Chromosome Arms 1p Research Articles

High‐resolution analysis of genomic alteration on chromosome arm 8p in urothelial carcinoma

Loss of chromosome arm 8p, sometimes in combination with amplification of proximal 8p, is found in urothelial carcinoma (UC) and other epithelial cancers and is associated with more advanced tumor stage. We carried out array comparative genomic hybridization on 174 UC and 33 UC cell lines to examine breakpoints and copy number. This was followed by a detailed analysis of the cell lines using fluorescence in situ hybridization (FISH) and, in some cases, M-FISH, to refine breakpoints and determine translocation partners, heterozygosity analysis, and analysis of expression of selected genes. We showed an overall pattern of 8p loss with reduced heterozygosity and reduced gene expression. Amplification was seen in some samples and shown in the cell line JMSU1 to correlate with overexpression of ZNF703, ERLIN2, PROSC, GPR124, and BRF2. Apart from the centromere, no single breakpoint was overrepresented, and we postulate that frequent complex changes without consistent breakpoints reflect the need for alterations of combinations of genes. The region around 2 Mb, which was homozygously deleted in one cell line and includes the gene ARHGEF10 and the micro-RNA hsa-mir-596, is one candidate tumor suppressor gene region.

Recurrent genomic alterations in benign and malignant pheochromocytomas and paragangliomas revealed by whole-genome array comparative genomic hybridization analysis

Pheochromocytomas and abdominal paragangliomas are adrenal and extra-adrenal catecholamine-producing tumours. They arise due to heritable cancer syndromes, or more frequently occur sporadically due to an unknown genetic cause. The majority of cases are benign, but malignant tumours are observed. Previous comparative genomic hybridization (CGH) and loss of heterozygosity studies have shown frequent deletions of chromosome arms 1p, 3q and 22q in pheochromocytomas. We applied high-resolution whole-genome array CGH on 53 benign and malignant pheochromocytomas and paragangliomas to narrow down candidate regions as well as to identify chromosomal alterations more specific to malignant tumours. Minimal overlapping regions (MORs) were identified on 16 chromosomes, with the most frequent MORs of deletion (> or = 32%) occurring on chromosome arms 1p, 3q, 11p/q, 17p and 22q, while the chromosome arms 1q, 7p, 12q and 19p harboured the most common MORs of gain (> or = 14%). The most frequent MORs (61-75%) in the pheochromocytomas were identified at 1p, and the four regions of common losses encompassed 1p36, 1p32-31, 1p22-21 and 1p13. Tumours that did not show 1p loss generally demonstrated aberrations on chromosome 11. Gain of chromosomal material was significantly more frequent among the malignant cases. Moreover, gain at 19q, trisomy 12 and loss at 11q were positively associated with malignant pheochromocytomas, while 1q gain was commonly observed in the malignant paragangliomas. Our study revealed novel and narrow recurrent chromosomal regions of loss and gain at several autosomes, a prerequisite for identifying candidate tumour suppressor genes and oncogenes involved in the development of adrenal and extra-adrenal catecholamine-producing tumours.

