus (79) Google Scholar, 14Herrmann MA Hay ID Bartelt Jr, DH Ritland SR Dahl RJ Grant CS Jenkins RB Cytogenetic and molecular genetic studies of follicular and papillary thyroid cancers.J Clin Invest. 1991; 88: 1596-1604Crossref PubMed Scopus (130) Google Scholar, 15Herrmann ME Mohamed A Talpos G Wolman SR Cytogenetic study of a papillary thyroid carcinoma with a rearranged chromosome 10.Cancer Genet Cytogenet. 1991; 57: 209-217Abstract Full Text PDF PubMed Scopus (26) Google Scholar, 16Antonini P Vénuat A-M Caillou B Berger R Schlumberger M Bernheim A Parmentier C Cytogenetic studies on 19 papillary thyroid carcinomas.Genes Chromosomes & Cancer. 1992; 5: 206-211Crossref PubMed Scopus (20) Google Scholar, 17Sozzi G, Bongarzone I, Miozzo M, Cariani CT, Mondellini P, Calderone C, Pilotti S, Pierotti MA, Della Porta G: Cytogenetic and molecular genetic characterization of papillary thyroid carcinomas. Genes Chromosomes & Cancer, 5:212–218Google Scholar, 18Roque L Clode AL Gomes P Rosa-Santos J Soares J Castedo S Cytogenetic findings in 31 papillary thyroid carcinomas.Genes Chromosomes & Cancer. 1995; 13: 157-162Crossref PubMed Scopus (29) Google Scholar, 19Lehmann L Greulich KM Zitzelsberger H Negele T Spelsberg F Bauchinger M Weier HUG Cytogenetic and molecular genetic characterization of a chromosome 2 rearrangement in a case of human papillary thyroid carcinoma with radiation history.Cancer Genet Cytogenet. 1997; 96: 30-36Abstract Full Text PDF PubMed Scopus (20) Google Scholar The karyotypic abnormalities are usually simple. An intrachromosomal rearrangement inv(10)(q11q21) resulting in the juxtaposition of sequences encoding the intracellular tyrosine kinase domain of RET with 5′ sequences from one of three unrelated genes has been considered as characteristic and has been identified in up to 30% of papillary carcinomas.12Jenkins RB Hay ID Herath JF Schultz CG Spurbeck JL Grant CS Goellner JR Dewald GW Frequent occurrence of cytogenetic abnormalities in sporadic nonmedullary thyroid carcinoma.Cancer. 1990; 66: 1213-1220Crossref PubMed Scopus (87) Google Scholar, 14Herrmann MA Hay ID Bartelt Jr, DH Ritland SR Dahl RJ Grant CS Jenkins RB Cytogenetic and molecular genetic studies of follicular and papillary thyroid cancers.J Clin Invest. 1991; 88: 1596-1604Crossref PubMed Scopus (130) Google Scholar, 15Herrmann ME Mohamed A Talpos G Wolman SR Cytogenetic study of a papillary thyroid carcinoma with a rearranged chromosome 10.Cancer Genet Cytogenet. 1991; 57: 209-217Abstract Full Text PDF PubMed Scopus (26) Google Scholar, 17Sozzi G, Bongarzone I, Miozzo M, Cariani CT, Mondellini P, Calderone C, Pilotti S, Pierotti MA, Della Porta G: Cytogenetic and molecular genetic characterization of papillary thyroid carcinomas. Genes Chromosomes & Cancer, 5:212–218Google Scholar, 20Grieco M Santoro M Berlingieri MT Melillo RM Donghi R Bongarzone I Pierotti MA Della Porta G Fusco A Vecchio G PTC is a novel rearranged form of the ret proto-oncogene and is frequently detected in vivo in human thyroid papillary carcinomas.Cell. 1990; 60: 557-563Abstract Full Text PDF PubMed Scopus (891) Google Scholar In many cases, it has been the only anomaly. Activation of other receptor tyrosine kinase genes, such as the proto-oncogenes TRK (encodes a cell surface receptor for nerve growth factor) and MET have also been reported in papillary carcinoma.17Sozzi G, Bongarzone I, Miozzo M, Cariani CT, Mondellini P, Calderone C, Pilotti S, Pierotti MA, Della Porta G: Cytogenetic and molecular genetic characterization of papillary thyroid carcinomas. Genes Chromosomes & Cancer, 5:212–218Google Scholar, 21Di Renzo MF Olivero M Ferro S Prat M Bongarzone I Pilotti S Belfiore A Constantino A Vigneri R Pierotti MA Comoglio PM Overexpression of the c-MET/HGF receptor gene in human thyroid carcinomas.Oncogene. 1992; 7: 2549-2553PubMed Google Scholar, 22Bongarzone I Pierotti MA Monzini N Mondellini P Manenti G Donghi R Pilotti S Grieco M Santoro M Fusco A Vecchio G Della Porta G High frequency of activation of tyrosine kinase oncogenes in human papillary thyroid carcinoma.Oncogene. 1989; 4: 1457-1462PubMed Google Scholar, 23Santoro M Dathan NA Berlingieri MT Bongarzone I Paulin C Grieco M Pierotti MA Vecchio G Fusco A Molecular characterization of RET/PTC3, a novel rearranged version of the RET proto-oncogene in human thyroid papillary carcinoma.Oncogene. 1994; 9: 509-516PubMed Google Scholar Cytogenetic information is limited also in follicular carcinoma, and only a few tumors have been examined.11Bondeson L Bengtsson A Bondeson A-G Dahlenfors R Grimelius L Wedell B Mark J Chromosome studies in thyroid neoplasia.Cancer. 1989; 64: 680-685Crossref PubMed Scopus (83) Google Scholar, 12Jenkins RB Hay ID Herath JF Schultz CG Spurbeck JL Grant CS Goellner JR Dewald GW Frequent occurrence of cytogenetic abnormalities in sporadic nonmedullary thyroid carcinoma.Cancer. 1990; 66: 1213-1220Crossref PubMed Scopus (87) Google Scholar, 13Teyssier J-R Liautaud-Roger F Ferre D Patey M Dufer J Chromosomal changes in thyroid tumours: relation with DNA content, karyotypic features, and clinical data.Cancer Genet Cytogenet. 1990; 50: 249-263Abstract Full Text PDF PubMed Scopus (79) Google Scholar, 14Herrmann MA Hay ID Bartelt Jr, DH Ritland SR Dahl RJ Grant CS Jenkins RB Cytogenetic and molecular genetic studies of follicular and papillary thyroid cancers.J Clin Invest. 1991; 88: 1596-1604Crossref PubMed Scopus (130) Google Scholar, 24van den Berg E van Doormaal JJ Oosterhuis JW de Jong B Wiersema J Vos AM Dam A Vermeij A Chromosomal aberrations in follicular thyroid carcinoma: case report of a primary tumor and its metastasis.Cancer Genet Cytogenet. 1991; 54: 215-222Abstract Full Text PDF PubMed Scopus (21) Google Scholar, 25Roque L Castedo S Clode A Soares J Deletion of 3p25-pter in a primary follicular thyroid carcinoma and its metastasis.Genes Chromosomes & Cancer. 1993; 8: 199-203Crossref PubMed Scopus (40) Google Scholar, 26Grebe SKG McIver B Hay ID Wu PSC Maciel LMZ Drabkin HA Goellner JR Grant CS Jenkins RB Eberhardt NL Frequent loss of heterozygosity on chromosomes 3p and 17p without VHL or p53 mutations suggests involvement of unidentified tumor suppressor genes in follicular thyroid carcinoma.J Clin Endocrinol Metab. 1997; 82: 3684-3691Crossref PubMed Scopus (109) Google Scholar The short arm of chromosome 3 has been reported to contain rearrangements, and a minimal common deleted region at 3p25-pter has been detected.14Herrmann MA Hay ID Bartelt Jr, DH Ritland SR Dahl RJ Grant CS Jenkins RB Cytogenetic and molecular genetic studies of follicular and papillary thyroid cancers.J Clin Invest. 1991; 88: 1596-1604Crossref PubMed Scopus (130) Google Scholar, 25Roque L Castedo S Clode A Soares J Deletion of 3p25-pter in a primary follicular thyroid carcinoma and its metastasis.Genes Chromosomes & Cancer. 1993; 8: 199-203Crossref PubMed Scopus (40) Google Scholar Jenkins et al12Jenkins RB Hay ID Herath JF Schultz CG Spurbeck JL Grant CS Goellner JR Dewald GW Frequent occurrence of cytogenetic abnormalities in sporadic nonmedullary thyroid carcinoma.Cancer. 1990; 66: 1213-1220Crossref PubMed Scopus (87) Google Scholar reported aberrations that were mainly deletions in three follicular carcinomas investigated, and we found a loss of chromosome 22 in 7 of the 13 follicular carcinomas studied.27Hemmer S Wasenius V-M Knuutila S Joensuu H Franssila K Comparison of benign and malignant follicular thyroid tumours by comparative genomic hybridization.Br J Cancer. 1998; 78: 1012-1017Crossref PubMed Scopus (54) Google Scholar Similarly, only few anaplastic carcinomas with an abnormal karyotype have been described,12Jenkins RB Hay ID Herath JF Schultz CG Spurbeck JL Grant CS Goellner JR Dewald GW Frequent occurrence of cytogenetic abnormalities in sporadic nonmedullary thyroid carcinoma.Cancer. 1990; 66: 1213-1220Crossref PubMed Scopus (87) Google Scholar, 28Mark J Ekedahl C Dahlenfors R Westermark B Cytogenetical observations in five anaplastic thyroid carcinomas.Hereditas. 1987; 107: 163-174Crossref PubMed Scopus (61) Google Scholar and the molecular mechanisms involved in the genesis of this type of thyroid carcinoma are unknown. Point mutations in the p53 tumor suppressor gene appear to be frequent in anaplastic carcinoma, but not in differentiated thyroid carcinomas.29Ito T Seyama T Mizuno T Tsuyama N Hayashi T Hayashi Y Dohi K Nakamura N Akiyma M Unique association of p53 mutations with undifferentiated but not with differentiated carcinomas of the thyroid gland.Cancer Res. 1992; 52: 1369-1371PubMed Google Scholar This paucity of cytogenetic data on thyroid neoplasms may reflect difficulties in performing karyotype studies on solid tumors, such as finding complex karyotypes and having only a low mitotic index of neoplastic cells in short-term cultures. In the present study we screened genetic imbalances in a series of thyroid carcinomas by comparative genomic hybridization (CGH). This method does not require tumor cell cultures, and it may be performed even from DNA extracted from formalin-fixed and deparaffinized tissue.30Isola JJ de Vries S Chu LW Ghazvini S Waldman FM Analysis of changes in DNA sequence copy number by comparative genomic hybridization in archival paraffin-embedded tumor samples.Am J Pathol. 1994; 145: 1301-1308PubMed Google Scholar To our knowledge, this is the first study where DNA copy number changes of different types of thyroid carcinomas have been investigated by this method. Papillary and follicular thyroid carcinomas are often grouped together as differentiated thyroid carcinomas, but the present results show that the DNA copy number change patterns are widely different in these two types of thyroid cancer. The series consists of 69 patients with thyroid carcinoma diagnosed in the Department of Pathology, Helsinki University Central Hospital, between 1981 and 1997. The patients were chosen for analysis based on the availability of fresh-frozen tumor tissue (n = 53) or randomly whenever paraffin-embedded tissue was used (n = 16). Twenty-six of the carcinomas were of the papillary, twenty follicular, ten medullary, and thirteen of the anaplastic histological type. The histological sections were re-examined and the tumors were reclassified according to the World Health Organization (WHO) classification31Hedinger C Williams ED Sobin LH The WHO histological classification of thyroid tumours: a commentary on the second edition.Cancer. 1989; 63: 908-911Crossref PubMed Scopus (520) Google Scholar by one of us (K. Franssila). The clinical stage was determined according to the International Union Against Cancer (UICC) TNM classification.32Union Internationale Contrele Cancer TNM Classification of Malignant Tumours. ed 4. Springer-Verlag, Berlin1987Google Scholar The clinical data are shown in Table 1, Table 2.Table 1Clinical Data and Comparative Genomic Hybridization Findings in 23 Papillary and 20 Follicular Carcinomas of the ThyroidCaseAge (years)SexTNMCopy number changesFollow-upPapillary carcinoma1 to 236, 18, 21,F n = 18T1N0M0, n = 2Normal21 alive,33, 34, 34, 35,T2N0M0, n = 104–9 years37, 39, 42, 44,M, n = 5T2N1M0, n = 21 DOD 4 years44, 46, 49, 49,T3N0M0, n = 11 DOD 7 years50, 52, 54, 55T3N1M0, n = 363, 76, 78, 80T4N0M0, n = 3T4N1M0, n = 1TXNXM0, n = 72478FT3N1M0+5q14-23.3DOD 5 years2581FT3N1M0+5q23.3-qter,+7,+17, +21q22.1-qterAlive 5 years2673FT2N0M0+1q23-qterAlive 8 yearsFollicular carcinomaMinimally invasive134FT2N0M0NormalAlive 1 year278FT3N0M0NormalDOD 2 years353MT2N0M0+17qAlive 4 years457FT2N0M1NormalAlive 4 years, M+533MT3N0M0+9q11.1-p13Alive 1 year673FT3N0M0+12q11-q16,+12q22-qter,+19p, +19q13.2-qter,−13q21-q22.1Alive 1 year766FT2N0M1+17q,−22q12.3-qterAlive 3 yearsWidely invasive879MT1N0M0+1q22-qter, −1p21-22Alive 5 years976MT4N1M1−13, −22Alive 4 years077FT1N0M1−22DOD 1 year1181FT3N0M0+1q22-qter,−1p,−22DOD 2 years1271FT3N0M0NormalAlive 4 years1379FT2N0M0−22Alive 3 years1485FT3N0M0−1p13-23,−21,−22Alive 1 year1568FT2N0M0+1q24-qter,−1p,−9,−13,−18, −22DOD 10 years1668FT3N0M0−13,−22Alive 6 years, M+1779MT4N0M0+17q12-qter, −13q12.3-qterAlive 1 year1874FT3N0M0+1q11-q41Alive 3 years, M+1953FTXN0M0+1,+4,+5,+7,+12,+14, high14q11.2-q22,+18, high18q12.1- q21,−11q14-qterAlive 1 year2066MT2N0M0+5,+X,+7,+12,+18,+19,+20DOI 4 yearsF, female; M, male; DOD, dead of disease; DOI, dead of intercurrent disease; M+, alive with distant metastases. Open table in a new tab Table 2Comparative Genomic Hybridization Findings in Medullary and Anaplastic Carcinomas of the ThyroidCaseAge (years)SexCopy number changesMedullary carcinoma1–518, 34, 34, 38, 55F, n = 3NormalM, n = 2638M+1q21-qter,+6,+7p,+11q,+21q21-qter745M−18p,−22838F−1p31.1-p32, −3,−13,−18q21-qter948M−3q26.2-q29,−131064M−22Anaplastic carcinoma1–286FNormal77F383F+6p12-pter,+5p13-pter474F+3p12-p21548M+8q21-qter674M+9q34-qter,+21q22-qter787F+Xp11.2-pter,+7p21-pter878F−18p,−22960M+2q11.1-q24, +5q34-qter,+8q21.3-qter,+19q13.1-q13.2,+7p22-pter1074M+9q22.3-qter1172M+3q,−18q1269M+X,+7p21-pter1364F+3q13.3-qter, +5p,+Xq12-qter,+7p13-pter,+8q22-qter,+9,+10q25-qter, +11p11.2-q23.3,+12q21-qter,+15q22-qter,+20q,−4q34-qter, −13F, female; M, male. Open table in a new tab F, female; M, male; DOD, dead of disease; DOI, dead of intercurrent disease; M+, alive with distant metastases. F, female; M, male. Histological sections from frozen tissue were cut, stained with toluidine blue, and examined for the presence of representative tumor tissue. In all cases at least 70% of the cells analyzed were cancer cells. From each tumor specimen 20 to 30 5-μm sections were cut, and genomic DNA was isolated as described elsewhere.30Isola JJ de Vries S Chu LW Ghazvini S Waldman FM Analysis of changes in DNA sequence copy number by comparative genomic hybridization in archival paraffin-embedded tumor samples.Am J Pathol. 1994; 145: 1301-1308PubMed Google Scholar, 33Sambrook J Fritsch EF Maniatis T Molecular Cloning: A Laboratory Manual. ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar In one case of anaplastic carcinoma (case 8, Table 2) the sample did not contain enough DNA for a CGH analysis, and we multiplied the genomic DNA by using degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR). DNA of peripheral blood lymphocytes obtained from healthy male and female donors was extracted according to standard procedures and was used as a reference in the CGH analyses. CGH was performed according to the method of Kallioniemi et al34Kallioniemi O-P Kallioniemi A Piper J Isola J Waldman FM Gray JW Pinkel D Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in solid tumour.Genes Chromosomes & Cancer. 1994; 10: 231-243Crossref PubMed Scopus (948) Google Scholar with a modification using fluorochromes conjugated to a mixture of dCTP and dUTP for standard nick translation.35El-Rifai W Larramendy ML Björkqvist A-M Hemmer S Knuutila S Optimization of comparative genomic hybridization by fluorochrome conjugated to dCTP and dUTP nucleotides.Lab Invest. 1997; 77: 699-700PubMed Google Scholar Tumor DNA was labeled with fluorescein isothiocyanate (FITC)-dUTP and FITC-dCTP (Dupont, Boston, MA), and the normal reference DNA (extracted from the blood of a healthy man or a woman) was labeled with Texas-Red-dUTP and Texas-Red-dCTP (Dupont) in a standard nick-translation reaction. Equal amounts (1 μg) of the labeled test and reference probes were used for hybridization with 10 μg of unlabeled human Cot-1 DNA to block the binding of repetitive sequences in 10 μl of the hybridization buffer (50% formamide, 10% dextran sulfate, 2X SSC (1X SSC is 0.15 mol/L sodium chloride/0.015 mol/L sodium citrate, pH 7)). The DNA was then denatured for 5 minutes at 75°C before applying it to normal lymphocyte preparations. Before hybridization, the metaphase preparations were dehydrated in a series of 70%, 80%, and 100% ethanol concentrations and denatured at 65°C for 2 minutes in a formamide solution (70% formamide/2X SSC). The slides were then dehydrated on ice as described above. They were then treated with proteinase K at 37°C for 7.5 minutes (0.2 μg/ml in 20 mmol/L Tris/HCl, 2 mmol/L CaCl2, pH 7) and once again dehydrated in a series of increasing ethanol concentrations as indicated above. Hybridization was performed in a moist chamber at 37°C for 48 hours. Post-hybridization washes were as follows: three times in 50% formamide/2X SSC, pH 7, twice in 2X SSC, and once in 0.1X SSC at 45°C followed by 2X SSC and 0.1 mol/L NaH2 PO4/0.1 mol/L Na2HPO4/0.1% Nonidet P-40, pH 8, and distilled water at room temperature for 10 minutes each. The slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) at a concentration of 0.1 μg/ml in an anti-fade solution. To confirm the CGH results, additional hybridization experiments using the reverse-labeling system, ie, tumor DNA labeled with Texas Red and reference DNA with FITC, were performed on some specimens. The hybridizations were analyzed using an Olympus fluorescence microscope and an ISIS digital image analysis system (Metasystem, Altlussheim, Germany) based on an integrated high-sensitivity monochrome CCD camera and automated CGH analysis software. The three-color images with red, green, and blue were acquired from 8 to 10 metaphases. Only metaphases of good quality with strong uniform hybridization were included in the analysis. Chromosomes not suitable for CGH were excluded from the analysis (eg, chromosomes that were heavily bent or overlapping or those that had overlying artifacts). Chromosomal regions were interpreted as over-represented (gained) when the red-to-green ratio exceeded 1.17 and under-represented (lost) when the ratio was less than 0.85 (Figure 1). Ninety-nine percent confidence limits with 1% error probability were used to confirm the interpretation. The cut-off values were taken from negative control experiments by using differentially labeled male DNA versus female DNA. A positive control with known chromosomal aberrations and a negative control were included in each hybridization to verify the reliability of the method. Chromosomal regions in the centromeric areas of chromosomes 1, 9, 16, and Y and the p-arms of acrocentric chromosomes were discarded from the analysis because of their large heterochromatic areas. A 5-μl volume of the extracted DNA was used as a template for DOP-PCR with a universal primer (5′-CCGACTCGAGNNNNNNATGTGG-3′, with N = A, C, G, or T) with some modifications.36Kuukasjärvi T Tanner M Pennanen S Karhu R Visakorpi T Isola J Optimizing DOP-PCR for universal amplification of small DNA samples in comparative genomic hybridization.Genes Chromosomes & Cancer. 1997; 18: 94-101Crossref PubMed Scopus (117) Google Scholar, 37Tapper J Sarantaus L Vahteristo P Nevanlinna H Hemmer S Seppälä M Knuutila S Butzow R Genetic changes in inherited and sporadic ovarian carcinomas by comparative genomic hybridization: extensive similarity except for a difference at chromosome 2q24–q32.Cancer Res. 1998; 58: 2715-2719PubMed Google Scholar We applied Thermosequenase enzyme (Amersham, Cleveland, OH) in 1:10 dilution (3 U/reaction) in a dilution buffer (10 mmol/L Tris/HCl, pH 8.0, 1 mmol/L 2-mercaptoethanol, 0.5% Tween-20, 0.5% Nonidet P-40) in a volume of 10 μl (26 mmol/L Tris/HCl, pH 9.5, 6.5 mmol/L MgCl2, 0.2 mmol/L dNTPs, 1 μmol/L universal primer) applying six cycles of 94°C for 1 minute, 30°C for 3 minutes, 65°C for 5 minutes, and final extension at 72°C for 10 minutes followed by high-stringency cycles consisting of an initial melting at 95°C for 3 minutes and 30 to 35 cycles of 94°C for 1 minute, 56°C for 1 minute, and 72°C for 2 minutes, with a final extension of 72°C for 5 minutes, in a volume of 50 μl using the same reaction conditions as above except 2 μmol/L primer. To confirm deletion of chromosome 22, two-color FISH was performed using an LSI DiGeorge/VCFS probe mixture (purchased from Vysis, Ahdiagnostics, Skarholmen, Sweden). The LSI DiGeorge/VCFS probe mixture contains a SpectrumOrange TUPLE 1 probe located at 22q11.2 and a SpectrumGreen LSI ARSA (arylsulfatase A gene) probe that maps close to the telomeric end of 22q at 22q13.3. Deparaffination of nuclei preparations was done in xylene for 15 minutes at +65°C and three times for 5 minutes each at room temperature, followed by dehydration in a 100%, 85%, 70%, and 50% ethanol series (5 minutes each at room temperature). To allow for penetration of the probe, the slides were treated in 1 mol/L sodium thiocyanate at +70°C for 15 minutes, followed by treatment in 0.05 N HCl at +37°C for 10 minutes and by 5 mg/ml pepsin in 0.05 N HCl at +37°C for 20 minutes. Hybridization was performed according to the standard protocols. The slides were denatured in 70% formamide/2X SSC, pH 7, at +75°C for 5 minutes and counterstained with 125 ng/ml DAPI in an anti-fade solution. From each preparation a minimum of 200 morphologically intact and non-overlapping nuclei were scored using an Olympus fluorescence microscope (Olympus, Tokyo, Japan). Normal lymphocytes were used as controls, and two hybridization signals for the LSI DiGeorge/VCFS probes at the expected locations were found in ∼90% of the interphase lymphocyte nuclei. Chromosome 22 was considered to be deleted if only one signal was detected in >20% of cells. Frequency tables were analyzed with Fisher's

