Gain Of 12p Research Articles

Abstract B16: Genomic, genetic, and gene expression profile characterization of a panel of primary human uveal melanoma xenografts.

Abstract Background: Uveal melanoma (UM), which is the most common primary malignancy of the eye in adults, is marked by the occurrence of non resectable liver metastases in about 50% of patients, without any curative treatment. New therapeutic strategies are therefore warranted, and require to be evaluated in relevant and predictive preclinical models in this rare tumor. Primary human cancer xenografts, directly obtained from patient's tumors, constitute such models available for pharmacological assessments. We have developed a panel of 16 UM xenografts obtained from intraocular or metastatic tumor lesions and performed a first comparative characterization with their corresponding patient's tumors (Némati et al, CCR 2010). In this study, we have completed this characterization by the determination of their genomic, genetic, and gene expression profiles. Materials and methods: Sixteen UM xenografts at very early (P1), early (P4), and late (P9) in vivo passages and their corresponding patient's tumors have been included in the study. After extraction, available DNA/RNA tumor samples were hybridized and scanned at the Institut Curie microarray core facility. Affymetrix GeneChip® Genome-Wide Human SNP6.0 arrays and protocols were used for DNAs, and Affymetrix GeneChip® Human Exon 1.0 ST arrays and protocols for RNA. Gene mutations of GNAQ, GNA11, and BRAF were detected on tumors, metastasis, xenografts, and derived cell lines by PCR, de novo sequencing using forward and reverse primers (BigDye v1.1, Life Technologies) and capillary electrophoresis on GNAS and GNA15 were screened on tumors and xenografts when mutation of GNAQ, and GNA11 were not seen. Results: Detection of copy number alterations revealed chromosomal aberrations of high risk data sets, including loss of chromosome 1p (46%), gain of 1q (56%), loss of 3 (73%), gain of 6p (37%), loss of 6q (75%), gain of 8q (80%), and multiple genomic alterations in 31/42 samples (73%). Comparison of patient's tumors and corresponding xenografts showed high similarities, with a total correlation score of 0.89. Among the 22517 studied genes, about 3% were differentially expressed between patient's tumors and their corresponding xenografts, in which 2 thirds due to the loss of the human stroma cells during the in vivo transplantation process. The remaining third is due to genes up-expressed in xenografts and related to mitosis, DNA repair, and kinase activity. No differentially expressed genes were found between xenografts at different passages. Finally, with total concordance between patient's tumors and xenografts, GNAQ, GNA11, and BRAF genes were found to be mutated in 31%, 62%, and 0%, respectively. Conclusion: Genomic, genetic, and gene expression profile characterization of our primary human UM xenografts showed a high conservation of their molecular features during their in vivo transplantation process and in vivo maintain into mice. We therefore consider that they constitute relevant preclinical tools for pharmacological experiments. Reference: 1. Némati et al. Clin Cancer Res 2010;16:2352–2362. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B16.

IDH1/2 gene status defines the prognosis and molecular profiles in patients with grade III gliomas

The discovery of isocitrate dehydrogenase 1 and 2 gene (IDH1/2) mutations has enabled grade III glioma to be divided into mutated and wild-type IDH1/2 groups, which are known to carry different prognosis and molecular features. However, detailed subgroup analysis of grade III glioma is limited. To address this, we investigated molecular and prognostic features of grade III glioma with and without IDH1/2 mutation. We retrospectively analyzed 115 grade III glioma patients. Clinical parameters were obtained from medical records. The mutation of IDH1/2 and TP53 was analyzed by direct sequencing. O(6)-methylguanine methyltransferase gene (MGMT) gene promoter methylation status was determined by methylation-specific polymerase chain reaction. Detection of chromosome copy number changes of 1p, 7p (EGFR), 9p (CDKN2A), 10q (PTEN), and 19q was carried out by multiple ligation-dependent probe amplification. Patients were divided into two groups, mutated IDH1/2 and wild-type IDH1/2, for correlation with the factors analyzed. In our series, as previously reported, IDH1/2 mutation was an independent prognostic marker for improved progression-free and overall survival (OS) (P < 0.0001 and P < 0.0001, respectively) in patients with grade III gliomas. Subgroup analysis found that incomplete resection, 7p gain, and TP53 mutation were independent prognostic factors of poor outcome in grade III glioma patients with mutated IDH1/2 (P = 0.0092, P = 0.015 and P = 0.026, respectively), while there were none in patients with wild-type IDH1/2. IDH1/2 gene status was significantly associated with prognosis in grade III gliomas. Subgroup analysis found that poor prognostic factors existed even in patients with IDH1/2 mutation.