Abstract 1781: Use of SNP arrays to assess loss of heterozygosity in gliomas

Abstract Introduction: Loss of heterozygosity (LOH) of chromosome arms 1p and 19q in gliomas has diagnostic, prognostic, and therapeutic implications. The deletion of 1p and the codeletion of chromosome arms 1p and 19q correlate with the diagnosis of oligodendroglioma, increased chemosensitivity and improved prognosis. Clinically, LOH is most frequently determined by Fluorescent In Situ Hybridization (FISH) and short tandem repeat (STR) analysis. Each approach suffers from the limitation that the probes/primers interrogate only a small portion of the chromosomes. Small deletions can be misinterpreted as LOH of the entire chromosome arm. We have investigated the use of a single nucleotide polymorphism (SNP) array for identifying LOH in gliomas. Methods: Genomic DNA was extracted from 5 formalin-fixed paraffin-embedded tissue samples following microdissection, and from peripheral blood. Samples were run on an ABI 3100 following multiplex PCR amplification of 5 STRs on chromosome 1p (D1S199, D1S186, D1S162, D1S312, D1S226) and 3 STRs on chromosome 19q (D19S918, D19S112, D19S206). Array analysis was performed using the Affymetrix genome-wide human SNP array 6.0 platform (906,600 SNPs) according to protocol. Data were analyzed with Partek Genomics Suite. Results: Cases 1 and 4 showed no LOH on chromosomes 1p and 19q by STR analysis and SNP array. Cases 2 and 5 showed LOH on chromosomes 1p and 19q by STR analysis and SNP array. Case 3 showed partial centromeric LOH on chromosome 1p by STR analysis which was confirmed by SNP array. Case 1 was a 48 year old male with anaplastic mixed oligoastrocytoma. Additional SNP array findings included a deletion of chromosome arm 13q. Case 4 was a 31 year old female with a low grade glioneural tumor, and no additional findings were made by SNP array. Case 2 was a 47 year old female with anaplastic oligodendroglioma. Additional SNP array findings included losses on chromosomes 5, 6, 9,15, and whole gain of chromosome 11. Case 5 was a 12 year old male with anaplastic oligodendroglioma. SNP array found additional losses on chromosomes 2, 3, 6, 8, 9, 10, and 20. Case 3 was a 47 year old female with an astrocytoma. Analysis of the 3 most centromeric STR loci on 1p demonstrated allelic imbalance that was consistent with LOH. The 2 most telomeric STR loci demonstrated allele ratios consistent with heterozygosity (negative for LOH). SNP array confirmed partial centromeric LOH on 1p and identified losses on chromosomes 2, 6, 10, 13, 16, and 22. Conclusion: Analysis of LOH on chromosome arms 1p and 19q by STR analysis and SNP array were concordant. SNP array identified additional gains or losses in each sample that were not detected by the STR assay. Interestingly, SNP array analysis also identified LOH on chromosome 13q, which contains the BRCA2 gene locus, in cases 1 and 3. If a second-hit can be identified, this may present a novel approach for identifying glioma patients who may potentially benefit from Poly (ADP-Ribose) Polymerase (PARP) inhibitor therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1781.

Abstract 4961: Analysis of genomic alterations in breast cancer using an automated aCGH workstation

Abstract The current sample preparation process for aCGH tests is tedious leading to errors that confound clinical results and increase re-test rates and costs. To overcome this problem, we have developed an aCGH workstation that automates the bench work from DNA labeling to array scanning. A robotic liquid handling and incubation system (ArrayPrep® Target Preparation System) automates the labeling and magnetic bead purification of 8-96 gDNA samples then loads them onto microarrays for hybridization using a rotating incubation system (Mai Tai® Hybridization System). Arrays are then post-processed using a robotic instrument (Little Dipper® Processor) for washing and drying. Previous studies have shown that the aCGH workstation generates high quality labeled gDNA and highly reproducible array data from batches of 8-96 samples. It requires less than 1hr hands-on technician time and can substantially enhance test reproducibility and lower cost (1). In this study, we examined the ability of the aCGH workstation to reproducibly detect genomic alterations in four well characterized breast cancer cell lines, MCF-7, SK-BR-3, BT-474 and MDA-MB-231 and in breast cancer tissue samples with known Her2/neu status. For each sample, replicate gDNA samples were processed using the aCGH workstation and Agilent 44K feature microarrays designed for genome-wide DNA copy number variation (CNV) profiling. The resulting array data were then analyzed for previously reported copy number alterations (CNAs) including loci within chromosomes 1q, 8q, 11q13, 17q and 20q13 (2, 3) and components of the epidermal growth factor receptor pathway affected by copy number changes (3). The results confirm that samples processed on the aCGH workstation reproducibly detect previously reported complex alterations involving multiple levels of change on chromosome arms 1p, 8q, 9p, 11q, 15q, 17q and 20q. Our data also confirmed that copy number alteration of multiple genetic loci involved in the EGF family of pathways is common in all four cell lines. The ERBB2 locus is highly amplified in the two known ERBB2-overexpressing cell lines (BT-474 and SK-BR-3). These two cell lines share amplifications at five additional gene loci, MAP2K6, CHN2, PRKCA, LIMK1 and cMYC, as previously reported (3). In summary, this initial study has shown the successful automation of the aCGH laboratory workflow for detection of genetic abnormalities at high resolution in breast cancer samples. This automated platform holds the potential to significantly improve test reliability and lower the cost of routine genetic analysis of clinical samples.