Comparative genomic hybridization (CGH) was used to detect copy number changes of DNA sequences in the Ewing family of tumours (ET). We analysed 20 samples from 17 patients. Fifteen tumours (75%) showed copy number changes. Gains of DNA sequences were much more frequent than losses, the majority of the gains affecting whole chromosomes or whole chromosome arms. Recurrent findings included copy number increases for chromosomes 8 (seven out of 20 samples; 35%), 1q (five samples; 25%) and 12 (five samples; 25%). The minimal common regions of these gains were the whole chromosomes 8 and 12, and 1q21-22. High-level amplifications affected 8q13-24, 1q and 1q21-22, each once. Southern blot analysis of the specimen with high-level amplification at 1q21-22 showed an amplification of FLG and SPRR3, both mapped to this region. All cases with a gain of chromosome 12 simultaneously showed a gain of chromosome 8. Comparison of CGH findings with cytogenetic analysis of the same tumours and previous cytogenetic reports of ET showed, in general, concordant results. In conclusion, our findings confirm that secondary changes, which may have prognostic significance in ET, are trisomy 8, trisomy 12 and a gain of DNA sequences in 1q.

DNA copy number changes were investigated in 29 leiomyosarcomas by comparative genomic hybridization. The most frequent losses were detected in 10q (20 cases, 69%) and 13q (17 cases, 59%). The most frequent gains were detected in 17p (16 cases, 55%). The most frequent high-level amplifications were detected in 17p (7 cases, 24%) and 8q (6 cases, 21%). A total of 137 losses and 204 gains were detected. Small tumors (less than 5 cm in diameter) displayed fewer changes per sample (3 to 11; mean, 7) than the other tumors (4 to 22; mean, 13). There was an increase in the number of gains from small tumors (mean, 4) to very large tumors (>20 cm; mean, 10). However, the number of losses was similar in small, large, and very large tumors (mean, 4.5). Tumor size-related aberrations were observed. Gains in 16p were detected in all small tumors but were infrequent in large and very large tumors (27% and 11%, respectively). Similarly, gains and high-level amplifications in 17p were more common in small (80%) than in very large tumors (33%). Gains in 1q, 5p, 6q, and 8q were not seen in any of the small tumors but were detected in large and very large tumors. Gains in 6q and 8q occurred in 8 of 9 cases (89%) of very large tumors, 5 of them with a high-level amplification in 8q.

The differential diagnosis of mesothelioma, primary adenocarcinomas and pleural metastases frequently causes problems. We have used the comparative genomic hybridization (CGH) technique on 34 malignant mesotheliomas and 30 primary lung carcinomas (adenocarcinoma, including bronchoalveolar carcinoma and large-cell anaplastic carcinoma) to compare their copy number changes and to evaluate the use of CGH to distinguish between these two types of tumour. In mesothelioma, gains of genetic material occurred as frequently as losses, whereas gains predominated over losses in carcinoma. In mesothelioma, the most frequent changes were losses in 4q, 6q and 14q and gains in 15q and 7p, whereas gains in 8q, 1q, 7p, 5p and 6p were the most common changes in carcinoma. Amplification of KRAS2 was detected in two adenocarcinomas by Southern blot analysis. CGH showed gains in 12p in the same tumours. Statistically significant differences between the two types of tumour were detected in chromosomes X, 1, 2p, 4, 8q, 10q, 12p, 14q, 15q and 18q. When comparing the frequency of gains and losses between mesothelioma and lung carcinoma using discriminant analysis, the sensitivity of CGH to differentiate mesotheliomas from lung carcinomas was 81% and the specificity 77%. The differences in DNA copy number changes between the two types of tumour suggest that they are genetically different tumour entities. Although CGH cannot be used as a definitive discriminatory method, we were able to distinguish between mesothelioma and lung carcinoma in a large proportion of the abnormal cases.

We studied DNA copy number changes in gastric cancer (GC) using comparative genomic hybridization (CGH) analysis on 35 resected gastric carcinomas (22 of the intestinal type and 13 of the diffuse type). Eighty-three percent of the cases showed DNA copy number changes. Gains were more common than losses (median of 3 and 1 in primary tumors of the intestinal and diffuse type, respectively). The most common gains were detected on 20q [46%; 12 intestinal type (55%) and four diffuse type (31%)], 8q [37%; 10 intestinal type (45%) and three diffuse type (23%)], and 17q12-21 [29%; all but one intestinal type (41%)]. The most frequent losses were detected on 18q [26%; all intestinal type (41%)] and on 4q [23%; all intestinal type (32%)]. High-level amplifications were observed in the intestinal type of tumors at 17q12-21 (three tumors), 20q (three tumors). 2q (one tumor), and 18q (one tumor). In the diffuse type, high-level amplification was detected once at 13q.

Chromosome analysis was performed on 304 samples of 249 consecutive patients examined for a possible bone tumor. The series consisted of 86 nonneoplastic disorders, 108 benign and 78 malignant primary bone tumors, and 32 other bone malignancies. In the group of nonneoplastic disorders, one sample from an infectious lesion demonstrated a clonal chromosome aberration, i.e., additional material in the short arm of chromosome 1. Simple clonal aberrations were noted in six of 75 successfully cultured benign tumors, e.g., a chondromyxoid fibroma with an insertion type translocation from 2p21p25 to 5q13 and 2p deletion and a nonossifying fibroma with del(4)(p14). Complex clonal aberrations were evident in 21 of 54 successfully cultured malignant primary bone tumors and eight of 21 secondary bone malignancies. The complexity of clonal aberrations correlated with the grade of malignancy as the osteosarcomas and chondrosarcomas of high-grade demonstrated chaotic abnormalities. Six Ewing's sarcomas demonstrated the t(11;22)(q24;q12); in one this was the sole abnormality, and in five additional changes were evident: der(1;16)(q10;p10) in one. Homogeneously staining elongated areas interpreted as HSR were observed in three patients, all of whom had a highly malignant tumor. The most frequent nonclonal abnormality was telomeric association, which was observed mainly in giant cell tumors.

Seven parosteal osteosarcoma (POS) samples, six of which were cytogenetically characterized, were studied by using comparative genomic hybridization (CGH). All samples showed DNA sequence copy number changes (mean, six aberrations/tumor; range, 1–13); gains were more frequent than losses. Gain of 12q13–15 sequences was found in every tumor and correlated with the presence of ring chromosomes. High-level amplification, which was detected in four tumors, was seen only in chromosome 12, with 12q13–14 as the minimal common region. By using chromosome painting, one of the rings of one case was shown to be composed entirely of chromosome 12 material. Together with previous data, our findings show that gain of 12q13–15 sequences is a characteristic feature of POS and that these sequences are contained within the ring chromosomes. Genes Chromosom Cancer 16:31–34 (1996). © 1996 Wiley-Liss, Inc.

Cytogenetic findings of our 30 previously reported and eight new patients with malignant pleural mesothelioma were summarised and correlated with asbestos fibre burden in lung tissue and survival. Successful cytogenetic analyses were performed on cells obtained from the tumours and/or pleural effusions of 34 of the 38 patients. Clonal chromosomal abnormalities were detected in 25 patients, 19 of them studied before treatment. Nine patients, seven of them studied before treatment, had normal karyotypes and/or non-clonal chromosomal abnormalities. Most of the karyotypic findings in the patients with clonal abnormalities were complex and heterogeneous, and no chromosome aberration specific to mesothelioma could be demonstrated. The following numerical abnormalities in decreasing order of frequency were preferentially present in karyotypic changes: -22, +7, -1, -3, -9, +11 and -14 (-/+ denoting partial or total loss or gain). Translocations and deletions involving a breakpoint at 1p11-p22 were the most frequent structural aberrations. Statistically significant correlations were found between high content of asbestos fibres in lung tissue and partial or total losses of chromosomes 1 and 4, and a breakpoint at 1p11-p22 (P = 0.0001, P = 0.003, P = 0.009, respectively). The number of copies of chromosome 7 short arms was inversely correlated with survival (P = 0.02). In this study no diagnostic cytogenetic markers of mesothelioma were found, instead the copy number of chromosome 7 short arms turned out to be a possible prognostic factor in malignant mesothelioma.

Although greater than 50% of Ewing tumours contain non-random cytogenetic aberrations in addition to the pathognomonic 22q12 rearrangements, little is known about their prognostic significance. To address this question, tumour samples from 134 Ewing tumour patients were analysed using a combination of classical cytogenetics, comparative genomic and fluorescence in situ hybridisation. The evaluation of the compiled data revealed that gain of chromosome 8 occurred in 52% of Ewing tumours but was not a predictive factor for outcome. Gain of 1q was associated with adverse overall survival and event-free survival in all patients, irrespective of whether the tumour was localised or disseminated (overall survival: P=0.002 and P=0.029; event-free survival: P=0.018 and P=0.010). Loss of 16q was a significant predictive factor for adverse overall survival in all patients (P=0.008) and was associated with disseminated disease at diagnosis (P=0.039). Gain of chromosome 12 was associated with adverse event-free survival (P=0.009) in patients with localised disease. These results indicate that in addition to a 22q12 rearrangement confirmation in Ewing tumours it is important to assess the copy number of 1q and 16q to identify patients with a higher probability of adverse outcome.

The impact of cytogenetic characterization based on chromosome banding analyses and fluorescence in situ hybridization on clinical decision making has increased dramatically during recent years. Therefore, laboratory techniques have to be optimized to provide reliable results for optimal patient care. In addition, quick and correct results save time and money by preventing unnecessary additional diagnostics and suboptimal treatment approaches. It was our aim to present proposals for standardized protocols to improve the diagnosis, and hence the treatment outcome, of hematologic malignancies.

Asbestos has been shown to cause chromosomal damage and DNA aberrations. Exposure to asbestos causes many lung diseases e.g. asbestosis, malignant mesothelioma, and lung cancer, but the disease-related processes are still largely unknown. We exposed the human cell lines A549, Beas-2B and Met5A to crocidolite asbestos and determined time-dependent gene expression profiles by using Affymetrix arrays. The hybridization data was analyzed by using an algorithm specifically designed for clustering of short time series expression data. A canonical correlation analysis was applied to identify correlations between the cell lines, and a Gene Ontology analysis method for the identification of enriched, differentially expressed biological processes.We recognized a large number of previously known as well as new potential asbestos-associated genes and biological processes, and identified chromosomal regions enriched with genes potentially contributing to common responses to asbestos in these cell lines. These include genes such as the thioredoxin domain containing gene (TXNDC) and the potential tumor suppressor, BCL2/adenovirus E1B 19kD-interacting protein gene (BNIP3L), GO-terms such as "positive regulation of I-kappaB kinase/NF-kappaB cascade" and "positive regulation of transcription, DNA-dependent", and chromosomal regions such as 2p22, 9p13, and 14q21. We present the complete data sets as Additional files.This study identifies several interesting targets for further investigation in relation to asbestos-associated diseases.