The hypermethylation of the O6-methylguanine-DNA methyltransferase gene promoter in gliomas-correlation with array comparative genome hybridization results and IDH1 mutation

The use of molecular markers in the diagnostics of gliomas aids histopathological diagnosis and allows their further classification into clinically significant subgroups. The aim of this study was to characterize the methylation pattern of the O(6) -methylguanine-DNA methyltransferase (MGMT) promoter, gene copy number aberrations, and isocitrate dehydrogenase I (IDH1) mutation in gliomas. We studied 51 gliomas (15 oligodendrogliomas, 18 oligoastrocytomas, 3 astrocytomas, and 15 glioblastomas) by pyrosequencing, array comparative genome hybridization (CGH), and immunohistochemistry. MGMT hypermethylation was observed in 100% of oligoastrocytomas, 93% of oligodendrogliomas, and 47% of glioblastomas. The most frequently altered chromosomal regions were deletions of 1p31.1/21.1-22.2 and 19q13.3qter in oligodendroglial tumors, and losses of 9p21.3, 10q25.3qter, and 10q26.13-26.2 in glioblastomas. Deletions on 9p and 10q, and gain of 7p were associated with the unmethylated MGMT phenotype, whereas deletion of 19q and oligodendroglial morphology was associated with MGMT hypermethylation. IDH1 mutation showed positive correlation with MGMT hypermethylation and loss of 1p/19q. Our results suggest that MGMT promoter methylation, analyzed by pyrosequencing, is a frequent event in oligodendroglial tumors, and it correlates with IDH1 mutation and 19q loss in gliomas. Pyrosequencing proved a good method for assessing the degree of MGMT methylation in formalin-fixed paraffin-embedded glioma samples. However, further studies are needed to confirm a clinically relevant cut-off point for MGMT methylation in gliomas.

Whole Genome Analysis From Microdissected Tissue Revealed Adult Supratentorial Grade II-III Gliomas Are Divided Into Clinically Relevant Subgroups by Genetic Profile

Gliomas are classified into pathologically defined subgroups. However, the tumors in the same entity could show varied 'clinical courses. Recently, various studies revealed that genetic analyses of gliomas can provide clinically relevant information. To investigate the correlation between genetic characterization and clinical information of adult supratentorial grade II-III gliomas to establish a genetic classification of these tumors. We analyzed the genetic profile of tumor samples microdissected from formalin-fixed archival tissue sections based on the defined selection criteria of tumor region using a comparative genomic hybridization method. We collected genetic and clinical data from 140 adult supratentorial gliomas of World Health Organization grade II-III and classified the tumors analyzed into subgroups according to chromosomal copy number aberrations. The relationship between histologic or genetic subclassification and clinical features was analyzed. The tumors were classified into subgroups based on the genetic profiles. Tumors with +7q and those with -1p/19q showed long progression-free survival, and loss of 10q in association with gain of 7p (+7/-10q) appeared to predict poor outcome. Most tumors with histologic diagnosis of glioblastoma showed +7/-10q; however, World Health Organization grade II-III gliomas contained a variety of genetic subgroups that correlated well with the clinical course, and this might explain why these tumors do not respond uniformly to therapies. Our pilot study suggests that care must be taken in clinical studies of these tumors because, unlike glioblastoma, grade II-III tumors may contain genetically distinct subgroups with different sensitivity to the therapies.