Oligodendroglioma cell lines containing t(1;19)(q10;p10)

Investigating the biology of oligodendroglioma and its characteristic combined deletion of chromosomal arms 1p and 19q, mediated by an unbalanced translocation, t(1;19)(q10;p10), has been hampered by the lack of cell lines that harbor these traits. We grew cells from 2 anaplastic oligodendrogliomas in serum-free conditions. Serial propagation and expansion led to the establishment of permanent cell lines that maintained the genetic signature of the parent oligodendrogliomas and displayed features of brain tumor stem cells in vitro. One line was established from a treatment-naïve tumor and the other from a temozolomide resistant recurrent tumor. These lines may be important tools for understanding the biology of oligodendrogliomas and the function of their defining genetic traits.

Genomic aberrations in borderline ovarian tumors

BackgroundAccording to the scientific literature, less than 30 borderline ovarian tumors have been karyotyped and less than 100 analyzed for genomic imbalances by CGH.MethodsWe report a series of borderline ovarian tumors (n = 23) analyzed by G-banding and karyotyping as well as high resolution CGH; in addition, the tumors were analyzed for microsatellite stability status and by FISH for possible 6q deletion.ResultsAll informative tumors were microsatellite stable and none had a deletion in 6q27. All cases with an abnormal karyotype had simple chromosomal aberrations with +7 and +12 as the most common. In three tumors with single structural rearrangements, a common breakpoint in 3q13 was detected. The major copy number changes detected in the borderline tumors were gains from chromosome arms 2q, 6q, 8q, 9p, and 13q and losses from 1p, 12q, 14q, 15q, 16p, 17p, 17q, 19p, 19q, and 22q. The series included five pairs of bilateral tumors and, in two of these pairs, informative data were obtained as to their clonal relationship. In both pairs, similarities were found between the tumors from the right and left side, strongly indicating that bilaterality had occurred via a metastatic process. The bilateral tumors as a group showed more aberrations than did the unilateral ones, consistent with the view that bilaterality is a sign of more advanced disease.ConclusionBecause some of the imbalances found in borderline ovarian tumors seem to be similar to imbalances already known from the more extensively studied overt ovarian carcinomas, we speculate that the subset of borderline tumors with detectable imbalances or karyotypic aberrations may contain a smaller subset of tumors with a tendency to develop a more malignant phenotype. The group of borderline tumors with no imbalances would, in this line of thinking, have less or no propensity for clonal evolution and development to full-blown carcinomas.

Deletion at chromosome arm 9p in relation to BRAF/NRAS mutations and prognostic significance for primary melanoma

We report an investigation of gene dosage at 9p21.3 and mutations in BRAF and NRAS, as predictors of relapse and histological markers of poor melanoma prognosis. Formalin-fixed primary melanomas from 74 relapsed and 42 nonrelapsed patients were sequenced for common BRAF and NRAS mutations (N = 71 results) and gene dosage at 9p21.3 including the genes CDKN2A (which encodes CDKN2A and P14ARF), CDKN2B (CDKN2B), and MTAP was measured using multiplexed ligation-dependant probe amplification (MLPA), (N = 75 results). BRAF/NRAS mutations were detected in 77% of relapsers and 58% of nonrelapsers (Fisher's exact P = 0.17), and did not predict ulceration or mitotic rate. There was no relationship between BRAF/NRAS mutations and gene dosage at 9p21.3. Reduced gene dosage at MTAP showed a borderline association with BRAF mutation (P = 0.04) and reduced gene dosage at the interferon gene cluster was borderline associated with wild type NRAS (P = 0.05). Reduced gene dosage in the CDKN2A regions coding for CDKN2A was associated with an increased risk of relapse (P = 0.03). Reduced gene dosage across 9p21.3 was associated with increased tumor thickness, mitotic rate, and ulceration (P = 0.02, 0.02, and 0.002, respectively), specifically in coding regions impacting on CDKN2B and P14ARF and CDKN2A. Loss at MTAP (P = 0.05) and the interferon gene cluster (P = 0.03) on 9p21 was also associated with tumor ulceration. There was no association between reduced gene dosage at 9p21.3 and subtype or site of tumor. This study presents supportive evidence that CDKN2B, P14ARF, and CDKN2A may all play a tumor suppressor role in melanoma progression. © 2010 Wiley-Liss, Inc.