Deletion in chromosome 9p involving the CDKN2A locus (9p21.3) is known in many malignancies. To detect this deletion in adolescent ALL patients we used oligo array CGH and studied 54 patients aged 10-25 years. Deletion rate was 25/54 (46%), of these 19/25 (76%) were homozygous. Small deletions (<200 kb) were found in 8/25 (32%) and the smallest deletion was <30 kb. The only gene affected in all deletions was CDKN2A. We were unable to demonstrate prognostic value of the deletion, however patients with deletion belonged more often (P=0.06) to unfavorable biological category. Our results indicate that CDKN2A deletions <200 kb may not be detected by conventional methods.

Ewing sarcoma family of tumors (ESFT), characterized by t(11;22)(q24;q12), is one of the most common tumors of bone in children and young adults. In addition to EWS/FLI1 gene fusion, copy number changes are known to be significant for the underlying neoplastic development of ESFT and for patient outcome. Our genome-wide high-resolution analysis aspired to pinpoint genomic regions of highest interest and possible target genes in these areas.Array comparative genomic hybridization (CGH) and expression arrays were used to screen for copy number alterations and expression changes in ESFT patient samples. A total of 31 ESFT samples were analyzed by aCGH and in 16 patients DNA and RNA level data, created by expression arrays, was integrated. Time of the follow-up of these patients was 5-192 months. Clinical outcome was statistically evaluated by Kaplan-Meier/Logrank methods and RT-PCR was applied on 42 patient samples to study the gene of the highest interest.Copy number changes were detected in 87% of the cases. The most recurrent copy number changes were gains at 1q, 2, 8, and 12, and losses at 9p and 16q. Cumulative event free survival (ESFT) and overall survival (OS) were significantly better (P < 0.05) for primary tumors with three or less copy number changes than for tumors with higher number of copy number aberrations. In three samples copy number imbalances were detected in chromosomes 11 and 22 affecting the FLI1 and EWSR1 loci, suggesting that an unbalanced t(11;22) and subsequent duplication of the derivative chromosome harboring fusion gene is a common event in ESFT. Further, amplifications on chromosomes 20 and 22 seen in one patient sample suggest a novel translocation type between EWSR1 and an unidentified fusion partner at 20q. In total 20 novel ESFT associated putative oncogenes and tumor suppressor genes were found in the integration analysis of array CGH and expression data. Quantitative RT-PCR to study the expression levels of the most interesting gene, HDGF, confirmed that its expression was higher than in control samples. However, no association between HDGF expression and patient survival was observed.We conclude that array CGH and integration analysis proved to be effective methods to identify chromosome regions and novel target genes involved in the tumorigenesis of ESFT.

Background. Unlike in most adult-onset cancers, an association between typical paediatric neoplasms and inflammatory triggers is rare. We studied whether immune system-related genes are activated and have prognostic significance in Ewing's sarcoma family of tumors (ESFTs). Method. Data analysis was performed on gene expression profiles of 44 ESFT patients, 11 ESFT cell lines, and 18 normal skeletal muscle samples. Differential expression of 238 inflammation and 299 macrophage-related genes was analysed by t-test, and survival analysis was performed according to gene expression. Results. Inflammatory genes are activated in ESFT patient samples, as 38 of 238 (16%) inflammatory genes were upregulated (P < 0.001) when compared to cell lines. This inflammatory gene activation was characterized by significant enrichment of macrophage-related gene expression with 58 of 299 (19%) of genes upregulated (P < 0.001). High expression of complement component 5 (C5) correlated with better event-free (P = 0.01) and overall survival (P = 0.004) in a dose-dependent manner. C5 and its receptor C5aR1 expression was verified at protein level by immunohistochemistry on an independent ESFT tumour tissue microarray. Conclusion. Immune system-related gene activation is observed in ESFT patient samples, and prognostically significant inflammatory genes (C5, JAK1, and IL8) for ESFT were identified.

The report of cases of lung squamous cell cancers harboring anaplastic lymphoma kinase (ALK) gene rearrangements raises the question whether this histologic subtype should be also evaluated for such molecular predictive test.A consecutive series of 40 lung pure squamous cell carcinomas were analyzed for ALK gene status by fluorescence in situ hybridization. Squamous differentiation was validated using an immunohistochemical panel including n-p63 (p40), cytokeratin (CK) 5/6, sex-determining region Y (SRY)-Box2 (SOX2), thyroid transcription factor 1, CK7, and Napsin-A.Squamous differentiation was confirmed in all tumors as they stained positive for n-p63 and CK5/6 and negative for thyroid transcription factor 1 and Napsin-A. One of 40 cases (2.5%) showed an ALK rearrangement on fluorescence in situ hybridization analysis.ALK translocation may be found in lung pure squamous cell carcinomas. Our data suggest the opportunity to test ALK rearrangements on biopsy samples harboring squamous cell cancer differentiation.

Comparative genomic hybridization (CGH) analyses were performed on 27 human pleural mesothelioma tumour specimens, consisting of 18 frozen tumours and nine paraffin-embedded tumours, to screen for gains and losses of DNA sequences. Copy number changes were detected in 15 of the 27 specimens with a range from one to eight per specimen. On average, more losses than gains of genetic material were observed. The loss of DNA sequences occurred most commonly in the short arm of chromosome 9 (p21-pter), in 60% of the abnormal specimens. Other losses among the abnormal specimens were frequently detected in the long arms of chromosomes 4 (q31.1-qter, 20%), 6 (q22-q24, 33%), 13 (33%),14 (q24-qter, 33%) and 22 (q13, 20%). A gain in DNA sequences was found in the long arm of chromosome 1 (cen-qter) in 33% of the abnormal specimens. Our analysis is the first genome-wide screening for gains and losses of DNA sequences using comparative genomic hybridization in malignant pleural mesothelioma tumours. The recurrent DNA sequence changes detected in this study suggest that the corresponding chromosomal areas most probably contain genes important for the initiation and progression of mesothelioma.

Our aim was to identify chromosomal regions that are likely to harbor previously unknown genes with an important role in the genesis of osteosarcoma. Comparative genomic hybridization was used to screen for losses and gains of DNA sequences along all chromosome arms in 11 tumors. Extensive genetic aberrations, with an average of 11 changes/tumor (range, 1-20), were found in 10 of the 11 specimens. High level amplifications of small chromosomal regions were detected in eight tumors. These involved the 12q12-q13 region (known to contain the SAS-MDM2 locus) and several previously unreported amplification sites such as 17p11-p12, 3q26, and Xq12. When all DNA sequence gains were evaluated, the gains at 8q and Xp were most common (45%). The most common losses of DNA sequences were seen at 2q, 6q, 8p, and 10p (36%). In conclusion, despite the very complex pattern of genetic changes in osteosarcomas, certain chromosomal regions appear to be affected more often than others. Most of these regions have not previously been reported to be implicated in osteosarcomas and may thus highlight locations of novel genes with an important role in the development and progression of these tumors.

DNA copy number changes were characterized by comparative genomic hybridization (CGH) in 18 breast cancer cell lines. In 5 of these, the results were comparable with those from the primary tumors of which the cell lines were established. All of the cell lines showed extensive DNA copy number changes, with a mean of 16.3 +/- 1.1 aberrations per sample (range 7-26). All of the cell lines had a gain at 8q22-qter. Other common gains of DNA sequences occurred at 1q31-32 (89%), 20q12-q13.2 (83%), 8q13 (72%), 3q26.1-qter (67%), 17q21-qter (67%) 5p14 (61%), 6p22 (56%), and 22pter-qter (50%). High-level amplifications were observed in all cell lines; the most frequent minimal common regions were 8q24.1 (89%), 20q12 (61%), 1q41 (39%), and 20p11.2 (28%). Losses were observed less frequently than gains and the minimal common regions of the most frequent losses were Xq11-q12 (56%), Xp11.2-pter (50%), 13q21 (50%), 8p12-pter (44%), 4p13-p14 (39%), 6q15-q22 (39%), and 18q11.2-qter (33%). Although the cell lines showed more DNA copy number changes than the primary tumors, all aberrations, except one found in a primary tumor, were always present in the corresponding cell line. High-level amplifications found both in primary tumors and cell lines were at 1q, 8q, 17q, and 20q. The DNA copy number changes detected in these cell lines can be valuable in investigation of tumor progression in vitro and for a more detailed mapping and isolation of genes implicated in breast cancer.

Abstract Several chromosomal regions are recurrently amplified or deleted in lung tumors, but little is known about the underlying genes, which could be important mediators in tumor formation or progression. In lung cancer, the RB1–CCND1–CDKN2A pathway, involved in the G1–S transition, is damaged in nearly all tumors. In the present study, we localized a novel amplicon in lung tumors to a fragment of less than 0.5 Mb at 12q13.3–q14.1 by using comparative genomic hybridization (CGH) on cDNA microarrays. This approach enabled us to identify 10–15 genes with the most consistent amplifications. Semiquantitative RT‐PCR analyses of 13 genes in this region showed that four of them ( CDK4 , CYP27B1 , METTL1 , and TSFM ) were also highly up‐regulated. Immunohistochemical (IHC) analysis of 141 tumor samples on a tissue microarray showed that CDK4 was expressed at a high level in 23% of lung tumors. Six (21.4%) of the tumors with high CDK4 expression ( n = 28) were shown by fluorescence in situ hybridization (FISH) to contain the 12q13.3–q14.1 amplification. For CDK4 , a positive correlation was found between gene copy number (FISH and CGH array), mRNA expression (RT‐PCR), and level of protein expression (IHC). CDK4 expression did not correlate with CDKN2A methylation status. Amplification of CDK4 has been described in other tumor types, but its role in lung cancer remains to be elucidated. Although CDK4 amplification seems to be a relatively rare event (4.3%) in lung tumors, it indicates the significance of the RB1–CCND1 pathway in lung tumorigenesis. © 2004 Wiley‐Liss, Inc.

DNA copy number losses at chromosome arm 14q are the most frequently occurring aberrations in gastrointestinal stromal tumors (GISTs). To characterize the deletion at 14q, we performed comparative genomic hybridization (CGH) and high-resolution deletion mapping using a panel of 32 polymorphic microsatellite markers in 30 GISTs. The GISTs were classified according to their metastatic potential and mitotic counts into 15 low-risk and 15 high-risk tumors. Losses with a minimal common overlapping region at 14q12-q24 were detected by CGH in 16 tumors (53) (nine low-risk and seven high-risk). Investigation with microsatellite markers was informative in 690 analyses (72%). Loss of heterozygosity (LOH) with at least one marker was detected in 279 analyses in 24 tumors (80%). Deletions were equally frequent in low-risk and high-risk GISTs. Two common deletion regions were identified at 14q11.1-q12 and 14q23-q24.3. The highest frequencies of deletions were seen in regions corresponding to markers D14S283 (20/28, 71%) at 14q11.1-q12 and D14S258 (17/27, 63%) at 14q23-q24, suggesting that these are two tumor suppressor loci.

Our previous comparative genomic hybridization (CGH) study of Ewing sarcoma and related tumors showed that DNA sequence copy number increases of 1q21-q22 and of chromosomes 8 and 12 were associated with trends toward poor survival (Armengol et al., Br J Cancer 1997, 75, 1403-1409). These trends were not statistically significant. In the present study, we analyzed 28 primary Ewing sarcomas and related tumors by CGH to study whether these (or other) changes have prognostic value in these tumors. Twenty-one tumors (75%) had changes with a mean of 1.9 changes per tumor. The most frequent aberration was gain of chromosome 8 in 10 tumors (36%). Five tumors (18%) had copy number increases at 1q21-22 and 5 had gain of 7q. Copy number increase of 6p21.1-pter, gain of chromosome 12, and loss of 16q were seen in 11%. Copy number increases of 1q21-q22 and of chromosomes 8 and 12 were associated with trends toward worse outcome, but the differences did not reach statistical significance. A novel finding is the association of copy number increase at 6p with worse distant disease-free (P = 0.04) and overall survival (P = 0.004). To confirm this finding and to see whether copy number increases of 1q21-q22 and of chromosomes 8 and 12 have definite prognostic value, a larger number of cases needs to be studied.

Chronological aging of women is clearly associated with an increase in both X-chromosome loss and micronuclei formation in peripheral lymphocytes. It has been suggested that micronucleus formation is an important mechanism of chromosome loss. In the present study, fluorescence in situ hybridization was used to study micronuclei content in two age groups (women below 30 and above 50 years old). A probe for centromeric alphoid consensus sequences (SO-αAllCen) and a cloned X-specific centromeric probe (pXBR) were separately used to detect the presence of any chromosomes and the X chromosome, respectively. The presence of centromere-positive micronuclei was significantly higher among the older donors (51.5%) than among the younger donors (34.3%). The X chromosome was highly overrepresented in the micronuclei, the older women showing a higher proportion of X-positive micronuclei (24.0%) than the younger women (14.0%). Assuming that the rest of the centromere-positive micronuclei contained autosomes, a significant age-dependent difference was also noted for micronuclei harboring autosomes (27.5 % among the older women and 20.3 % among the younger women). These findings suggest that both the X chromosome and autosomes are responsible for the age-dependent increase of micronuclei in women’s peripheral lymphocytes.

Genomic copy number changes are frequently found in cancers and they have been demonstrated to contribute to carcinogenesis; and it is widely accepted that tumors with microsatellite instability (MSI) are genetically stable and mostly diploid. In the present study we compared the copy number alterations and the gene-expression profiles of microsatellite stable (MSS) and MSI colorectal tumors. A total number of 31 fresh-frozen primary tumors (16 MSS and 15 MSI) were used. Twenty-eight samples (15 MSS and 13 MSI) were analyzed with metaphase comparative genomic hybridization (CGH), nine of which plus one additional sample (4 MSS and 6 MSI) were further analyzed by cDNA-based array-CGH. Gene expression analysis was performed with six samples [3 MSS and 3 MSI, four of these used in metaphase CGH (mCGH) analysis] to identify differentially expressed genes possibly located in the lost or amplified regions found by CGH, stressing the biological significance of copy number changes. Metaphase and array-CGH analysis of two colon cancer cell lines (HTC116 and SW480, reported as MSI and MSS archetypes) gave comparable results. Alterations found by mCGH in MSS tumors were +20, +8q, -8p and -18q. Interestingly, 1p22, 4q26 and 15q21 were found deleted preferentially in MSS tumors, while 22q13 was found gained in MSI tumors. The regions of alterations identified by array-CGH were gains at 8q24, 16q24.3 and 20q13, and the loss of 5q21, appearing in the both types of tumors. Gene expression analysis revealed genes with specific associations with the copy number changes of the corresponding genomic regions. As a conclusion, colorectal cancer is a heterogeneous disease, demonstrated by the genomic profiles of individual samples. However, our data shows that copy number changes do not occur exclusively in the MSS phenotypes.

Conventional cytogenetic analyses and comparative genomic hybridization have revealed a complex and even chaotic nature of chromosomal aberrations in pleural malignant mesothelioma (MM). We set out to describe the complex gene copy number changes and screen for novel genetic aberrations using a high-density oligonucleotide microarray platform for comparative genomic hybridization (aCGH) of a series of 26 well-characterized MM tumor samples. The number of copy number changes varied from zero to 40 per sample. Gene copy number losses predominated over gains, and the most frequent region of loss was 9p21.3 (17/26 cases), the locus of &lt;i&gt;CDKN2A&lt;/i&gt; and &lt;i&gt;CDKN2B&lt;/i&gt;, both known to be commonly lost in MM. The most recurrent minimal regions of losses were 1p31.1→ p13.2, 3p22.1→p14.2, 6q22.1, 9p21.3, 13cen→q14.12, 14q22.1→qter, and 22qcen→q12.3. Previously unreported gains included 9p13.3, 7p22.3→p22.2, 12q13.3, and 17q21.32→qter. The results suggest that gene copy number losses are a major mechanism of MM carcinogenesis and reveal a recurrent pattern of copy number changes in MM.

Olfactory neuroblastoma is an unusual neuroectodermal malignancy, which is thought to arise at the olfactory membrane of the sinonasal tract. Due to its rarity, little is understood regarding its molecular and cytogenetic abnormalities. The aim of the current study is to identify specific DNA copy number changes in olfactory neuroblastoma. Thirteen dissected tissue samples were analyzed using array comparative genomic hybridization. Our results show that gene copy number profiles of olfactory neuroblastoma samples are complex. The most frequent changes included gains at 7q11.22-q21.11, 9p13.3, 13q, 20p/q, and Xp/q, and losses at 2q31.1, 2q33.3, 2q37.1, 6q16.3, 6q21.33, 6q22.1, 22q11.23, 22q12.1, and Xp/q. Gains were more frequent than losses, and high-stage tumors showed more alterations than low-stage olfactory neuroblastoma. Frequent changes in high-stage tumors were gains at 13q14.2-q14.3, 13q31.1, and 20q11.21-q11.23, and loss of Xp21.1 (in 66% of cases). Gains at 5q35, 13q, and 20q, and losses at 2q31.1, 2q33.3, and 6q16-q22, were present in 50% of cases. The identified regions of gene copy number change have been implicated in a variety of tumors, especially carcinomas. In addition, our results indicate that gains in 20q and 13q may be important in the progression of this cancer, and that these regions possibly harbor genes with functional relevance in olfactory neuroblastoma.