Copy number, loss of heterozygosity and amplification detection in formalin-fixed paraffin-embedded melanocytic lesions using molecular inversion probes

s 473 GenArray: uncovering novel and recurrent chromosomal alterations with diagnostic and prognostic value in hematological malignancies Annette Leon , Joseph Marino , Kenneth Moreno , Rev Obrera , Swaroop Aradhya , Wendy Chung , Jim Weisberger a GenPath, BioReference Laboratories Inc., Elmwood Park, NJ, USA Traditional cytogenetic techniques such as chromosome analysis and FISH have provided valuable genetic information for the evaluation of hematological malignancies. However, many genomic abnormalities remain undetected due to the limitations intrinsic to these techniques. Recently, the introduction of DNA array-based technologies has enabled the identification of previously undetected copy number changes with an increased resolution and sensitivity and a more precise determination of genomic break points and gene content. We have designed, validated and clinically applied a combined targeted-whole genome custom oligonucleotide microarray for the evaluation of hematological malignancies. The use of this technology not only allowed us to confirm genetic alterations identified by chromosome analysis or FISH, but also enabled the detection of novel genomic imbalances with important diagnostic and prognostic value in myeloid, lymphoid and plasma cell disorders. Several copy number alterations were observed in karyotypically normal patients allowing the discovery of potential tumor suppressor genes or oncogenes important in the pathogenesis of several hematological neoplasms such as myelodysplastic syndrome, multiple myeloma and chronic lymphocytic lymphoma. Our work also emphasizes the importance of incorporating this technology into current algorithms for the diagnosis of cancer. Conflict of Interest: The authors are employees and stockholders in GenPath BioReference Laboratories, Inc. Copy number, loss of heterozygosity and amplification detection in formalin-fixed paraffinembedded melanocytic lesions using molecular inversion probes Leslie R. Rowe , Wells Chandler , Mona Jahromi , Joshua D. Schiffman , Sarah T. South a University of Utah and ARUP Laboratories, Salt Lake City, UT, USA A significant number of melanocytic lesions are called indeterminate for malignancy using standard diagnostic techniques. For these cases, a sensitive and robust molecular method that provides multiple data points predictive of tumorigenicity would be useful. Previous molecular studies have demonstrated characteristic copy number alterations in melanoma. We have used molecular inversion probe (MIP) technology (OncoScan , Affymetrix) to identify and characterize genomic alterations in formalin-fixed, paraffinembedded (FFPE) melanocytic lesions. Sixty-four melanocytic lesions (23 benign nevi, 11 melanocytic lesions of uncertain malignant potential (MLUMP), 27 primary melanoma, 3 metastatic melanoma) were identified for this study. Genomic DNA was isolated from tissue scrolls and processed onto a 330,000 feature MIP microarray. No benign nevi demonstrated copy number alterations associated with melanoma. However, copy number alterations characteristic of melanoma were identified in 24/27 primary (89%) melanoma samples, including gain of 1q, 6p, 7p, 7q and 11q and loss of 6q, 9p, 9q, 10p, 10q, 11q, and 17p. Copy number changes were also identified in 4/11 (36%) MLUMP samples including loss of 1p, 2p, 6q, 7p and chromosomes 3, 9 and 17. Loss of heterozygosity (LOH) was identified in both MLUMP and melanoma samples. Additionally, amplification of CCND1, CKS1B and MYC genes was identified in a subset of the melanoma samples. In summary, we have employed a single molecular platform, using the OncoScan array, to generate information on copy number changes, LOH and amplification in melanocytic lesions. This approach will be useful to classify future melanocytic lesions with unknown malignant potential.