Segregation distortion in chicken and the evolutionary consequences of female meiotic drive in birds

As all four meiotic products give rise to sperm in males, female meiosis result in a single egg in most eukaryotes. Any genetic element with the potential to influence chromosome segregation, so that it is preferentially included in the egg, should therefore gain a transmission advantage; a process termed female meiotic drive. We are aware of two chromosomal components, centromeres and telomeres, which share the potential to influence chromosome movement during meioses and make the following predictions based on the presence of female meiotic drive: (1) centromere-binding proteins should experience rapid evolution as a result of a conflict between driving centromeres and the rest of the genome; and (2) segregation patterns should be skewed near centromeres and telomeres. To test these predictions, we first analyze the molecular evolution of seven centromere-binding proteins in nine divergent bird species. We find strong evidence for positive selection in two genes, lending support to the genomic conflict hypothesis. Then, to directly test for the presence of segregation distortion, we also investigate the transmission of approximately 9000 single-nucleotide polymorphisms in 197 chicken families. By simulating fair Mendelian meioses, we locate chromosomal regions with statistically significant transmission ratio distortion. One region is located near the centromere on chromosome 1 and a second region is located near the telomere on the p-arm of chromosome 1. Although these observations do not provide conclusive evidence in favour of the meiotic drive/genome conflict hypothesis, they do lend support to the hypothesis that centromeres and telomeres drive during female meioses in chicken.

Differential proteome analysis of human gliomas stratified for loss of heterozygosity on chromosomal arms 1p and 19q

Combined deletion of chromosomal arms 1p and 19q is an independent prognostic marker in patients with oligodendroglial brain tumors, including oligodendrogliomas and oligoastrocytomas. However, the relevant genes in these chromosome arms and the molecular mechanisms underlying the prognostic significance of 1p/19q deletion are yet unknown. We used two-dimensional difference gel electrophoresis followed by mass spectrometry to perform a proteome-wide profiling of low-grade oligoastrocytomas stratified for the presence or absence of 1p/19q deletions. Thereby, we identified 22 different proteins showing differential expression in tumors with or without combined deletions of 1p and 19q. Four of the differentially expressed proteins, which are vimentin, villin 2 (ezrin), annexin A1, and glial fibrillary acidic protein, were selected for further analysis. Lower relative expression levels of these proteins in 1p/19q-deleted gliomas were confirmed at the protein level by Western blot analysis and immunohistochemistry. Furthermore, sequencing of sodium bisulfite-treated tumor DNA revealed more frequent methylation of 5'-CpG islands associated with the VIM and VIL2 genes in 1p/19q-deleted gliomas when compared with gliomas without these deletions. In summary, we confirm proteome-wide profiling as a powerful means to identify candidate biomarkers in gliomas. In addition, our data support the hypothesis that 1p/19q-deleted gliomas frequently show epigenetic down-regulation of multiple genes due to aberrant methylation of the 5'-CpG islands.

Renal carcinoid tumor: An immunohistochemical and molecular genetic study of four cases

Few genetic studies of renal carcinoid tumor have been conducted thus far. We performed immunohistochemical and genetic examinations on four renal carcinoid tumors. Histologically, the tumors consisted of neoplastic cells with round to oval nuclei. Various growth patterns such as tightly packed cords and trabeculae, ribbon-like, trabecular, sheet-like or solid growth were observed. Nuclear chromatin showed a coarse and granular pattern. Immunohistochemically, tumors were positive for chromogranin A and synaptophysin. In the fluorescence in situ hybridization study, three of four tumors revealed monosomy of chromosome 3 (D3Z1), but one tumor showed monosomy of chromosome 13 (D13S319/13q34). Using PCR amplification and fragment analysis of three microsatellite markers (D3S1300, D3S666 and D3S1768) of chromosome arm 3p, one tumor showed loss of heterozygosity at D3S1300 and D3S1768, one tumor was not informative and the analysis of two tumors failed due to low DNA quality. In three cases, the VHL gene status was tested. Two tumors showed wild-type, but the analysis of one tumor failed to provide adequate results. In conclusion, we suggest that the abnormality of chromosome 3 is involved in the pathogenesis of renal carcinoid tumor.