DNA copy number amplifications activate oncogenes and are found in the majority of advanced solid tumors. Cell-lineage specificity and oncogene affinity of DNA amplifications in cancer suggest that properties of precursor stem cells and selection pressure in the tissue micro-environment determine the genomic location of gene amplifications. Biological specificity and significance of gene amplifications make them potential targets for clinical applications. Here we discuss the specificity of non-randomly occurring DNA copy number amplifications as defining features for cancers, their selection in the tumor tissue, and significance in the clinical practice.

Abstract Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract. The tumors characteristically harbor KIT or PDGFRA mutations, and mutant tumors respond to imatinib mesylate (Glivec™). Chromosomal imbalances resulting in altered gene dosage are known to have a role in the molecular pathogenesis of these tumors, but the target genes remain to be identified. The present study aimed to identify some of these genes. In total, 35 GIST samples were screened for chromosomal imbalances by array‐based comparative genomic hybridization. A cDNA array was used to define the minimal common overlapping areas of DNA copy number change. Eight confirmative, replicate hybridizations were performed using an oligonucleotide array. The most recurrent copy number losses were localized to 14q, 22q, and 1p. Gains were less common with 8q being the most recurrent. Two recurrent deleted regions of 14q were 14q11.2 harboring the PARP2, APEX1 , and NDRG2 genes and 14q32.33 harboring SIVA . Additional target candidates were NF2 at chromosome 22, CDKN2A/2B at 9p, and ENO1 at 1p for copy number losses, and MYC at 8q for copy number gains. Array CGH proved to be an effective tool for the identification of chromosome regions involved in the development and progression of GISTs. © 2007 Wiley‐Liss, Inc.

The rare and highly aggressive adult soft tissue sarcomas leiomyosarcoma (LMS) and undifferentiated pleomorphic sarcoma (UPS) contain complex genomes characterized by a multitude of rearrangements, amplifications, and deletions. Differential diagnosis remains a challenge. MicroRNA (miRNA) profiling was conducted on a series of LMS and UPS samples to initially investigate the differential expression and to identify specific signatures useful for improving the differential diagnosis. Initially, 10 high‐grade LMS and 10 high‐grade UPS were profiled with a miRNA microarray. Two cultured human mesenchymal stem cell samples were used as controls. 38 and 46 miRNAs classified UPS and LMS samples, respectively, into separate groups compared to control samples. When comparing the two profiles, miR‐199b‐5p, miR‐320a, miR‐199a‐3p, miR‐126, miR‐22 were differentially expressed. These were validated by RT‐PCR on a further series of 27 UPS and 21 LMS for a total of 68 cases. The levels of miR‐199‐5p and miR‐320a, in particular, confirmed the microarray data, the former highly expressed in UPS and the latter in LMS. Immunohistochemistry was performed on all 68 cases to confirm original diagnosis. Recently reported LMS‐ and UPS‐associated genes were correlated with miRNA targets based on target algorithms of three databases. Several genes including IMP3 , ROR2 , MDM2 , CDK4 , and UPA , are targets of differentially expressed miRNAs. We identified miRNA expression patterns in LMS and UPS, linking them to chromosomal regions and mRNA targets known to be involved in tumor development/progression of LMS and UPS. © 2014 Wiley Periodicals, Inc.

Asbestos is a carcinogen linked to malignant mesothelioma (MM) and lung cancer. Some gene aberrations related to asbestos exposure are recognized, but many associated mutations remain obscure. We performed exome sequencing to determine the association of previously known mutations (driver gene mutations) with asbestos and to identify novel mutations related to asbestos exposure in lung adenocarcinoma (LAC) and MM. Exome sequencing was performed on DNA from 47 tumor tissues of MM (21) and LAC (26) patients, 27 of whom had been asbestos-exposed (18 MM, 9 LAC). In addition, 9 normal lung/blood samples of LAC were sequenced. Novel mutations identified from exome data were validated by amplicon-based deep sequencing. Driver gene mutations in BRAF, EGFR, ERBB2, HRAS, KRAS, MET, NRAS, PIK3CA, STK11, and ephrin receptor genes (EPHA1-8, 10 and EPHB1-4, 6) were studied for both LAC and MM, and in BAP1, CUL1, CDKN2A, and NF2 for MM. In asbestos-exposed MM patients, previously non-described NF2 frameshift mutation (one) and BAP1 mutations (four) were detected. Exome data mining revealed some genes potentially associated with asbestos exposure, such as MRPL1 and SDK1. BAP1 and COPG1 mutations were seen exclusively in MM. Pathogenic KRAS mutations were common in LAC patients (42 %), both in non-exposed (n = 5) and exposed patients (n = 6). Pathogenic BRAF mutations were found in two LACs. BAP1 mutations occurred in asbestos-exposed MM. MRPL1, SDK1, SEMA5B, and INPP4A could possibly serve as candidate genes for alterations associated with asbestos exposure. KRAS mutations in LAC were not associated with asbestos exposure.

Twenty lipomatous tumors, including eight lipoma-like liposarcomas and 12 benign lipomas, were analyzed using comparative genomic hybridization (CGH). DNA sequence copy number changes detected in five lipoma-like liposarcomas (mean, 1.1 aberrations/tumor; range, 0-2) consisted of gains of 12q13-21 (five tumors) and 1q21-23 (four tumors). Two of the tumors showed high-level amplification at 12q14-21 and one tumor at 1q21-22. No copy number changes were found in lipomas. Overrepresentation of 1q and 12q sequences was a recurrent finding in lipoma-like liposarcomas but not in lipomas. Thus, CGH may help in the differential diagnosis of low-grade or borderline adipose neoplasms.

We investigated the frequency of bcl-2 protein overexpression in 80 diffuse large B-cell lymphoma (DLBCL) patients using both Western blotting and immunohistochemistry (IHC). Fifty-nine percent of the DLBCLs overexpressed bcl-2 protein by Western blot and 52% by IHC. The two methods usually gave concordant results (p = 0.005), but 14 (21%) out of the 67 cases that were analyzed by both methods were positive by Western blot and negative by IHC, and 8 (12%) cases vice versa. Bcl-2 overexpression by IHC was associated with poor response to chemotherapy and poor survival, whereas these associations were not found when bcl-2 overexpression was determined by Western blotting. The molecular mechanisms leading to bcl-2 overexpression were evaluated by PCR, karyotype analysis, and comparative genomic hybridization (CGH). When studied by PCR and/or karyotype analysis, 12 (15%) of the 80 cases had translocation (14;18)(q32;q21). All 12 lymphomas with (14;18)(q32;q21) translocation had bcl-2 overexpression by Western blot as compared with 35 (51%) of the 68 lymphomas without translocation (p = 0.001). Ten (29%) out of 34 cases that were analyzed by CGH showed amplification of chromosome 18 in which the BCL2 gene is located, and all cases showed bcl-2 overexpression by both Western blot and IHC. The results suggest that gene amplification and translocation are at least equally common mechanisms causing bcl-2 protein overexpression in DLBCL. Bcl-2 protein overexpression as determined by IHC is associated with poor response to chemotherapy and poor survival.

The genetic changes underlying the development of resistance to the platinum-containing drugs are poorly defined. We analyzed six resistant cell lines using comparative genomic hybridization (CGH) in order to screen and identify possible genetic changes in common. We compared parental 2008 and A2780 human ovarian cancer cells to sublines selected for resistance to cisplatin (DDP) (2008/C8, 2008/C13*5.25, 2008/A, A2780/CP); we also compared 2008 cells to sublines selected for resistance to antimonite (2008/H) and arsenite (2008/I) which demonstrated cross-resistance to DDP. DNA samples from the resistant cell lines were hybridized against DNA from the parental cells rather than from normal human cells to permit detection of only those changes associated with the development of resistance. The DNA sequence copy number changes were surprisingly numerous in the DDP, antimony, and arsenite-resistant variants of the 2008 cell line and most of the chromosomes were affected. On the other hand, in the A2780/CP subline only a few changes were found and these were limited to just four chromosomes. The most common findings among the DDP-resistant cell lines were gains of material from chromosomes or chromosome arms 2q (5 out of 6 lines), 4 (4/6), 6q (5/6), and 8q (4/6). Deletions were observed on chromosomes or chromosome arms 2p (4/6), X (4/6), 7p (5/6), 11p (4/6), and 13 (4/6). The most frequently involved chromosomal regions, affected in the majority of cell lines, were: gain of 2q14.1-q33, 4p15.2-p13, 4q22-q25, 4q31.1-q34, 6q13-q16, 8q12-q21.2, and loss of Xp22.2-q21, 7p21-p14, 11cen-p14 in sublines of 2008, and loss of 2pter-p22 and 13q21 in sublines of 2008 and A2780. The results suggest that the acquired resistance and cross-resistance to DDP in these cell lines was associated with substantial genomic instability, quite unlike the changes observed in association with the development of resistance to drugs participating in the multidrug resistance phenotype.

Gene amplifications are essential features of advanced cancers and have prognostic as well as therapeutic significance in clinical cancer treatment. Models explaining the amplification process, such as breakage–fusion–bridge cycle and excision and unequal segregation of extrachromosomal DNA fragments, predict that independent DNA double-stranded breaks must occur to induce amplification formation. Many cellular, tissue and environmental factors induce DNA damage and amplifications. Also labile DNA sequence features like fragile sites facilitate amplifications. Although, databases and data mining tools of various genomic attributes are already available, extra-large scale systems biology endeavors to decipher dynamics, interactions and dependencies between different factors contributing to amplification process fail, because current databases of DNA copy number aberrations and fragile sites comprise conventional cytogenetics results obtained at far too coarse chromosome band resolution. Array comparative genomic hybridization (aCGH) enables genome-wide gene copy number measurements and amplification detection at molecular genetic resolution. Similarly, cloning and sequencing of fragile sites produce mapping information of vastly improved resolution. In conclusion, databases of aCGH and sequenced fragile sites are needed to resolve the mechanisms of gene amplifications in systems biology configuration.

DNA copy number amplification at the chromosomal region of 17q is frequent in gastric cancer. Recently 17q21 was identified as the critical region for the amplicon formation because this region harbors the ERBB2 oncogene and several other targets, such as TOP2A and DARPP32. In our study, we characterized the amplification (52 cases) and expression (29 cases) levels of ERBB2, TOP2A and DARPP32 in gastric cancer samples. These 3 genes were concomitantly amplified in 17% of the intestinal type of gastric adenocarcinoma. However, the expression levels were independent, showing overexpression of DARPP32 (48%), TOP2A (17%) and ERBB2 (3%) studied by quantitative real-time PCR. The most frequently overexpressed gene, DARPP32, exhibited strong protein overexpression in 45% (30/66) of the cases in immunohistochemical study of gastric cancer tumor tissue array. Additional studies are required to thoroughly understand the biological significance of these genes in gastric cancer.

Abstract Asbestos is a well-known lung cancer-causing mineral fiber. In vitro and in vivo experiments have shown that asbestos can cause chromosomal damage and aberrations. Lung tumors, in general, have several recurrently amplified and deleted chromosomal regions. To investigate whether a distinct chromosomal aberration profile could be detected in the lung tumors of heavily asbestos-exposed patients, we analyzed the copy number profiles of 14 lung tumors from highly asbestos-exposed patients and 14 matched tumors from nonexposed patients using classic comparative genomic hybridization (CGH). A specific profile could lead to identification of the underlying genes that may act as mediators of tumor formation and progression. In addition, array CGH analyses on cDNA microarrays (13,000 clones) were carried out on 20 of the same patients. Classic CGH showed, on average, more aberrations in asbestos-exposed than in nonexposed patients, and an altered region in chromosome 2 seemed to occur more frequently in the asbestos-exposed patients. Array CGH revealed aberrations in 18 regions that were significantly associated with either of the two groups. The most significant regions were 2p21-p16.3, 5q35.3, 9q33.3-q34.11, 9q34.13-q34.3, 11p15.5, 14q11.2, and 19p13.1-p13.3 (P &amp;lt; 0.005). Furthermore, 11 fragile sites coincided with the 18 asbestos-associated regions (P = 0.08), which may imply preferentially caused DNA damage at these sites. Our findings are the first evidence, indicating that asbestos exposure may produce a specific DNA damage profile. (Cancer Res 2006; 66(11): 5737-43)

Preeclampsia is a common, pregnancy-specific vascular disorder characterised by hypertension and proteinuria. A recent report suggested association of the STOX1 gene on chromosome 10q22.1 with preeclampsia in the Dutch population. Here, we present a comprehensive assessment of STOX1 as a candidate gene for preeclampsia in the Finnish population by re-examining our previous genetic linkage analysis results for both chromosome 10 and paralogous loci, by genotyping representative markers in a nationwide data set, and by studying STOX1 expression in placentas from preeclamptic and uncomplicated pregnancies. In conclusion, we are unable to validate STOX1 as a common preeclampsia susceptibility gene.

Kaposi's sarcoma (KS) is a mesenchymal tumor, caused by Human herpesvirus 8 (HHV8) with molecular and cytogenetic changes poorly understood. To gain further insight on the underlying molecular changes in KS, we performed microRNA (miRNA) microarray analysis of 17 Kaposi's sarcoma specimens. Three normal skin specimens were used as controls. The most significant differentially expressed miRNA were confirmed by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). We detected in KS versus normal skin 185 differentially expressed miRNAs, 76 were upregulated and 109 were downregulated. The most significantly downregulated miRNAs were miR-99a, miR-200 family, miR-199b-5p, miR-100 and miR-335, whereas kshv-miR-K12-4-3p, kshv-miR-K12-1, kshv-miR-K12-2, kshv-miR-K12-4-5p and kshv-miR-K12-8 were significantly upregulated. High expression levels of kshv-miR-K12-1 (p = 0.004) and kshv-miR-K12-4-3p (p = 0.001) was confirmed by RT-PCR. The predicted target genes for differentially expressed miRNAs included genes which are involved in a variety of cellular processes such as angiogenesis (i.e. THBS1) and apoptosis (i.e. CASP3, MCL1), suggesting a role for these miRNAs in Kaposi's sarcoma pathogenesis.

Renal cell carcinoma with prominent smooth muscle stroma is a rare neoplasm composed of an admixture of epithelial cell with clear cytoplasm arranged in small nest and tubular structures and a stroma composed of smooth muscle. In the epithelial component, loss of chromosome 3p detected by fluorescence in situ hybridization (FISH) has been reported and on this basis these neoplasms have been viewed as variants of clear cell renal cell carcinoma. To test the validity of this classification, we have evaluated the chromosome 3 and VHL status of three of these tumors using FISH, array comparative genomic hybridization, gene sequencing, and methylation-specific multiplex ligation-dependent probe amplification analysis. None of the tumors showed deletion of chromosome 3p, VHL mutation, a significant VHL methylation, or changes in VHL copy number and all three tumors demonstrated a flat profile in the comparative genomic hybridization analysis. We conclude that renal cell carcinoma with smooth muscle stroma should be considered as an entity distinct from clear cell renal cell carcinoma.

Sequestration of the tumor suppressor retinoblastoma protein (RB) by the Merkel cell polyomavirus (MCV) is a crucial step in the pathogenesis of Merkel cell carcinoma (MCC). RB expression is frequently lost, particularly in MCV-negative MCC tumors, through yet unknown mechanisms. We compared the genomic copy number changes of 13 MCV-positive and 13 -negative MCC tumors by array comparative genomic hybridization. The analysis revealed increased genomic instability, amplification of 1p34.3–1p34.2, and losses of 11p in the absence of MCV infection. Deletions of the RB1 locus were also detected at high rates in MCV-negative tumors. None of the tumors with heterozygous RB1 losses expressed RB in immunohistochemistry. RB1 promoter hypermethylation was studied with a methylation-specific multiplex ligation-dependent probe amplification technique. The RB1 promoter was methylated in all tumor specimens at CpG islands located close to the ATG start codon, albeit at low levels. The pattern of hypermethylation was similar in all MCC samples, despite RB expression, survival or MCV status. In conclusion, the frequent heterozygous losses of the RB1 locus could partly explain the decreased RB expression in MCV-negative MCC tumors, although the effects of RB1 mutations, coinciding promoter hypermethylation and, for example, miRNA regulation, cannot be excluded.