Genome-wide molecular characterization of central nervous system primitive neuroectodermal tumor and pineoblastoma

Central nervous system primitive neuroectodermal tumor (CNS PNET) and pineoblastoma are highly malignant embryonal brain tumors with poor prognoses. Current therapies are based on the treatment of pediatric medulloblastoma, even though these tumors are distinct at both the anatomical and molecular level. CNS PNET and pineoblastoma have a worse clinical outcome than medulloblastoma; thus, improved therapies based on an understanding of the underlying biology of CNS PNET and pineoblastoma are needed. To this end, we characterized the genomic alterations of 36 pediatric CNS PNETs and 8 pineoblastomas using Affymetrix single nucleotide polymorphism arrays. Overall, the majority of CNS PNETs contained a greater degree of genomic imbalance than pineoblastomas, with gain of 19p (8 [27.6%] of 29), 2p (7 [24.1%] of 29), and 1q (6[20.7%] of 29) common events in primary CNS PNETs. Novel gene copy number alterations were identified and corroborated by Genomic Identification of Significant Targets In Cancer (GISTIC) analysis: gain of PCDHGA3, 5q31.3 in 62.1% of primary CNS PNETs and all primary pineoblastomas and FAM129A, 1q25 in 55.2% of primary CNS PNETs and 50% of primary pineoblastomas. Comparison of our GISTIC data with publically available data for medulloblastoma confirmed these CNS PNET-specific copy number alterations. With use of the collection of 5 primary and recurrent CNS PNET pairs, we found that gain of 2p21 was maintained at relapse in 80% of cases. Novel gene copy number losses included OR4C12, 11p11.12 in 48.2% of primary CNS PNETs and 50% of primary pineoblastomas. Loss of CDKN2A/B (9p21.3) was identified in 14% of primary CNS PNETs and was significantly associated with older age among children (P = .05). CADPS, 3p14.2 was lost in 27.6% of primary CNS PNETs and was associated with poor prognosis (P = .043). This genome-wide analysis revealed the marked molecular heterogeneity of CNS PNETs and enabled the identification of novel genes and clinical associations potentially involved in the pathogenesis of these tumors.

Array Comparative Genomic Hybridization Analysis Identifies Recurrent Gain of Chromosome 2p25.3 Involving the ACP1 and MYCN Genes in Chronic Lymphocytic Leukemia

Chromosomal aberrations are independent prognostic markers in chronic lymphocytic leukemia (CLL). Recent studies using genomic arrays have shown recurrent gains of the short arm of chromosome 2 (2p) in a subset of CLL. We evaluated 178 CLL cases for 2p gains using custom-designed oligonucleotide array-based comparative genomic hybridization (aCGH). A high frequency of 2p gains was observed in 53 of 178 (30%) cases, which ranged from a small 29-kb region to large segments involving the entire short arm. Besides several common chromosomal aberrations associated with 2p gain, we demonstrated a novel observation that gain of the telomeric region 2p25.3 harboring the ACP1 gene is common in CLL (25%, 44 of 178 cases). The ACP1 gene has been previously shown to regulate T-cell receptor signaling through ZAP-70, and both genes are unfavorable clinical markers for CLL. Quantitative polymerase chain reaction (qPCR) confirmed the presence of 3-6 copies of ACP1 in 35 of 40 (88%) of these cases. Interestingly, none of the aCGH diploid CLL cases showed gain of ACP1. Assessment of 73 healthy individuals by qPCR revealed ACP1 copy number gain in only two cases (2.7%). Gain of 2p25.3 was associated with ZAP-70 expression (P < .002) and unmutated immunoglobulin heavy chain variable (IGHV) gene mutation (P < .0001). A high frequency of MYCN co-amplication with ACP1 was observed (14 of 40 cases, 35%). The frequent 2p25.3 gain involving the ACP1 and MYCN genes may help define the critical region of 2p that contributes to pathogenesis of CLL together with other chromosomal abnormalities.