Focal 9p instability in hematologic neoplasias revealed by comparative genomic hybridization and single‐nucleotide polymorphism microarray analyses

Copy number losses in chromosome arm 9p are well-known aberrations in malignancies, including leukemias. The CDKN2A gene is suggested to play a key role in these aberrations. In this study overviewing 9p losses in hematologic neoplasias, we introduce the term focal 9p instability to indicate multiple areas of copy number loss or homozygous loss within a larger heterozygous one in 9p. We have used microarray comparative genomic hybridization to study patients with acute lymphoblastic leukemia (ALL, n = 140), acute myeloid leukemia (n = 50), chronic lymphocytic leukemia (n = 20), and myelodysplastic syndromes (n = 37). Our results show that 9p instability is restricted to ALL. In total, 58/140 (41%) patients with ALL had a loss in 9p. The 9p instability was detected in 19% of the patients with ALL and always included homozygous loss of CDKN2A along with loss of CDKN2B. Other possibly important genes included MTAP, IFN, MLLT3, JAK2, PTPLAD2, and PAX5. 13/27 (48%) patients with the instability had the BCR/ABL1 fusion gene or other oncogene-activating translocation or structural aberrations. Two patients had homozygous loss of hsa-mir -31, a microRNA known to regulate IKZF1. IKZF1 deletion at 7p12.1 was seen in 10 (37%) patients with the 9p instability. These findings suggest that, in ALL leukemogenesis, loss of CDKN2A and other target genes in the instability region is frequently associated with BCR/ABL1 and IKZF1 dysfunction. The multiple mechanisms leading to 9p instability including physical or epigenetic loss of the target genes, loss of the microRNA cluster, and the role of FRA9G fragile site are discussed.

“Clinical and Molecular Heterogeneity of Eosinophilia-Associated Myeloproliferative Neoplasms with a Translocation t(5;12)”.

Abstract 4983Chromosomal or molecular aberrations in eosinophilia-associated myeloproliferative neoplasms (Eos-MPN) often involve receptor (e.g. PDGFRA at 4q12, PDGFRB at 5q31, FGFR1 at 8p11) or intracellular (e.g. ABL1 at 9q34, JAK2 at 9p24) tyrosine kinases (TK). Fusion genes with rearrangement of PDGFRA or PDGFRB are known to be associated with rapid and durable complete hematological and clinical remission on treatment with imatinib; many of these cases also achieve complete molecular remission (undetectable fusion gene transcripts by nested reverse transcriptase polymerase chain reaction, RT-PCR). The most common fusion genes in Eos-MPN are FIP1L1-PDGFRA caused by a cytogenetically invisible interstitial deletion on 4q12, and ETV6-PDGFRB as consequence of a reciprocal translocation t(5;12)(q31-33;p13). Due to restricted availability of molecular diagnostic tools, a substantial proportion of Eos-MPN patients with t(5;12) are treated with imatinib based on karyotype but without confirmation of the underlying fusion gene by fluorescence-in-situ-hybridization (FISH) or RT-PCR. We report here on three patients (male, n=2; female, n=1) (median age 58 years, range 38-71) with Eos-MPN (median absolute eosinophil numbers in peripheral blood 8,100/μL, range 2,300-62,000) in chronic (n=2) or secondary acute myeloid leukemia (AML)/blast phase (n=1). Cytogenetic analysis revealed a t(5;12) (n=2) or a complex karyotype with involvement of chromosome arms 5q and 12p (n=1). Despite the fact that the clinical and hematological phenotype was resembling ETV6-PDGFRB positive disease (e.g. leukocytosis, eosinophilia, hypercellular marrow, splenomegaly), all three cases tested negative for ETV6-PDGFRB. A fourth patient with t(5;12)(q31;p13) but negative for ETV6-PDGFRB had identical clinical characteristics except basophilia of 7% without eosinophilia. Further molecular analyses revealed an ETV6-ACSL6 fusion gene in all four patients. Two patients received imatinib (400 mg/d) without knowledge of the molecular status and two patients were treated with sorafenib (400-800 mg/d) due to its multitargeted activity towards signal transduction molecules. No responses were observed to imatinib or sorafenib. Two male patients received an allogeneic stem cell transplantation (SCT) from a related or a matched unrelated donor, respectively. The first patient died on day +67 due to relapse of secondary AML/blast phase while the second patient died on day +64 due to transplant related complications. After failure to imatinib, the female patient with secondary AML/blast crisis only received supportive care because of comorbidity and died 7 months after diagnosis due to cytopenia-related complications. The remaining chronic phase male patient is alive 5 months after diagnosis. In addition to patients known from the literature (n=6), primary AML or rapid progression to secondary AML/blast phase has been observed in 7 of overall 10 patients with t(5;12) and an ETV6-ACSL6 fusion gene indicating a potentially aggressive clinical course. Consistent with the lack of involvement of a TK, the disease is primarily resistant to currently available TK-inhibitors and allogeneic SCT should be considered in eligible patients. In conclusion, treatment with TK-inhibitors in patients with myeloproliferative neoplasms and a t(5;12) should only be initiated if involvement of PDGFRB is confirmed by FISH or PCR analysis. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Prognostic Significance of Molecular Markers and Extent of Resection in Primary Glioblastoma Patients