Recurrent and clonal genetic alterations are characteristic of different subtypes of T- and B-cell lymphoblastic leukemia (ALL), and several subtypes are strong independent predictors of clinical outcome. A next-generation sequencing-based multiplex ligation-dependent probe amplification variant (digitalMLPA) has been developed enabling simultaneous detection of copy number alterations (CNAs) of up to 1000 target sequences. This novel digitalMLPA assay was designed and optimized to detect CNAs of 56 key target genes and regions in ALL. A set of digital karyotyping probes has been included for the detection of gross ploidy changes, to determine the extent of CNAs, while also serving as reference probes for data normalization. Sixty-seven ALL patient samples (including B- and T-cell ALL), previously characterized for genetic aberrations by standard MLPA, array comparative genomic hybridization, and/or single-nucleotide polymorphism array, were analyzed single blinded using digitalMLPA. The digitalMLPA assay reliably identified whole chromosome losses and gains (including high hyperdiploidy), whole gene deletions or gains, intrachromosomal amplification of chromosome 21, fusion genes, and intragenic deletions, which were confirmed by other methods. Furthermore, subclonal alterations were reliably detected if present in at least 20% to 30% of neoplastic cells. The diagnostic sensitivity of the digitalMLPA assay was 98.9%, and the specificity was 97.8%. These results merit further consideration of digitalMLPA as a valuable alternative for genetic work-up of newly diagnosed ALL patients.

Clear cell papillary renal cell carcinoma (CCPRCC) is a recently recognised neoplasm with a broad spectrum of morphological characteristics, thus representing a challenging differential diagnosis, especially with the low malignant potential multicystic renal cell neoplasms and clear cell renal cell carcinoma. We selected 14 cases of CCPRCC with a wide spectrum of morphological features diagnosed on morphology and CK7 immunoreactivity and analysed them using a panel of immunohistochemical markers, focusing on 34βE12 and related CKs 1,5,10 and 14 and several molecular analyses such as fluorescence in situ hybridisation (FISH), array comparative genomic hybridisation (aCGH), VHL methylation, VHL and TCEB1 sequencing and multiplex ligation-dependent probe amplification (MLPA). Twelve of 13 (92%) CCPRCC tumours were positive for 34βE12. One tumour without 3p alteration by FISH revealed VHL mutation and 3p deletion at aCGH; thus, it was re-classified as clear cell RCC. We concluded that: (1) immunohistochemical expression of CK7 is necessary for diagnostic purposes, but may not be sufficient to identify CCPRCC, while 34βE12, in part due to the presence of CK14 antigen expression, can be extremely useful for the recognition of this tumour; and (2) further molecular analysis of chromosome 3p should be considered to support of CCPRCC diagnosis, when FISH analysis does not evidence the common loss of chromosome 3p.

Comparative genomic hybridization (CGH) was performed using DNAs pooled from numerous specimens from tumor categories studied case-by-case. The series of six DNA pools consisted of 28 diffuse centroblastic lymphomas (DCL), 28 gastrointestinal stromal tumors (GIST), 21 primary chondrosarcomas (CS), 17 samples from the Ewing family of tumors (ET), 14 liposarcomas (LS), and 14 mesotheliomas (MS). Losses and gains present in at least 50% of the individual specimens were always detected in the pooled DNAs. The loss of the whole p-arm of chromosome 1 was observed even when the affected proportion of individual specimens was only 25%. Gains were also detected at frequencies lower than 50%, but with a high-level amplification in one or more specimens. In conclusion, the present pooled DNA study revealed the following changes: DCL had a gain at 18q22-qter; GIST had losses at 14 and 22q12, and gains at 5p, 8q22-24, 17q22-qter, and 19q13; ET had gains at 1q and 8q13-qter; LS had gains at 1q21-25 and 12q; and MS had a loss at 9p22-pter. No changes were observed in the CS DNA pool. The results from individual specimens also stressed the importance of these chromosomal regions to the tumorigenesis in the corresponding malignancies. This pooled DNA approach can thus be used for fast screening of recurrent DNA copy number in a specific tumor entity.

We analyzed eight samples of xenografted human pancreatic tumors and two metastases developed in mice by comparative genomic hybridization (CGH). The most recurrent changes were: gains on chromosomes 8 (8q24∼qter; 7/8 cases), 15 (15q25∼q26; 6/8 cases), 16 (16p in 6/8 cases; 16q in 5/8 cases), 20 (20q; 6/8 cases), and 19 (19q; 5/8 cases); and losses on chromosomes 18 (18q21; 6/8 cases), 6 (6q16∼q21 and 6q24∼qter; 5/8 cases each), and 9 (9p23∼pter; 5/8 cases). The two metastases maintained the aberrations of the original pancreatic tumor plus gain of 11q12∼q13 and 22q. Loss of heterozygosity analysis was carried out for 10p14∼pter, a region that was lost in 3/8 samples. All of them presented allelic imbalance for all the informative loci. Fluorescence in situ hybridization and Southern analysis were performed to test some candidate oncogenes in 8q24 (MYC) and 15q25∼qter (IGF1R and FES). Two of seven tumors showed high-level amplification of MYC relative to the centromere (>3-fold), another two tumors had low-level amplification (1.5- to 3.0-fold), and one displayed 5.5 MYC signals/cell. In relation to the FES gene, low-level amplification was found in three tumors. Southern analysis showed five cases with a low-level amplification of IGF1R. Our data suggest that either few extra gene copies may be enough for cancer progression or other genes located in these regions are responsible for the amplifications found by CGH.

DNA sequence copy number changes were studied by comparative genomic hybridization (CGH) along all chromosomes in 58 samples of malignant fibrous histiocytoma (MFH). The material consisted of 43 primary tumors (9 of myxoid and 34 of storiform-pleomorphic subtype), 13 local recurrences (2 myxoid and 11 storiform-pleomorphic), and 2 metastases (1 myxoid and 1 storiform-pleomorphic). Genetic aberrations, with a mean of 5.5 changes per sample (range, 0 to 22), were detected in 47 of 58 samples (81%). The minimal common regions of the most frequent gains were 1p31 (33%), 9q31 (29%), 5p14-pter (26%), 7q32 (24%), and 7p15-pter (22%). High-level amplifications were detected in 16 of the 58 samples (28%). High-level amplification of 13q31-qter was seen in four tumors (7%); other high-level amplifications were more sporadic. Losses of DNA sequences were less frequent than gains. The minimal common regions of the most common losses were 13q21 (21%) and 13q22 (21%). Statistically significant correlation was found between gain of 7q32 and the rates of worse metastasis-free survival (P = 0.01) and overall survival (P = 0.004). The gain of 7q32 retained its prognostic significance also in a multivariate analysis with tumor size and grade. Gain of 1p31 was associated with a trend to decreased overall survival. Gains of 5p14-pter and 9q31 and losses of 13q21 and/or 13q22 did not have any prognostic value; neither did the total number of aberrations, total number of gains, or total number of losses per sample.

Well-differentiated liposarcomas (WDLPS), especially those located in the retroperitoneum, may occasionally undergo dedifferentiation. Although this process is associated with a more aggressive clinical course, dedifferentiated liposarcomas rarely produces metastases. The case reported here is rather uncommon: A retroperitoneal WDLPS gave lung metastases that were diagnosed as highly malignant osteosarcomas. We used comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), and Southern blot analyses to characterize the copy number changes and genetic aberrations occurring at different stages of the disease. In the primary tumor, the only detectable aberration was amplification of 12q13–q14, which was present only in a fraction of the cells and revealed by FISH analysis. High-level amplification of 12q13–q14, involving CDK4, MDM2, and HMGIC, was seen both in the relapse and the metastases. The second most common change, gain or high-level amplification of 1q22–q24, was detectable by CGH only in the osteogenic metastases, as was loss of the distal 2q. FISH analyses revealed considerable heterogeneity in the samples, and the percentage of cells showing aberrations was significantly higher in the metastatic samples. In particular, increased copy numbers of 789f2, a marker for 1q21 amplification in sarcomas, was observed in more than 65% of the cells in the metastatic samples, but in less than 10% of the cells from the recurrent samples. These observations could indicate that 1q amplification, in particular, may be indicative of a more malignant phenotype and ability of metastasis in WDLPS, as has also been suggested by others.

Cell proliferation and chromosomal imbalances, important parameters in relation to tumor progression, were studied in ameloblastoma (n=20), a benign odontogenic tumor of locally recurrent nature. Immunocytochemical staining with MIB-1 antibody and comparative genomic hybridization (CGH) were performed on formalin-fixed paraffin-embedded ameloblastomas. The mean follow-up time was 12.4 years. An MIB-1-index was formed by counting 5000 tumor-cell nuclei in 10-15 randomly chosen high-power fields and calculating percentages of positively stained cells. CGH involved hybridization of FITC-dUTP-labeled tumor DNA with Texas-red-labeled normal DNA. Images were digitally analyzed. The MIB-1-index (range 0-2.51) was low for all tumors. No statistically significant correlation between MIB-1 index and tendency to recurrence was found. Chromosomal aberrations were detected in 2 of 17 cases. The results suggest that formation of an MIB-1 index is not helpful in assessing future clinical behavior of an ameloblastoma and that chromosomal imbalances are uncommon.

Dermatofibrosarcoma protuberans (DFSP) is a tumor of low or intermediate malignant potential with a tendency for recurrence, but low rate of metastasis. The tumorigenesis of DFSP has recently been shown to be associated with the fusion of the collagen type I alpha 1 (COL1A1) and platelet-derived growth factor B-chain (PDGFB) genes, often as a consequence of translocation t(17;22)(q22;q13). Cytogenetically, DFSP is often characterized by supernumerary ring chromosomes containing material from chromosomes 17 and 22. A subset of DFSPs undergo fibrosarcomatous transformation de novo or upon recurrence, and contain components indistinguishable from fibrosarcoma (FS-DFSP). The fibrosarcomatous transformation appears to carry an increased risk for recurrence and metastasis, and is considered to represent tumor progression. The molecular cytogenetic events contributing to tumor progression are unknown. We used comparative genomic hybridization to analyze DNA copy number changes in 11 cases of typical DFSP and 10 cases of FS-DFSP. All cases in both groups were found to exhibit a gain or high-level amplification on chromosome 17q and the majority also on 22q. This finding is in line with previous studies, and suggests further that not only the COL1A1/PDGFB fusion gene formation but also the role of DNA copy number gains in the 17q and 22q regions is crucial per se in the pathogenesis of DFSP. Even though FS-DFSPs displayed a trend toward increase in the number of DNA copy number changes, the difference was not statistically significant, which indicates that mechanisms other than copy number changes are important in the transformation process of DFSP.

Translocation-associated gene fusions are well recognized in acute myeloid leukemia. Other molecular genetic changes are less well known. The novel cDNA technology has opened the avenue to large-scale gene expression analysis. Our aim was to perform cDNA microarray analysis of acute myeloid leukemia (AML).We performed cDNA microarray analysis using the Clontech hematology filter (containing 406 genes) on 15 patients to study gene expression profiling in AML. As reference, we used whole bone marrow from 5 healthy donors.Our results revealed 50 differentially expressed genes in at least 3 out of 15 patients. Twenty-two genes were upregulated (ratio > or =4), whereas 28 genes were downregulated (ratio < or =0.25). All but one of the 13 genes tested by real-time polymerase chain reaction (PCR) showed the same expression profiles. Among the overexpressed genes, several were those earlier associated with chromosomal translocations and gene fusions. These genes were FGFR1, MYC, NPM1, DEC, and BCL2. The expression of two upregulated genes, HOXA4 and CSF1R, was significantly higher in patients with a white blood cell count higher than 30 x 10(9)/L cells. In patients whose white blood cell count was higher than 100 x 10(9)/L cells, both CLC and GRN were significantly underexpressed, whereas HOXA4 and DAPK1 were overexpressed. FGFR1 and CAMLG were more frequently significantly overexpressed in patients with CD56 immunophenoytpe.Clinical and prognostic significance of differential gene expression should be studied with a larger series of patients by using other techniques, such as quantitative real-time PCR.

Abstract Gene amplifications occur rarely in hematologic neoplasms. We characterized two cases of acute myeloid leukemia (AML) with marker chromosomes and 11q23–25 amplicons. Case 1 was a 14‐year‐old male with an additional ring of chromosome 11 material as the sole karyotypic abnormality, as determined by G‐banding and multicolor fluorescence in situ hybridization. Standard comparative genomic hybridization (CGH) showed amplification in 11q23–qter. However, the MLL gene, in 11q23, was not amplified by FISH. Case 2 was a 38‐year‐old male with the G‐banding karyotype 51,XY,+8,+19,+3mar and with 11q22–qter amplification by standard CGH. This patient also had the MLL – LARG fusion gene. We used microarray‐based CGH (array‐CGH) to characterize the amplicons. In case 1, the amplified region in 11q24.3–25 (5.5 Mb) was continuous, and MLL was not amplified, as expected. In case 2, the amplicon was divided into two distinct parts, in 11q23.3 (1.2 Mb) and in 11q23.3–25 (13.3 Mb). It contained a loss (∼1 Mb) in 11q23.3, and the amplicon breakpoint was in the middle of MLL . Although the amplicon size varied, the patients had a common amplified region in 11q24–25 that comprised 14 genes. Expression microarray of case 1 revealed that three of these genes, FLI1 , NFRKB , and SNX19 , were also overexpressed. The results indicate that the 11q24–q25 region may harbor new candidate oncogenes. In addition, the complex amplicon of case 2 suggests some intriguing chromosomal mechanisms. © 2005 Wiley‐Liss, Inc.

Leiomyosarcoma (LMS) is a rare malignant mesenchymal tumor of smooth muscle cells. Chromosomal aberrations in LMS have been studied, but the cytogenetic data that have been published so far are complex, limited, and incomplete. Here, we performed for the first time a high-resolution genome-wide array comparative genomic hybridization (CGH) analysis (aCGH) on a pool of 14 low- and high-grade LMS cases to obtain gene-level information about the amplified and deleted regions that may play a role in the development and progression of LMS. Our aCGH results indicated that 2,218 genes were involved in 25 altered chromosomal regions; 9 regions in low-grade LMS, 12 regions in high-grade LMS, and 4 minimal common regions shared by low- and high-grade LMS. The frequency of DNA copy number gains in high-grade LMS was threefold compared to low-grade LMS, whereas losses in low-grade LMS were almost twice as frequent as in high-grade LMS. Both low- and high-grade tumors shared two minimal common regions of gain (15q26∼qter and 17p13.1∼q11) and loss (6p12∼p21.3 and 13q14.3∼qter). Moreover, our findings indicated that low- and high-grade LMS and osteosarcoma share 12 genes located in the 17p amplicon. In conclusion, by using aCGH, we were able to define the precise location of the altered chromosomal areas and to identify putative tumor suppressor genes and oncogenes therein. The list of altered genes in the minimal common regions is available as Appendix 1 at our web site (http://www.helsinki.fi/cmg/microarray_data).

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.

Deletion of the CDKN2A locus at 9p21.3 has been reported to be a poor prognostic sign in the Ewing sarcoma family of tumours. In clinical applications CDKN2A deletion is primarily detected using fluorescent in situ hybridisation (FISH) with a commercial probe, size approximately 190 kb. Due to limitations in resolution, FISH analysis may fail to detect microdeletions smaller than 190 kb. In the present study, we performed 44K array comparative genomic hybridisation (CGH) on eleven Ewing sarcoma cell lines and 26 tissue samples in order to define the sizes of 9p21.3 deletions. Microarray CGH analysis revealed 9p21.3 deletions encompassing the CDKN2A locus in eight cell lines (73%) and in six tumours (23%). In four cases (two cell lines and two tissue samples) the deletion was less than 190 kb in size. In one cell line sample, we detected a microdeletion of approximately 58 kb in 9p21.3 harbouring the CDKN2A locus. We confirmed this result using 244K microarray CGH and TaqMan quantitative RT-PCR analysis and further performed FISH analysis on this cell line sample. Here, we show that CDKN2A FISH analysis can give false negative results in cases with small microdeletions. Our results suggest that new and more accurate FISH methods should be developed for detection of deletions in the CDKN2A locus.

Abstract The oculoauriculovertebral anomaly (OAV) or Goldenhar syndrome is a malformation complex that has been described in several chromosomal rearrangements. Among them a deletion of the terminal 5p has recurred in seven previous patients. We wish to report on an additional such patient in order to reinforce the significance of this genomic region in the cause of at least a subgroup of OAV cases. Future studies, particularly in the OAV patients with a lateral facial cleft, might define one genetic background of the disorder. Our patient had a complex translocation chromosome 45,XX, inv(2) (q32q37)mat, dic(5;21) (p15.3;q22.3)dn, resulting in a terminal 5p deletion, a terminal deletion of 21q demonstrated by FISH studies, and a duplication of 21q22.11–q22.12 documented by molecular karyotyping. In addition to OAV she developed myelodysplasia treated with bone marrow transplantation. We discuss her clinical findings with reference to her karyotype findings and review the patients with OAV and a terminal deletion of 5p. © 2008 Wiley‐Liss, Inc.