Copy number profiling in von hippel‐lindau disease renal cell carcinoma

Germline mutations in the VHL tumor suppressor gene cause von Hippel-Lindau (VHL) disease and somatic VHL mutations occur in the majority of clear cell renal cell carcinoma (cRCC). To compare copy number abnormalities (CNAs) between cRCC from VHL patients and sporadic cRCC cases without detectable somatic VHL mutations, we analyzed 34 cRCC with Affymetrix 250K arrays. To increase the power of the study, we then combined our results with those of a previously published study and compared CNAs in VHL and non-VHL related cRCC using the genomic identification of significant targets in cancer (GISTIC) program. In VHL, cRCC GISTIC analysis identified four statistically significant regions of copy number gain and four statistically significant regions of deletion that occurred in >10% of tumors analyzed. Sporadic cRCC without detectable VHL mutations had, on average, more copy number abnormalities than VHL cRCC though the most common regions of loss/gain (e.g., 3p and 14q loss and 5q gain) were present in both tumor sets. However, CNAs on chromosome arms 7p (gain) and 8p (loss) were only detected in VHL RCC. Although individual copy number abnormality peaks contained clear candidate cancer genes in some cases (e.g., the 3p loss peak in VHL cRCC contained only six genes including VHL), most peaks contained many genes. To date, only a minority of the candidate genes included in these peaks have been analyzed for mutation or epigenetic inactivation in cRCC but TNFRSF10C and DUSP4 map to the 8p region deleted in VHL cRCC and TP53 and HIF2A (EPAS1) mapped to CNA loss and gain peaks (chromosomes 17 and 2, respectively) detected in sporadic VHL wild-type cRCC.

Clinicopathological and prognostic implications of genetic alterations in oral cancers.

This study evaluated the clinicopathological and prognostic implications of genetic alterations characterizing oral squamous cell carcinoma(OSCC). Comparative genomic hybridization(CGH) was used to identify chromosomal alterations present in primary OSCCs obtained from 97 pateints. In this population, tobacco use was a significant risk factor for OSCC. By contrast, all 97 of our samples are negative for human papillomavirus (HPV) DNA integration, which is another known risk factor for OSCC in certain populations. Results of the Fisher's exact test followed by Benjamini-Hochberg correction for multiple testing, showed a correlation of 7p gain and 8p loss with node-positive OSCC (p≤0.04 for both genetic alterations) and association of 11q13 gain with high-grade OSCC (p≤0.05). Univariate Cox-proportional hazard models, also corrected for multiple testing, showed significant association of 11q13 gain and 18q loss with decreased survival (p≤0.05). These findings were supported by multivariate analysis which revealed that 11q13 gain and 18q loss together serve as a strong bivariate predictor of poor prognosis. In conclusion, our study has identified genetic alterations that correlate significantly with nodal status, grade, and poor survival status of OSCC. These potential biomarkers may aid the current TNM system for better prediction of clinical outcome.

Pulmonary O2 uptake and muscle deoxygenation kinetics are slowed in the upper compared with lower region of the moderate-intensity exercise domain in older men