Despite multimodal aggressive treatment glioblastoma patients still face a rather poor prognosis. Recent data indicate that certain molecular markers, in particular MGMT promoter hypermethylation, are associated with response to alkylating chemotherapy and longer survival. The clinical significance of other glioblastoma-associated molecular aberrations and their relationship to MGMT promoter hypermethylation is still poorly understood. We conducted a translational study involving 67 newly diagnosed glioblastoma patients treated at our institution from 1998 to 2004. All patients were treated by open resection, followed by radiotherapy and adjuvant temozolomide chemotherapy. The tumors were investigated for MGMT promoter methylation, mRNA and protein expression, as well as presence of MGMT sequence polymorphisms. In addition, we screened for genetic aberrations of the EGFR, TP53, CDK4, MDM2, and PDGFRA genes as well as allelic losses on chromosomal arms 1p, 10q, and 19q. Correlation of molecular findings with clinical data revealed significantly longer time to progression after onset of chemotherapy and longer overall survival of patients with MGMT-hypermethylated tumors. In contrast, MGMT protein expression, MGMT polymorphisms, and aberrations in any of the other genes and chromosomes were not significantly linked to patient outcome. Multivariate analysis identified MGMT promoter hypermethylation and near-complete tumor resection as the most important parameters associated with better prognosis. Our study provides novel insights into the significance of molecular and clinical markers in predicting the prognosis of glioblastoma patients, which may improve stratification of patients into distinct prognostic subgroups.

Establishment and conventional cytogenetic characterization of three gastric cancer cell lines

Gastric cancer is the fourth most frequent type of cancer and the second most frequent cause of cancer mortality worldwide. Only a modest number of gastric carcinoma cell lines have been isolated thus far. Here we describe the establishment and cytogenetic characterization of three new gastric cancer cell lines obtained from primary gastric adenocarcinoma (ACP02 and ACP03) and cancerous ascitic fluid (AGP01) of individuals from northern Brazil. ACP02, ACP03, and AGP01 cell lines are presently in the 60th passage. The cell lines grew in a disorganized single layer with some agglomerations and heterogeneous divisions (bipolar and multipolar). All cell lines exhibited a composite karyotype with several clonal chromosome alterations. Trisomy 8 was the most frequent alteration. Chromosome 8 aneusomy was confirmed by fluorescence in situ hybridization. All cell lines also exhibited trisomy 7 and deletion of chromosome arm 17p. These results suggest that, although frequent chromosome alterations are commonly observed due to culture process, the ACP02, ACP03, and AGP01 cell lines and primary gastric cancer from individuals of northern Brazil share genetic alterations, supporting use of these cell lines as a model of gastric carcinogenesis in this population.

Polysomy for Chromosomes 1 and 19 Predicts Earlier Recurrence in Anaplastic Oligodendrogliomas with Concurrent 1p/19q Loss

Loss of chromosome arms 1p and 19q is a molecular feature of oligodendroglial tumors characterized by responsiveness to chemotherapy and a favorable prognosis. The purpose of this study was to evaluate the prognostic significance of polysomy of chromosomes 1 and 19 in the setting of 1p/19q codeletion. We analyzed 64 anaplastic oligodendrogliomas with 1p/19q loss or maintenance diagnosed at Massachusetts General Hospital and Brigham and Women's Hospital from 1996 to 2005; fluorescence in situ hybridization for 1p/19q and Ki-67 immunohistochemistry was done. Polysomy was defined as more than two 1q and 19p signals in >30% of the cells with concurrent 1p/19q deletion. Tumors were divided into groups based on their 1p/19q status and compared for progression-free survival, overall survival, and 5-year survival probabilities. Forty-six tumors (72%) in our cohort had 1p/19q loss and 18 (28%) had 1p/19q maintenance. Of those with loss, 19 (41%) had concurrent polysomy and 27 (59%) lacked polysomy. In agreement with previous studies, the group of anaplastic oligodendrogliomas with 1p/19q loss had significantly better progression-free survival and overall survival than anaplastic oligodendrogliomas with 1p/19q maintenance (P = 0.0009 and P < 0.0003, respectively). Among anaplastic oligodendrogliomas with 1p/19q loss, those with polysomy showed shorter progression-free survival than those with 1p/19q loss without polysomy (P = 0.0048). Overall survival was similar in tumors with and without polysomy. The Ki-67 labeling index was not associated with polysomy and did not have prognostic significance. The presence of polysomy in anaplastic oligodendrogliomas with deletion of 1p/19q is a marker of earlier recurrence.