Xenografts have been shown to provide a suitable source of tumor tissue for molecular analysis in the absence of primary tumor material. We utilized ES xenograft series for integrated microarray analyses to identify novel biomarkers.Microarray technology (array comparative genomic hybridization (aCGH) and micro RNA arrays) was used to screen and identify copy number changes and differentially expressed miRNAs of 34 and 14 passages, respectively. Incubated cells used for xenografting (Passage 0) were considered to represent the primary tumor. Four important differentially expressed miRNAs (miR-31, miR-31*, miR-145, miR-106) were selected for further validation by real time polymerase chain reaction (RT-PCR). Integrated analysis of aCGH and miRNA data was performed on 14 xenograft passages by bioinformatic methods.The most frequent losses and gains of DNA copy number were detected at 9p21.3, 16q and at 8, 15, 17q21.32-qter, 1q21.1-qter, respectively. The presence of these alterations was consistent in all tumor passages. aCGH profiles of xenograft passages of each series resembled their corresponding primary tumors (passage 0). MiR-21, miR-31, miR-31*, miR-106b, miR-145, miR-150*, miR-371-5p, miR-557 and miR-598 showed recurrently altered expression. These miRNAS were predicted to regulate many ES-associated genes, such as genes of the IGF1 pathway, EWSR1, FLI1 and their fusion gene (EWS-FLI1). Twenty differentially expressed miRNAs were pinpointed in regions carrying altered copy numbers.In the present study, ES xenografts were successfully applied for integrated microarray analyses. Our findings showed expression changes of miRNAs that were predicted to regulate many ES associated genes, such as IGF1 pathway genes, FLI1, EWSR1, and the EWS-FLI1 fusion genes.

The association of microRNA alterations with progression and treatment outcome has been revealed in different types of cancers. To find miRNAs involved in imatinib response we performed miRNA microarray followed by RT-qPCR verification of 9 available diagnostic bone marrow core biopsies from 9 CML patients including 4 imatinib-resistant and 5 imatinib-responder patients. Only one differentially expressed miRNA, miR-181c, was found when the imatinib-resistant group was compared with imatinib-responders. Significant down-regulation of miR-181c in imatinib-resistant versus imatinib-responders was confirmed by qRT-PCR. Some miR-181c target genes such as PBX3, HSP90B1, NMT2 and RAD21 have been associated with drug response.

Merkel cell polyomavirus (MCV) infection underlies most Merkel cell carcinoma (MCC), a primary neuroendocrine carcinoma of the skin. While previous research has focused on MCV-positive MCC tumors, less is known about the oncogenesis in MCV-negative tumors. In this study, we analyzed mutational status of 27 MCC tumors with known MCV status for hotspot regions of 50 cancer-related genes by targeted next-generation sequencing using the Ion AmpliSeq Cancer Hotspot Panel. In addition to previously reported TP53, KIT, and PIK3CA gene mutations, we found somatic mutations in the tyrosine kinase domain of the EGFR gene in a small proportion of the cells in six tumor tissues. RB1 mutations were seen only in virus negative tumors. Hotspot mutations were more frequent in MCV-negative tumors, although the difference was not statistically significant. No clear hotspot mutation profile was observed. Novel RB1 mutations were detected only in MCV-negative tumors. Merkel cell polyomavirus (MCV) infection underlies most Merkel cell carcinoma (MCC), a primary neuroendocrine carcinoma of the skin. While previous research has focused on MCV-positive MCC tumors, less is known about the oncogenesis in MCV-negative tumors. In this study, we analyzed mutational status of 27 MCC tumors with known MCV status for hotspot regions of 50 cancer-related genes by targeted next-generation sequencing using the Ion AmpliSeq Cancer Hotspot Panel. In addition to previously reported TP53, KIT, and PIK3CA gene mutations, we found somatic mutations in the tyrosine kinase domain of the EGFR gene in a small proportion of the cells in six tumor tissues. RB1 mutations were seen only in virus negative tumors. Hotspot mutations were more frequent in MCV-negative tumors, although the difference was not statistically significant. No clear hotspot mutation profile was observed. Novel RB1 mutations were detected only in MCV-negative tumors.

The genetic alterations underlying the development of gastric and gastro-esophageal carcinoma remain largely undefined. DNA copy number changes were determined by comparative genomic hybridization in eight xenografts of proximal gastric and gastro-esophageal junction adenocarcinomas of the intestinal type. All tumors exhibited DNA copy number changes, with a total of 139 changes detected (range, 11-24 per tumor; mean = 17), indicating numerous and widespread alterations within these cancers. Gains (65%) in DNA copy number were more frequent than losses (35%). Our most striking finding was gain (all eight cases) or high-level amplification (four cases) in 20q, with a minimal common overlapping region at 20q13. Other frequent gains were observed at 6p, 7q, and 17q (six cases each) and at 1q, 2q, and 8q (five cases each). Frequent losses were observed at 4q and 5q (six cases each) and at 9p (five cases). No differences in DNA copy number changes were seen in tumors arising from the gastro-esophageal junction compared to those of the proximal stomach. The presence of common and consistent DNA copy number changes in these tumors implicate a number of chromosomal regions that may harbor important genes that are involved in tumorigenesis of the proximal stomach and gastro-esophageal junction.

We used comparative genomic hybridization (CGH) to evaluate DNA sequence copy number changes in 67 synovial sarcomas of both monophasic and biphasic histological subtypes. Changes (mean among aberrant cases: 4.7 aberrations/tumor; range: 1–17), affecting most often entire chromosomes or chromosome arms, were detected in 37 sarcomas (55%). Gains and losses were distributed equally, but different chromosomes were affected with variable frequencies. The most frequent aberrations, each detected in 9–11 of 67 tumors, were gain of 8q and gain at 12q (12q14-15 and 12q23-qter), loss of 13q21-31, and loss of 3p. Other frequent changes (in 7 or 8 cases) included gains at 2p, 1q24-31, and 17q22-qter, and losses at 3cen-q23 and 10q21. High-level amplifications were seen in 7 cases. A total of 16 regions were detected. Two of them, 8p12-qter and 21q21-qter, seen in 4 and 2 tumors, respectively, were recurrent. No aberrations specific to histological subtype were identified. However, genetic changes in the monophasic tumors were more complex and numerous (mean among aberrant cases: 5.3 aberrations/tumor; range: 1–17) than in the biphasic tumors (mean: 2.5 aberrations/tumor; range: 1–5), and high-level amplifications occurred more frequently. All but 1 of the sarcomas showing high-level amplification were of the monophasic subtype. These findings may reflect differences in the pathogenesis and biological behavior of both histological subtypes of synovial sarcoma. Genes Chromosomes Cancer 23:213–219, 1998. © 1998 Wiley-Liss, Inc.

Frequent DNA copy number gain at 3q, with minimal overlapping area at 3q24-qter, has previously been reported in squamous cell carcinoma of the lung (SQCC), implicating the importance of genes at 3q in the tumorigenesis of SQCC. To further characterize the gain of DNA sequences at 3q, we performed interphase fluorescence in situ hybridization (FISH) analysis on 16 paraffin-embedded SQCC tumor samples that had previously been studied by comparative genomic hybridization (CGH). Eleven yeast artificial chromosome (YAC) probes located at 3q25-q27 and a chromosome 3-specific centromeric probe were used in the analysis. All SQCC tumors showed increase in DNA sequence copy number with 9-11 probes. In 5 tumors (31%) the number of centromeric signals varied from 3 to 5 and the YAC/centromeric signal ratio was 1.0, suggesting that the increase in DNA sequence copy number at 3q in these cases resulted from polysomy of chromosome 3. In 11 tumors (69%), the YAC/centromeric signal ratio varied between 1.5 and 4.7, indicating that the increase in DNA sequence copy number was due to intrachromosomal gain of DNA sequences at 3q. In each case, several YACs showed increased number of signals, demonstrating that the gained area was relatively large. Our findings therefore suggest that multiple genes located at 3q25-q27 are involved in the tumorigenesis of SQCC.