Molecular Predictors of Progression-Free and Overall Survival in Patients With Newly Diagnosed Glioblastoma: A Prospective Translational Study of the German Glioma Network

The prognostic value of genetic alterations characteristic of glioblastoma in patients treated according to present standards of care is unclear. Three hundred one patients with glioblastoma were prospectively recruited between October 2004 and December 2006 at the clinical centers of the German Glioma Network. Two hundred fifty-eight patients had radiotherapy, 199 patients had temozolomide, 189 had both, and seven had another chemotherapy as the initial treatment. The tumors were investigated for TP53 mutation, p53 immunoreactivity, epidermal growth factor receptor, cyclin-dependent kinase CDK 4 or murine double minute 2 amplification, CDKN2A homozygous deletion, allelic losses on chromosome arms 1p, 9p, 10q, and 19q, O(6)-methylguanine methyltransferase (MGMT) promoter methylation, and isocitrate dehydrogenase 1 (IDH1) mutations. Median progression-free (PFS) and overall survival (OS) were 6.8 and 12.5 months. Multivariate analysis revealed younger age, higher performance score, MGMT promoter methylation, and temozolomide radiochemotherapy as independent factors associated with longer OS. MGMT promoter methylation was associated with longer PFS (relative risk [RR], 0.5; 95% CI, 0.38 to 0.68; P < .001) and OS (RR, 0.39; 95% CI, 0.28 to 0.54; P < .001) in patients receiving temozolomide. IDH1 mutations were associated with prolonged PFS (RR, 0.42; 95% CI, 0.19 to 0.91; P = .028) and a trend for prolonged OS (RR, 0.43; 95% CI, 0.15 to 1.19; P = .10). No other molecular factor was associated with outcome. Molecular changes associated with gliomagenesis do not predict response to therapy in glioblastoma patients managed according to current standards of care. MGMT promoter methylation and IDH1 mutational status allow for stratification into prognostically distinct subgroups.

Deletions of chromosome arms 7p and 7q in adult acute myeloid leukemia: a marker chromosome confirmed by array comparative genomic hybridization

Acute myeloid leukemia (AML) cases with monosomy 7 (−7) and del(7q) comprise a heterogeneous subgroup. The association of losses in 7q with myeloid leukemia suggests that this region contains a tumor suppressor gene or genes whose loss of function contributes to leukemic transformation or tumor progression. The −7/del(7q) aberrations frequently coexist with complex karyotypes such as −5/del(5q) and trisomy 8. In the present case, we identified a rare abnormality involving deletion of both arms of chromosome 7 presenting with a marker chromosome-like appearance in an AML patient. Bone marrow aspiration and biopsy revealed acute myelomonocytic leukemia. Immunophenotyping study showed CD13, CD14, CD33, CD117, and myeloperoxidase positivities. Analysis of 20 metaphases indicated a diploid cell number with an unidentifiable tiny marker chromosome instead of one normal chromosome 7, described as 46,XY,−7,+mar[20]. Array comparative genomic hybridization test confirmed the chromosome 7 origin of the marker chromosome with deletions of 7p and 7q. The evaluation after remission induction treatment indicated morphological and cytogenetic remissions. There is evidence that the outcome is better when −7 or del(7q) occurs in patients with a simple karyotype, compared with a complex karyotype.

Is the 1p/19q deletion a diagnostic marker of oligodendrogliomas?

The diagnosis and classification of diffusely infiltrative gliomas are based on their histopathological appearance; however, histopathological delineation of diffuse gliomas can be difficult because of vague and subjective histopathological criteria. Combined loss of chromosome arms 1p and 19q (denoted as 1p−/19q−) has proven to be a powerful predictor of chemotherapeutic response and survival in oligodendrogliomas. We undertook retrospective and prospective studies of brain tumor patients originally diagnosed as oligodendrogliomas or oligoastrocytomas patients followed at our institution using molecular genetic techniques. Fluorescence in situ hybridization using probes specific for chromosomes 1 and 19 was performed on 22 paraffin-embedded tissues retrospectively; 15 touch-preparation smear samples were studied prospectively; and loss of heterozygosity (LOH) screening was performed on 11 samples with microsatellite markers specific to chromosome 1 and chromosome 19. Of the 37 cases, 24 had 1p−/19q−, 1 case had 1p− only, 2 cases had 19q− only, and 10 cases had no deletion. The length of the largest deletion was mapped between markers D1S2795 (1p36.31 locus) and D1S2722 (1p34.2 locus) and between markers D19S416 (19q13.11 locus) and D19S397 (19q13.14 locus), using LOH. All of the pure oligodendrogliomas ( n = 7) harbored 1p−/19q−. In light of previous findings, the 1p−/19q− combination appears to be an objective diagnosis marker of classic oligodendrogliomas, one that can be used, in combination with histological examination, to improve the diagnosis of oligodendroglioma. Fluorescence in situ hybridization on touch preparations is a simple way to obtain information on 1p−/19q− in 24 hours.

Interphase Cytogenetics for 1p19q and t(1;19)(q10;p10) may Distinguish Prognostically Relevant Subgroups in Extraventricular Neurocytoma

Co-deletion of chromosome arms 1p and 19q, characteristic of oligodendroglial tumors, was recently found to be mediated by t(1;19)(q10;p10). To evaluate the prevalence of 1p19q co-deletion and t(1;19) in extraventricular neurocytomas (EVN), we studied tumors from 23 patients, including 13 females and 10 males (median age at diagnosis 34 years, range 2-76 years). Fluorescence in situ hybridization (FISH) studies were performed with probes targeting 1p36/1q25 and 19q13/19p13 to assess for 1p19q co-deletion, as well as chromosome 1 alpha-satellite and 19p12 to detect t(1;19)(q10;p10). FISH was successful in 21 (91%) cases and demonstrated 1p19q co-deletion in five cases (24%) or isolated 1p loss in two cases (10%). Evidence for t(1;19) was found in four (of five) cases with 1p19q co-deletion. Three tumors with 1p19q loss and t(1;19) demonstrated atypical histologic features, compared with one (of 17) tumors without 1p19q co-deletion (P = 0.01, Fisher exact test). In addition, tumors with t(1;19) showed increased mitotic activity compared with tumors without t(1;19) (P = 0.045; Wilcoxon rank sum test). The four patients with t(1;19) developed tumor recurrence (n = 3), or expired (n = 2) 3.5 to 5.5 years after first resection. These results suggest that 1p19q loss and t(1;19) occur in a subset of EVN, and may be associated with aggressive histology in these tumors.

High density DNA array analysis reveals distinct genomic profiles in a subset of gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) generally harbor activating mutations in KIT or platelet-derived growth facter receptor (PDGFRA). Mutations in these receptor tyrosine kinases lead to dysregulation of downstream signaling pathways that contribute to GIST pathogenesis. GISTs with KIT or PDGFRA mutations also undergo secondary cytogenetic alterations that may indicate the involvement of additional genes important in tumor progression. Approximately 10-15% of adult and 85% of pediatric GISTs do not have mutations in KIT or in PDGFRA. Most mutant adult GISTs display large-scale genomic alterations, but little is known about the mutation-negative tumors. Using genome-wide DNA arrays, we investigated genomic imbalances in a set of 31 GISTs, including 10 KIT/PDGFRA mutation-negative tumors from nine adults and one pediatric case and 21 mutant tumors. Although all 21 mutant GISTs exhibited multiple copy number aberrations, notably losses, eight of the 10 KIT/PDGFRA mutation-negative GISTs exhibited few or no genomic alterations. One KIT/PDGFRA mutation-negative tumor exhibiting numerous genomic changes was found to harbor an alternate activating mutation, in the serine-threonine kinase BRAF. The only other mutation-negative GIST with significant chromosomal imbalances was a recurrent metastatic tumor found to harbor a homozygous deletion in chromosome arm 9p. Similar findings in several KIT-mutant GISTs identified a minimal overlapping region of deletion of approximately 0.28 Mbp in 9p21.3 that includes only the CDKN2A/2B genes, which encode inhibitors of cell-cycle kinases. These results suggest that GISTs without activating kinase mutations, whether pediatric or adult, generally exhibit a much lower level of cytogenetic progression than that observed in mutant GISTs.