DNA copy number changes were investigated in 69 samples of schistosoma-associated (SA) and non-schistosoma-associated (NSA. squamous cell carcinoma (SCC) and transitional cell carcinoma (TCC) of the bladder by comparative genomic hybridization (CGH). DNA copy number changes were detected in 47 tumors. SA tumors had more changes than NSA tumors (mean, 7 vs. 4), whereas the number of changes in SCC and TCC tumors was similar. SA tumors displayed more gains than losses (1.7:1), whereas NSA tumors showed an equal number of gains and losses. Changes that were observed at similar frequencies in SCC and TCC, irrespective of the schistosomal status, included gains and high-level amplifications at 1q, 8q, and 20q and losses in 9p and 13q. These changes may be involved in a common pathway for bladder tumor development and progression independent of schistosomal status or histological subtype. Losses in 3p and gains at 5p were seen only in SCC (P < 0.01) and losses in 5q were more frequent in SA-SCC than in other tumors (P < 0.05). However, changes that were more frequent in TCC than those in SCC included gains at 17q (P < 0.01) and losses in 4q (P < 0.05) and 6q (P < 0.01). Gains and high-level amplifications at 5p were seen only in SA-SCC (P < 0.01), whereas gains and high-level amplifications with minimal common overlapping regions at 11q13 were more frequently seen both in SA-SCC and SA-TCC tumors (P < 0.01). In addition to the above mentioned alterations, several other changes were also seen at lower frequencies. The variations in the DNA copy number changes observed in TCC, SCC, SA, and NSA bladder carcinomas suggest that these tumors have different genetic pathways. DNA copy number changes were investigated in 69 samples of schistosoma-associated (SA) and non-schistosoma-associated (NSA. squamous cell carcinoma (SCC) and transitional cell carcinoma (TCC) of the bladder by comparative genomic hybridization (CGH). DNA copy number changes were detected in 47 tumors. SA tumors had more changes than NSA tumors (mean, 7 vs. 4), whereas the number of changes in SCC and TCC tumors was similar. SA tumors displayed more gains than losses (1.7:1), whereas NSA tumors showed an equal number of gains and losses. Changes that were observed at similar frequencies in SCC and TCC, irrespective of the schistosomal status, included gains and high-level amplifications at 1q, 8q, and 20q and losses in 9p and 13q. These changes may be involved in a common pathway for bladder tumor development and progression independent of schistosomal status or histological subtype. Losses in 3p and gains at 5p were seen only in SCC (P < 0.01) and losses in 5q were more frequent in SA-SCC than in other tumors (P < 0.05). However, changes that were more frequent in TCC than those in SCC included gains at 17q (P < 0.01) and losses in 4q (P < 0.05) and 6q (P < 0.01). Gains and high-level amplifications at 5p were seen only in SA-SCC (P < 0.01), whereas gains and high-level amplifications with minimal common overlapping regions at 11q13 were more frequently seen both in SA-SCC and SA-TCC tumors (P < 0.01). In addition to the above mentioned alterations, several other changes were also seen at lower frequencies. The variations in the DNA copy number changes observed in TCC, SCC, SA, and NSA bladder carcinomas suggest that these tumors have different genetic pathways. In Western countries, more than 90. of primary bladder carcinomas (BC) are transitional cell carcinoma (TCC), whereas squamous cell carcinoma (SCC) comprises less than 10%.1Gibas Z Gibas L Cytogenetics of bladder cancer.Cancer Genet Cytogenet. 1997; 95: 108-115Abstract Full Text PDF PubMed Scopus (28) Google Scholar Carcinoma of the urinary bladder is the most common malignancy in many tropical and subtropical countries due to endemic infection by Schistosoma hematobium. Schistosoma-associated bladder carcinoma (SA-BC) defines a characteristic pathology that differs from non-schistosoma-associated bladder carcinoma (NSA-BC).2Badawi AF Molecular and genetic events in schistosomiasis-associated human bladder cancer: role of oncogenes and tumor suppressor genes.Cancer Lett. 1996; 105: 123-138Abstract Full Text PDF PubMed Scopus (39) Google Scholar Bladder cancer complicating schistosomiasis constitutes 30.8% of all cancers in Egypt, ranking first among the reported malignancies in Egyptians.3Ibrahim S Site distribution of cancer in Egypt: twelve years experience (1970–1981).in: Cancer Prevention in Developing Countries. Pergamon Press, Oxford1986: 20-32Google Scholar Egypt has the highest frequency of bladder cancer in the world. In contrast to Western countries, more than two-thirds of bladder cancer in Egypt are SCC with a peak incidence at around 50 years of age. The chromosomal alterations in Western TCC have been extensively studied. About 140 tumors have been studied by banding cytogenetics1Gibas Z Gibas L Cytogenetics of bladder cancer.Cancer Genet Cytogenet. 1997; 95: 108-115Abstract Full Text PDF PubMed Scopus (28) Google Scholar, 4Mitelman F Catalog of Chromosome Aberrations in Cancer. th ed. Wiley-Liss, New York1994Google Scholar and 212 tumors by comparative genomic hybridization (CGH).5Kallioniemi A Kallioniemi OP Citro G Sauter G DeVries S Kerschmann R Caroll P Waldman F Identification of gains and losses of DNA sequences in primary bladder cancer by comparative genomic hybridization.Genes Chromosomes Cancer. 1995; 12: 213-219Crossref PubMed Scopus (204) Google Scholar, 6Richter J Jiang F Gorog JP Sartorius G Egenter C Gasser TC Moch H Mihatsch MJ Sauter G Marked genetic differences between stage pTa and stage pT1 papillary bladder cancer detected by comparative genomic hybridization.Cancer Res. 1997; 57: 2860-2864PubMed Google Scholar, 7Voorter C Joos S Bringuier P-P Vallinga M Poddighe P Schalken J du Manoir S Ramaekers F Lichter P Hopman A Detection of chromosomal imbalances in transitional cell carcinoma of the bladder by comparative genomic hybridization.Am J Pathol. 1995; 146: 1341-1354PubMed Google Scholar, 8Hovey RM Chu L Balazs M DeVries S Moore D Sauter G Carroll PR Waldman FM Genetic alterations in primary bladder cancers and their metastases.Cancer Res. 1998; 58: 3555-3560PubMed Google Scholar, 9Bruch J Wöhr G Hautmann R Mattfeldt T Brüderlein S Möller P Sauter S Hameister H Vogel W Paiss T Chromosomal changes during progression of transitional cell carcinoma of the bladder and delineation of the amplified interval on chromosome arm 8q.Genes Chromosomes Cancer. 1998; 23: 167-174Crossref PubMed Scopus (42) Google Scholar, 10Simon R Burger H Brinkschmidt C Bocker W Hertle L Terpe HJ Chromosomal aberrations associated with invasion in papillary superficial bladder cancer.J Pathol. 1998; 185: 345-351Crossref PubMed Scopus (137) Google Scholar These studies have shown several numerical and structural chromosomal aberrations involving mainly chromosomes 3, 5, 7, 8, 9, 17, and 20. In contrast, only four cases of SCC have been analyzed cytogenetically11Lundgren R Elfving P Heim S Kristoffersson U Mandahl N Mitelman F A squamous cell bladder carcinoma with karyotypic abnormalities reminiscent of transitional cell carcinoma.J Urol. 1989; 142: 374-376PubMed Google Scholar, 12Fadl-Elmula I Gorunova L Lundgren R Mandahl N Forsby N Mitelman F Heim S Chromosomal abnormalities in two bladder carcinomas with secondary squamous cell differentiation.Cancer Genet Cytogenet. 1998; 102: 125-130Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 13Vanni R Scarpa R Nieddu M Usai E Cytogenetic investigation on 30 bladder carcinomas.Cancer Genet Cytogenet. 1988; 30: 35-42Abstract Full Text PDF PubMed Scopus (64) Google Scholar and there are no cytogenetic reports on SA-BC. The most common genetic alteration identified in TCC is loss of heterozygosity (LOH) on 9p21, where the tumor suppressor gene p16 is located.14Reeder JE Morreale JF O'Connell MJ Stadler WM Olopade OF Messing EM Wheeless LL Loss of the CDKN2A/p16 locus detected in bladder irrigation specimens by fluorescence in situ hybridization.J Urol. 1997; 158: 1717-1721Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 15Cairns JP Chiang PW Ramamoorthy S Kurnit DM Sidransky D A comparison between microsatellite and quantitative PCR analyses to detect frequent p16 copy number changes in primary bladder tumors.Clin Cancer Res. 1998; 4: 441-444PubMed Google Scholar, 16Ozen H Bladder cancer.Curr Opin Oncol. 1998; 10: 273-278Crossref PubMed Scopus (16) Google Scholar Mutations and nuclear accumulation of p53 are frequently seen in NSA-TCC17Chaturvedi V Li L Hodges S Johnston D Ro JY Logothetis C von Eschenbach AC Batsakis JG Czerniak B Superimposed histologic and genetic mapping of chromosome 17 alterations in human urinary bladder neoplasia.Oncogene. 1997; 14: 2059-2070Crossref PubMed Scopus (44) Google Scholar, 18Abdel-Fattah R Challen C Griffiths TR Robinson MC Neal DE Lunec J Alterations of TP53 in microdissected transitional cell carcinoma of the human urinary bladder: high frequency of TP53 accumulation in the absence of detected mutations is associated with poor prognosis.Br J Cancer. 1998; 77: 2230-2238Crossref PubMed Scopus (56) Google Scholar and were demonstrated in SA-SCC.19Kamel D Soini Y Nuorva K Khalifa A Mangoud A Vahakangas K Paakko P p53 and c-erbB-2 expression in schistosomal urinary bladder carcinomas and schistosomal cystitis with premalignant lesions.Virchows Arch. 1994; 424: 349-355Crossref PubMed Scopus (21) Google Scholar, 20Cooper K Haffajee Z Taylor L Human papillomavirus and schistosomiasis associated bladder cancer.Mol Pathol. 1997; 50: 145-148Crossref PubMed Scopus (25) Google Scholar However, the proportion of TP53 mutations of basepair substitution at CpG dinucleotides was significantly higher in SA-BC than in NSA-BC.21Warren W Biggs PJ el-Baz M Ghoneim MA Stratton MR Venitt S Mutations in the p53 gene in schistosomal bladder cancer: a study of 92 tumours from Egyptian patients and a comparison between mutational spectra from schistosomal and non-schistosomal urothelial tumours.Carcinogenesis. 1995; 16: 1181-1189Crossref PubMed Scopus (102) Google Scholar The cytogenetic data available from some studies on Western NSA-TCC have been obtained using fluorescent in situ hybridization on SA-BC with probes for chromosomes 7, 9, and 17.22Ghaleb AH Pizzolo JG Melamed MR Aberrations of chromosomes 9 and 17 in bilharzial bladder cancer as detected by fluorescence in situ hybridization.Am J Clin Pathol. 1996; 106: 234-241PubMed Google Scholar, 23Pycha A Mian C Posch B Haitel A El-Baz M Ghoneim MA Marberger M Numerical aberrations of chromosomes 7, 9 and 17 in squamous cell and transitional cell cancer of the bladder: a comparative study performed by fluorescence in situ hybridization.J Urol. 1998; 160: 737-740Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Although these studies have shown differences between frequencies of chromosomal changes in invasive SA-SCC and SA-TCC or NSA-TCC, they do not provide an overview of the chromosomal alterations in SA-BC. CGH enables the screening of entire tumor genomes for gains and losses of DNA copy number and consequent mapping of aberrations to chromosomal subregions.24Knuutila S Aalto Y Autio K Björkqvist A-M El-Rifai W Hemmer S Huhta T Kettunen E Kiuru-Kuhlefelt S Larramendy M Lushnikova T Monni O Pere H Tapper J Tarkkanen M Varis A Wasenius V-M Wolf M Zhu Y DNA copy number losses in human neoplasms (review).Am J Pathol. 1999; 155: 683-694Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 25Knuutila S Björkqvist A-M Autio K Tarkkanen M Wolf M Monni O Szymanska J Larramendy ML Tapper J Pere H El-Rifai W Hemmer S Wasenius V-M Vidgren V Zhu Y DNA copy number amplifications in human neoplasms: review of comparative genomic hybridization studies.Am J Pathol. 1998; 152: 1107-1123PubMed Google Scholar So far, only NSA-TCC has been studied by this technique. In this study, we used CGH to compare the DNA copy number changes in SA-TCC, NSA-TCC, and SCC of the bladder. A total of 69 cases of primary bladder carcinomas were obtained. Thirty-eight cases were SA-BC and 31 NSA-BC. All SA-BC and 16 of the NSA-BC were collected from the files of the Pathology Department, National Cancer Institute (Cairo, Egypt) and 14 cases of the NSA-BC were collected from the files of The Institute of Pathology, The Royal London Hospital (London, UK). All of the material consisted either of cystectomy specimens or surgical biopsies and was obtained either as frozen tissue sections (41 samples) or fixed in 10% buffered formalin and paraffin-embedded (28 samples) as shown in Table 1 Table 1A. The diagnosis, classification, and tumor grading were based on light microscopy examination using the criteria of the World Health Organization (WHO) classification of urinary bladder carcinomas.26Mostofi FK Sobin LH Torloni H Histological typing of urinary bladder tumours.International Histological Classification of Tumours, No. World Health Organization, 10. Geneva1973Google Scholar Of the 38 SA-BC, 28 were SCC and 10 were TCC, and of the 31 NSA-BC, 18 were SCC and 13 were TCC. The SCCs had squamous cell differentiation in the entire tumor. SA-BC was histologically verified by the presence of schistosomal cystitis in the bladder mucosa close to the tumor. The stages of paraffin-embedded tumors were comparable to those of frozen tumors. Sixty-three tumors (91%) were staged as pT2-pT4, one tumor as pT1, and five tumors as noninvasive pTa (NSA-TCC; Table 1).Table 1Histopathology and CGH Karyotype in Squamous and Transitional Cell Bladder CarcinomasPathologyCGH karyotypeNo./age/sex/sample codesTissueGradeStageLossesGainsSchistosoma-associated SCC1/26/F B11PFIT32q31-q33, 3p12-q13, 4q, 8p, 9p21-pter2p22-pter, 4p, 6p, 8q, 9q, 11q13, 15q22-qter, 20q12-qter2/35/M B03PFIIT33p, 4q11-q285p15, 8q, 11q13-q223/60/M B05PFIIT34q24-qter, 13q21-q225p4/42/M B14PFIIT34, 5q, 13q21-q311q24-qter, 7q5/38/M B16PFIIT33p14-q13, 4, 5q15-q23, 9p2p22-pter, 2q14-q21, 8q24, 11q11-q21, 15q14-qter6/52/M B25PFIIT38p, 13q21-q311q21-q24, 4p, 8q7/74/M B32PFIIT35q11-q22, 6q, 9p21-pter, 13q21.2-q31, 141q21-q24, 2q12-q21, 3p21-pter, 5p, 7q33-qter, 8q22, 11q11-q13, 12q (12q14-q15), 17q, 20q12-qter8/45/M B37PFIIT34q, 13q21.1-q226p, 79/51/M B53PFIIT313q13-qter11q11-q1310/35/M B24PFIIT3−11q1311/55/M BC-22 89-1412AFZIIT35q17q12/49/F BC-49 89-1294AFZIIT35q11-q318q22-qter, 11q11-q1313/60/M BC-16 89-1294AFZIIT3−5p14-pter, 11q13-q14.214/53/M BC-6 89-1394AFZIIT3−1q21-q2415/35/M BC-33 89-1822AFZIIT33p, 11q22-qter11q1316/54/M B29FZIIIT313q21-q3111q11-q1317/65/M B56PFIIIT33p, 4, 5q, Xp, 8, 9, 10q22.2-qter, 11p13-pter, 13q13-q325p, Xq, 10pter-q22.1, 11q11-q14, 12, 17q, 19, 20 (20q), 2218/41/M B21PFIIIT39p21-pter5p, 6p, 8p12-qter, 11q13, 11q22-q23.2, 20q12-q13.219/51/M BC-50 89-1821AFZIT43p, 13, 18q3q, 6p22-pter, 8q21-qter, 17q20/39/M B13PFIIT4184p15, 6p, 8q (8q24), 11q13, 14q21-qter, 15, 17q, 20q, 2221/55/M B34PFIIT413q13-q31, 9p21-pter1q21-q25, 6p, 8p21-pter, 9q34, 12q13-q21 (12q14-q15), 17q, 20q22/50/M B52PFIIIT45q14-q23, 6q, 13q21-q315p, 7pter-q21, 9, 11q, 20Non-Schistosoma-Associated SCC23/56/M BC-5 SD 71/94FZIT213q−24/53/M B20PFIT3−8q (8q24)25/48/M B38PFIIT36q11-q15, 13q21-q31−26/77/F BC-11 SD-2004/94FZIIT313q13-q31, 18q12.2-q22.2−27/45/M B26PFIIIT3−1q, 8q22-qter, 9q22-qter, 20q12-qter28/42/F B02PFIIIT3−20q12-qter29/61/F B01PFIIT4−8, 1730/70/M B19PFIIT42q31-q353q, 4p16, 6p22-pter, 8q23-qter (8q24)31/72/F BC-39 SD 5833/93FZIIT44q21-qter, 6q14-q22, 13q14.2-q31.21q21-q2332/85/F BC-13 SD 4773/88FZIIIT43p, 4, 5q, 9p21-pter, 10p, 18q1q, 2p, 3q, 7q, 11q11-q21(11q13), 17q11-q22, 18p, 20q, 2233/78/F BC-48 SD 2031/88FZIIIT43p13-q13, 13q21-q3211q13, 20qSchistosoma-Associated TCC34/63/M B28PFIIIT34, 6q11-q22, 8q11-q13, 10q11-q21, T1p, 13q11-q32, 18q21-qter3p21-pter, 3q, 7, 8q21.3-qter, 9p, 11q11-q14, 11q23-qter, 12q23-qter, 2035/60/M BC-43 89-1660AFZIIIT3−7q, 11q13-qter, 17q, 20q36/46/M BC-41 89-1854AFZIIIT34, 9p3p14-pter, 8q22-qter, 17q, 20q37/66/M BC-21 89-1315AFZIIIT44p1q21-q31.2, 10p, 11q11-q13Non-Schistosoma-Associated TCC38/90/M BC-31 SD 4194/94FZIITa2q23-qter, 4q22-qter, 11p13-pter, 131q21-q31 (1q22-q23), 4p15-pter, 6p, 10p, 18p39/70/M BC-15 SD 637/94FZIITa8p, 9p21-pter, 11q22.2-qter, 18q−40/77/F BC-32 SD 4535/94FZIITa2q23-q32,4, 13q21-qter1q22-q24, 11q1341/76/F BC-17 SD 3401/94FZIITa18q17q, 20q (20q13.1)42/78/M BC-30 SD 3158/94FZIIT14q13-qter, 9p21-pter, 13q21-q311q21-q25, 15, 19, 20 (20q13.1-qter), 2243/64/F B18PFIIT32q24-q332p22-pter, 6p, 8q23-qter, 17q44/53/M B04PFIIIT36q11-q2311q1345/81/F BC-29 SD 5877/94FZIIIT36q13-q21, 13q21-q3117q, 2046/46/M B27PFIIT46q11-q217p13-p21, 8q24, 11q13-qter47/83/F BC-47 SD 3766/94FZIIIT44q23-qter, 6q16-q23, 9p, 13q13-q221q22-q24, 8q23-qter, 17qFZ, frozen tissue; PF, formalin-fixed paraffin-embedded tissue; Ta, papillary, non-invasive; T1, invasion limited to lamina propria; T2, invasion limited to inner half of muscularis propria; T3, invasion into outer half of muscularis propria or the perivesical fat; T4, invasion to contiguous viscera or pelvic organs; SCC, squamous cell carcinoma; TCC, transitional cell carcinoma.High-level amplifications are in bold type. Open table in a new tab FZ, frozen tissue; PF, formalin-fixed paraffin-embedded tissue; Ta, papillary, non-invasive; T1, invasion limited to lamina propria; T2, invasion limited to inner half of muscularis propria; T3, invasion into outer half of muscularis propria or the perivesical fat; T4, invasion to contiguous viscera or pelvic organs; SCC, squamous cell carcinoma; TCC, transitional cell carcinoma. High-level amplifications are in bold type. DNA was extracted from frozen tissue sections following standard methods, whereas DNA from paraffin-embedded tissue sections was extracted as described earlier.27Miller SA Dykes DD Polesky HF A simple salting out procedure for extracting DNA from human nucleated cells.Nucleic Acids Res. 1988; 16: 1215Crossref PubMed Scopus (17970) Google Scholar CGH was performed according to standard procedures28Kallioniemi OP Kallioniemi A Piper J Isola J Waldman FM Gray JW Pinkel D Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in solid tumors.Genes Chromosomes Cancer. 1994; 10: 231-243Crossref PubMed Scopus (944) Google Scholar with a modification using a mixture of fluorochromes conjugated to dCTP and dUTP nucleotides for nick translation.29El-Rifai W Larramendy ML Björkqvist A-M Hemmer S Knuutila S Optimization of comparative genomic hybridization using fluorochrome conjugated to dCTP and dUTP nucleotides.Lab Invest. 1997; 77: 699-700PubMed Google Scholar Hybridizations, washings, and ISIS digital image analysis (Metasystems GmbH, Altlussheim, Germany) were performed as described elsewhere.30El-Rifai W Sarlomo-Rikala M Miettinen M Knuutila S Andersson LC DNA copy number losses in chromosome 14: an early change in gastrointestinal stromal tumors.Cancer Res. 1996; 56: 3230-3233PubMed Google Scholar Three-color images (red for reference DNA, green for tumor DNA, and blue for counterstaining) were acquired from 8 to 10 metaphases per sample. Only metaphases of good quality with strong uniform hybridization were included in the analysis. Chromosomes not suitable for CGH analysis (ie, chromosomes heavily bent, overlapping, or with overlying artifacts) were excluded. Based on our earlier reports and the control results, we used 1.17 and 0.85 as cut-off levels for gains and losses, respectively. In each CGH experiment, a negative control (peripheral blood DNA from a healthy donor) and a positive control were included. The positive control was a gastric tumor with known DNA copy number changes. All of the CGH results were confirmed using a 99% confidence interval. Briefly, intraexperiment standard deviations for all positions in the CGH ratio profiles were calculated from the variation of the ratio values of all homologous chromosomes within the experiment. Confidence intervals for the ratio profiles were then computed by combining them with an empirical interexperiment SD and by estimating error probabilities based on the t distribution. For the analysis of the frequencies of DNA copy number changes in BC histological subtypes, we used Fisher's exact two-tailed test. P values <0.05 were considered significant. Changes in DNA copy numbers were detected in 47 tumors, 26 SA-BC, and 21 NSA-BC. A total of 149 gains and 96 losses was detected. Tumors that had no CGH changes included 6 SA-SCC, 7 NSA-SCC, 6 SA-TCC, and 3 NSA-TCC. SA-BC had more changes than NSA-BC (mean, 7 and 4, respectively), whereas SCC and TCC showed a comparable number of changes. SA-BC displayed more gains than losses (1.7:1), whereas NSA-BC had an equal number of gains and losses. No differences were noticed in the CGH results between DNA extracted from frozen and paraffin-embedded tissue sections. Because CGH sensitivity requires at least 50% of tumor material within a sample, tumors without alterations (Table 1) were excluded from the interpretation of the results and from the statistical analysis as they may reflect a high contamination by normal cells within the tumor material. In addition, four pTa tumors that had CGH changes (Table 1) were excluded from the statistical analysis as they may represent an entirely different tumor entity from invasive bladder carcinomas. Among the abnormal cases, the common overlapping regions of the most frequent changes were defined as follows. Gains and high-level amplifications at 11q13 were seen in 65% of SA tumors compared to 23% of NSA tumors (P < 0.01). Gains and high-level amplifications at 5p (21%) and losses in 3p (24%) were only seen in SCC tumors (P < 0.01). The gains at 5p were limited to SA-SCC (32%, 7 tumors, P < 0.01) and losses in 5q were more frequent in SA-SCC (P < 0.05). Changes that were more frequent in TCC than SCC tumors included gains at 17q11-q22 (50% vs. 24%, P < 0.01) and losses in 4q24-qter (40% vs. 27%, P < 0.05) and 6q11-q21 (50% vs. 12%, P < 0.01). Gains and high level-amplifications at 1q, 8q24, and 20q12-q13, and losses in 9p and 13q21-qter were seen equally in both SCC and TCC irrespective of the schistosomal status. Other changes were seen as gains at 1q, 2p, 3q, 7, 9, 12q, 14, 15, and 22, and losses in 2q, 3p, 5q, and 18q. The details of DNA copy number changes are shown in Table 1 and Figure 1. Figure 2 shows the relative frequencies of the aberrations among abnormal cases.Figure 2Comparative frequencies of common losses (−) and gains (+) detected in bladder tumors. Tumors with normal CGH and pTa tumors are excluded.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We undertook to compare, for the first time, the DNA copy number changes in SCC and TCC in both SA and NSA tumors. Although most of the tumors were high-grade/high-stage, our results indicate that some of the recurrent changes were common for all BC subtypes, whereas others were more frequent in a certain histological subtype. The higher number of copy number changes observed in SA-BC than in NSA-BC may be explained by the chromosome instability mediated by either reactive oxygen species or urinary nitrosamines as a result of chronic inflammation and irritation in the urinary bladder by schistosomal infection.31Rosin MP Anwar WA Ward AJ Inflammation, chromosomal instability, and cancer: the schistosomiasis model.Cancer Res. 1994; 54: 1929-1933Google Scholar Schistosomal infection has been reported to be directly involved in increased chromosomal breakage in the urothelial cells at the micronuclei level.32Rosin MP Anwar W Chromosomal damage in urothelial cells from Egyptians with chronic Schistosoma haematobium infections.Int J Cancer. 1992; 50: 539-543Crossref PubMed Scopus (40) Google Scholar Gains and high-level amplifications at 11q13 were significantly higher in SA-BC than in NSA-BC, indicating that 11q13 gains may be related to the schistosomal status irrespective of the histological subtype. Possible involvement of loci in chromosome 11 in controlling the level of chromosomal breakage caused by oxidative damage due to chronic schistosomal infection has been suggested earlier.31Rosin MP Anwar WA Ward AJ Inflammation, chromosomal instability, and cancer: the schistosomiasis model.Cancer Res. 1994; 54: 1929-1933Google Scholar Among all reports of CGH studies on TCC, 11q13 gains have been rare (28 tumors, 15%) and the rate is thus comparable to NSA-BC in our material. In one study, 11q13 gains were more frequently seen in pT1 tumors than in pTa tumors.10Simon R Burger H Brinkschmidt C Bocker W Hertle L Terpe HJ Chromosomal aberrations associated with invasion in papillary superficial bladder cancer.J Pathol. 1998; 185: 345-351Crossref PubMed Scopus (137) Google Scholar However, all other CGH studies5Kallioniemi A Kallioniemi OP Citro G Sauter G DeVries S Kerschmann R Caroll P Waldman F Identification of gains and losses of DNA sequences in primary bladder cancer by comparative genomic hybridization.Genes Chromosomes Cancer. 1995; 12: 213-219Crossref PubMed Scopus (204) Google Scholar, 6Richter J Jiang F Gorog JP Sartorius G Egenter C Gasser TC Moch H Mihatsch MJ Sauter G Marked genetic differences between stage pTa and stage pT1 papillary bladder cancer detected by comparative genomic hybridization.Cancer Res. 1997; 57: 2860-2864PubMed Google Scholar, 7Voorter C Joos S Bringuier P-P Vallinga M Poddighe P Schalken J du Manoir S Ramaekers F Lichter P Hopman A Detection of chromosomal imbalances in transitional cell carcinoma of the bladder by comparative genomic hybridization.Am J Pathol. 1995; 146: 1341-1354PubMed Google Scholar, 8Hovey RM Chu L Balazs M DeVries S Moore D Sauter G Carroll PR Waldman FM Genetic alterations in primary bladder cancers and their metastases.Cancer Res. 1998; 58: 3555-3560PubMed Google Scholar, 9Bruch J Wöhr G Hautmann R Mattfeldt T Brüderlein S Möller P Sauter S Hameister H Vogel W Paiss T Chromosomal changes during progression of transitional cell carcinoma of the bladder and delineation of the amplified interval on chromosome arm 8q.Genes Chromosomes Cancer. 1998; 23: 167-174Crossref PubMed Scopus (42) Google Scholar have shown this gain to be rare and not associated with tumor stage. Gains and high-level amplifications at 5p were seen only in SA-SCC. This gain has been less frequently reported (17%, 32 tumors) in CGH studies of TCC5Kallioniemi A Kallioniemi OP Citro G Sauter G DeVries S Kerschmann R Caroll P Waldman F Identification of gains and losses of DNA sequences in primary bladder cancer by comparative genomic hybridization.Genes Chromosomes Cancer. 1995; 12: 213-219Crossref PubMed Scopus (204) Google Scholar, 6Richter J Jiang F Gorog JP Sartorius G Egenter C Gasser TC Moch H Mihatsch MJ Sauter G Marked genetic differences between stage pTa and stage pT1 papillary bladder cancer detected by comparative genomic hybridization.Cancer Res. 1997; 57: 2860-2864PubMed Google Scholar, 7Voorter C Joos S Bringuier P-P Vallinga M Poddighe P Schalken J du Manoir S Ramaekers F Lichter P Hopman A Detection of chromosomal imbalances in transitional cell carcinoma of the bladder by comparative genomic hybridization.Am J Pathol. 1995; 146: 1341-1354PubMed Google Scholar, 8Hovey RM Chu L Balazs M DeVries S Moore D Sauter G Carroll PR Waldman FM Genetic alterations in primary bladder cancers and their metastases.Cancer Res. 1998; 58: 3555-3560PubMed Google Scholar, 9Bruch J Wöhr G Hautmann R Mattfeldt T Brüderlein S Möller P Sauter S Hameister H Vogel W Paiss T Chromosomal changes during progression of transitional cell carcinoma of the bladder and delineation of the amplified interval on chromosome arm 8q.Genes Chromosomes Cancer. 1998; 23: 167-174Crossref PubMed Scopus (42) Google Scholar, 10Simon R Burger H Brinkschmidt C Bocker W Hertle L Terpe HJ Chromosomal aberrations associated with invasion in papillary superficial bladder cancer.J Pathol. 1998; 185: 345-351Crossref PubMed Scopus (137) Google Scholar and has been detected mainly in advanced TCC (pT2 and higher).9Bruch J Wöhr G Hautmann R Mattfeldt T Brüderlein S Möller P Sauter S Hameister H Vogel W Paiss T Chromosomal changes during progression of transitional cell carcinoma of the bladder and delineation of the amplified interval on chromosome arm 8q.Genes Chromosomes Cancer. 1998; 23: 167-174Crossref PubMed Scopus (42) Google Scholar Cytogenetic data on TCC have shown isochromosome 5p to be the underlying mechanism of 5p gain.1Gibas Z Gibas L Cytogenetics of bladder cancer.Cancer Genet Cytogenet. 1997; 95: 108-115Abstract Full Text PDF PubMed Scopus (28) Google Scholar One of our cases (no. 17) had a high level-amplification at 5p with a loss of whole 5q, which is likely to be an isochromosome 5p. Changes involving other chromosomal regions were almost equally distributed among SA-BC and NSA-BC indicating that they may be related to bladder tumors rather than to schistosomal status. In the present study, gains and high-level amplifications at 5p and losses in 3p were seen only in SCC tumors. Although gains at 5p were exclusively seen in SA-SCC, a similar finding has been reported in advanced TCC.6Richter J Jiang F Gorog JP Sartorius G Egenter C Gasser TC Moch H Mihatsch MJ Sauter G Marked genetic differences between stage pTa and stage pT1 papillary bladder cancer detected by comparative genomic hybridization.Cancer Res. 1997; 57: 2860-2864PubMed Google Scholar Because secondary SCC can in rare instances develop on top of advanced TCC, gains at 5p may be one of the changes required for SCC differentiation. Alternatively, the high frequency of 5p in SCC may be explained by the higher stage of S

The molecular basis of malignant mesothelioma is poorly known. We examined genetic changes in 11 mesothelioma specimens by comparative genomic hybridization (CGH). Five DNA specimens originated from uncultured tumor tissues and six from cell lines established from the same patients. Findings from the classical karyotypic characterization of both primary tumors and cell lines have been reported previously. In the CGH analyses the most common genetic alterations in the 11 mesothelioma specimens were losses of chromosomal regions in 1p, 8p, 14q, and 22q and gains of 5p, 6p, 8q, 15q, 17q, and 20. The cell lines had on average a much higher total number of genetic changes than the uncultured tumor specimens. Clonal relationship between the cell lines and the uncultured tissue specimens could not usually be demonstrated even though they originated from the same patient. The observed differences may partly be due to high frequency of chromosomal rearrangements, which CGH cannot detect, partly due to contamination of tumor specimens with normal tissue, and partly due to genetic evolution in tumor cell lines.

In the periphery, B cells differentiate in germinal centres (GCs) of secondary lymphoid organs. Isolated GC cells die quickly in vitro by apoptosis. Therefore, cell lines originating from follicular lymphomas, which are the malignant counterparts of GC B cells, would provide a stable in vitro model to study the immunobiology of GC B cells. We have established three novel human follicular lymphoma cell lines that were characterized with special reference to immunophenotypic features, response to B-cell receptor (BCR) triggering, response to cytokines and cytokine mRNA expression. One of the cell lines, HF-1A3, has a phenotype of a centrocyte. It expresses surface immunoglobulin G (sIgG) and dies by apoptosis following BCR cross-linking. Co-stimulation with interleukin-6 (IL-6), IL-15 or interferon-gamma (IFN-gamma) rescues HF-1A3 cells from BCR-induced apoptosis. The second cell line, HF-28, also represents phenotypically an IgG+ centrocyte. Ligation of its BCR leads to the cell-cycle arrest at G1 instead of apoptosis. HF-28 cells express both CD45RA and RO isoforms, which is unusual in B lymphocytes apart from plasma cells, thus suggesting a transition to plasma cell phenotype. The third cell line, HF-4.9, which phenotypically represents an sIgM+ centroblast, responds by proliferation to BCR cross-linking. These cell lines offer a unique in vitro model to study antigenic selection and cytokine-mediated growth regulation of human GC B cells.

Gene expression profiles were studied in 13 cases of salivary gland carcinoma including mucoepidermoid carcinoma (MEC), acinic cell carcinoma (ACC), and salivary duct carcinoma (SDC) using a cDNA array. A total of 162 genes were deregulated. Only 5 genes were overexpressed in all carcinomas including fibronectin 1 (FN1), tissue metalloproteinase inhibitor 1 (TIMP1), biglycan (BGN), tenascin-C (HXB), and insulin-like growth factor binding protein 5 (IGFBP5), whereas 16 genes were underexpressed. The small number of similarly deregulated genes in these carcinoma entities suggests an extensive genetic variation between them. This result agrees with the great histopathological diversity of different entities of salivary gland carcinoma. Furthermore, diversity in gene expression between the carcinoma types was identified also by hierarchical clustering. Each carcinoma entity was clustered together but MEC, SDC, and ACC were separated from each other. Significance analysis of microarrays identified 27 genes expressed differently between the groups. In MEC, overexpressed genes included those of cell proliferation (IL-6 and SFN) and cell adhesion (SEMA3F and COL6A3), whereas many underexpressed genes were related to DNA modification (NTHL1 and RBBP4). Apoptosis-related genes CASP10 and MMP11 were overexpressed in SDC, in accordance with the typical tumor necrosis seen in this entity. An intermediate filament protein of basal epithelial cells, cytokeratin 14 (KRT14) was clearly differently expressed between the 3 types of carcinoma, and can be used as an aid in their differential diagnosis. The array results were validated by RT-PCR and immunohistochemistry.

Genomic instability can be divided into 2 categories: chromosomal instability (CIN) and microsatellite instability (MSI). CIN has been linked to aneuploidy and chromosomal aberrations, and high-level loss of heterozygosity (LOH-H) has been suggested to be an indicator of CIN. High-level MSI (MSI-H), which results from nonfunctional mismatch repair, has previously been suggested to be mutually exclusive with CIN. Four MSI-H and three LOH-H primary gastric tumors of intestinal histology were used for copy number analysis by array-based comparative genomic hybridization (aCGH) with 13,000 cDNA targets. The MSI-H group showed fewer gains (0-12, average 4.5) and losses (0-10, average 2.5) per tumor as compared to the LOH-H group (9-15 gains, average 11.6 and 1-6 losses, average 4). Two MSI-H tumors did not show any copy number changes and one showed only gains of whole chromosomes. The most common alterations were gains of 20q (5/7 samples), 1q, 8, and 10p (3/7 samples) and losses of 1p and 5p (3/7 samples). The minimal amplified regions in 1q and 20q were localized to 1q21.1∼q21.2, 1q21.3, 20q11.2, 20q13.12, and 20q13.3∼qter. No copy number change was found to be specific for MSI-H or LOH-H. The results suggest that the LOH-H phenotype revealed by microsatellite analysis predicts reliably copy number abnormalities on aCGH and that a subset of MSI-H and all LOH-H tumors share the CIN phenotype.

DNA amplification at 17q is frequently detected in upper gastrointestinal adenocarcinomas (UGC; stomach and esophagus). In this study, we did fluorescence in situ hybridization on a tissue microarray that contained 304 UGCs and 89 normal stomach samples using a approximately 168-kb BAC clone (CTD-2019C10) that maps to 17q12-q21.1. This 168-kb region contains the following genes: PPP1R1B/DARPP-32, STARD3, TCAP, PNMT, PERLD1, ERBB2, C17orf37, and GRB7 as well as the first two exons of ZNFN1A3. DNA amplification (> or =5 signals) was detected in 85 of 282 (30%) of UGCs, and high-level amplification (> or =10 signals) was seen in 28 of 282 (10%) of all tumors. Adenocarcinomas of gastroesophageal junction and lower esophagus had the highest frequency of amplification (45%) compared with stomach tumors (27%; P = 0.04). On the other hand, 38% of tumors with intestinal-type morphology had amplification compared with 26% of diffuse-type tumors (P = 0.02). We further did quantitative real-time reverse transcription-PCR on 74 frozen tissue samples from UGCs for 11 genes located within or adjacent to the boundaries of this approximately 168-kb genomic region. These genes include all 9 genes that are fully or partially located inside the CTD-2019C10 clone as well as 2 additional adjacent genes (NEUROD and TOP2A). Overexpression of PPP1R1B/DARPP-32, TCAP, and TOP2A was seen in approximately half of the tumors, whereas STARD3 and ZNFN1A3 were rarely overexpressed (12%). Interestingly, there was a statistical correlation between expression of all 8 genes that map between PPP1R1B/DARPP-32 and GRB7, whereas expression of NEUROD, ZNFN1A3, and TOP2A that are partially inside or adjacent to the boundaries of the CTD-2019C10 clone did not correlate with the expression of any of these 8 genes. These data show a transcriptionally active oncogenomic region bounded by PPP1R1B/DARPP-32 and GRB7 in UGCs and provide further insight into expression levels of several critical genes.

The use of genome-wide and high-throughput screening methods on large sample sizes is a well-grounded approach when studying a process as complex and heterogeneous as tumorigenesis. Gene copy number changes are one of the main mechanisms causing cancerous alterations in gene expression and can be detected using array comparative genomic hybridization (aCGH). Microarrays are well suited for the integrative systems biology approach, but none of the existing microarray databases is focusing on copy number changes. We present here CanGEM (Cancer GEnome Mine), which is a public, web-based database for storing quantitative microarray data and relevant metadata about the measurements and samples. CanGEM supports the MIAME standard and in addition, stores clinical information using standardized controlled vocabularies whenever possible. Microarray probes are re-annotated with their physical coordinates in the human genome and aCGH data is analyzed to yield gene-specific copy numbers. Users can build custom datasets by querying for specific clinical sample characteristics or copy number changes of individual genes. Aberration frequencies can be calculated for these datasets, and the data can be visualized on the human genome map with gene annotations. Furthermore, the original data files are available for more detailed analysis. The CanGEM database can be accessed at http://www.cangem.org/.

DNA amplifications alter gene dosage in cancer genomes by multiplying the gene copy number. Amplifications are quintessential in a considerable number of advanced cancers of various anatomical locations. The aims of this study were to classify human cancers based on their amplification patterns, explore the biological and clinical fundamentals behind their amplification-pattern based classification, and understand the characteristics in human genomic architecture that associate with amplification mechanisms.We applied a machine learning approach to model DNA copy number amplifications using a data set of binary amplification records at chromosome sub-band resolution from 4400 cases that represent 82 cancer types. Amplification data was fused with background data: clinical, histological and biological classifications, and cytogenetic annotations. Statistical hypothesis testing was used to mine associations between the data sets.Probabilistic clustering of each chromosome identified 111 amplification models and divided the cancer cases into clusters. The distribution of classification terms in the amplification-model based clustering of cancer cases revealed cancer classes that were associated with specific DNA copy number amplification models. Amplification patterns - finite or bounded descriptions of the ranges of the amplifications in the chromosome - were extracted from the clustered data and expressed according to the original cytogenetic nomenclature. This was achieved by maximal frequent itemset mining using the cluster-specific data sets. The boundaries of amplification patterns were shown to be enriched with fragile sites, telomeres, centromeres, and light chromosome bands.Our results demonstrate that amplifications are non-random chromosomal changes and specifically selected in tumor tissue microenvironment. Furthermore, statistical evidence showed that specific chromosomal features co-localize with amplification breakpoints and link them in the amplification process.

Exposure to asbestos is known to induce lung cancer, and our previous studies have suggested that specific chromosomal regions, such as 19p13, are preferentially aberrant in lung tumours of asbestos-exposed patients. Here, we further examined the association between the 19p region and exposure to asbestos using array comparative genomic hybridization and fluorescence in situ hybridization (FISH) in lung tumours and FISH characterization of asbestos-induced micronuclei (MN) in human bronchial epithelial BEAS 2B cells in vitro. We detected an increased number of 19p losses in the tumours of asbestos-exposed patients in comparison with tumours from non-exposed subjects with similar distribution of tumour histology in both groups (13/33; 39% versus 3/25; 12%, P = 0.04). In BEAS 2B cells, a 48 h exposure to crocidolite asbestos (2.0 microg/cm(2)) was found to induce centromere-negative MN-harbouring chromosomal fragments. Furthermore, an increased frequency of rare MN containing a 19p fragment was observed after the crocidolite treatment in comparison with untreated controls (6/6000 versus 1/10 000, P = 0.01). The results suggest that 19p has significance in asbestos-associated carcinogenesis and that asbestos may be capable of inducing specific chromosome aberrations.

The frequency of aberrations in normal human bone-marrow chromosomes was measured in 20 healthy subjects aged 24–37 years. The normal limits of indices for chromosome and chromatid aberrations, for aneuploidy and for polyploidy are extremely narrow in young healthy subjects. Of 2914 cells in which chromosomal aberration were studied such aberrations were seen in 0.4%. Most of them were chromatid breaks with the breakpoints randomly distributed. The percentage of hypodiploidy was 0–16.7. The number of hypodiploid mitoses correlated significantly with the number of broken metaphases. Moreover, because in all subjects the missing chromosome(s) was not consistently absent from every metaphase, hypodiploidy was probably a result of preparation technique. Hyperdiploidy was never observed. Among 29,062 dividing cells the frequency of polyploidy was 0.33%. Of these polyploid divisions 60% were tetraploid, 25% octoploid, 8% 16-ploid and 7% polyploids of other multiples. In only one subject an abnormal clone was found. In this clone there was an extra marker and one G chromosome was missing.