This study sought to determine the effect of the pre-transition work rate (WR) and WR transition magnitude on the adjustment of pulmonary oxygen uptake (VO(2p) kinetics) in older men. Seven men (69 ± 5 years; mean ± SD) each performed 4-6 cycling transitions from 20 W to either a WR corresponding to 90% estimated lactate threshold (full step, FS) or 2 equal-step transitions (lower step, LS; upper step, US) to the same end-exercise WR as in FS. Gas exchange was analysed breath-by-breath and muscle deoxygenation (∆[HHb]) was measured with NIRS. The time constant (τ) for VO(2p) was greater in US (53 ± 17 s) and FS (44 ± 11 s) compared to LS (37 ± 9 s); τVO(2p) for US also trended (p = 0.05) towards being greater than FS. The VO(2p) gain in US (9.97 ± 0.41 mL/min/W) was greater than LS (9.06 ± 1.17; p = 0.06) and FS (9.13 ± 0.54; p < 0.05). The O(2) deficit was greater in US (0.25 ± 0.08 L) than LS (0.19 ± 0.06 L); yet the 'accumulated O(2) deficit' (0.44 ± 0.13 L; O(2) deficit from LS + US) was similar to that of FS (0.42 ± 0.13 L; p = 0.38). The effective Δ[HHb] response time (τ'∆[HHb]) for US (36 ± 12 s) was greater than LS (27 ± 6 s; p = 0.07) and FS (26 ± 4 s; p < 0.05), suggesting that the slowed adjustment of muscle O(2) extraction was associated with the slowed VO(2) kinetics of the US. Despite already slowed VO(2p) kinetics, older men exhibit further slowing when small WR transitions are initiated from an elevated pre-transition WR, yet this results in no cumulative impact on O(2) deficit. This slowing in US compared to LS does not appear to be related to local O(2) availability.

Genotypic Profiling of 452 Choroidal Melanomas with Multiplex Ligation-Dependent Probe Amplification

Metastasis from uveal melanoma occurs almost exclusively with tumors showing chromosome 3 loss. We used multiplex ligation-dependent probe amplification (MLPA) to detect chromosome 1p, 3, 6p, 6q, 8p, and 8q abnormalities in uveal melanomas. The purpose of this study was to correlate our MLPA results with other risk factors and metastatic death. Patients were included if they had a uveal melanoma involving choroid. Correlations between baseline risk factors were analyzed using the chi-square test (without Yates's adjustment) and the Mann-Whitney test, with log-rank analysis for associations with metastatic death. The patients (194 female; 258 male) had a median age of 59.4 years and a median follow-up of 1.89 years. MLPA abnormalities occurred in a wide variety of combinations. Ten-year disease-specific mortality was 0% in 133 tumors with no chromosome 3 loss, 55% in tumors with chromosome 3 loss but no chromosome 8q gain, and 71% in 168 tumors showing combined chromosome 3 loss and 8q gain. In tumors with both these abnormalities, epithelioid melanoma cytomorphology, closed loops, and high mitotic rate correlated with poor survival as did lack of chromosome 6p gain. These results support the use of MLPA for routine clinical prognostication, especially if the genetic data are considered together with clinical and histologic risk factors. We showed a wide variety of MLPA results, which suggests that chromosomal abnormalities in uveal melanoma accumulate in a variable sequence.

Screening for DNA copy number aberrations in mucinous adenocarcinoma arising from the minor salivary gland: two case reports

Mucinous adenocarcinoma (MAC) is a rare malignancy in the minor salivary gland. To our knowledge, genomic alterations in this tumor have not been reported previously. To identify DNA copy number aberrations, we applied comparative genomic hybridization (CGH) to four samples of MAC in minor salivary gland derived from two patients: a primary tumor and two cervical metastatic lymph nodes from one patient, and a primary tumor from the other patient. Copy number increases were commonly detected in 1q21∼q31 and 20q13, and these may play an important role in MAC carcinogenesis. Copy number increases in 1q, 12p, 12q, and 20q were commonly detected in all three samples derived from patient 1, and gain of 7p and loss of chromosome 4 were additionally detected in the two samples derived from metastatic lymph nodes. Amplifications were also detected in the chromosomal regions 8q22∼qter, 12p11∼p12, 12q11∼q21, and 20q13. Amplification of MDM2 (12q15) and of AURKA (20q13) was detected with fluorescence in situ hybridization. The DNA copy number aberrations detected in MAC in minor salivary glands were different from those reported for colorectal MAC. The present findings are novel in identifying genomic alterations of MAC arising from the minor salivary gland.

Genomic differences between estrogen receptor (ER)‐positive and ER‐negative human breast carcinoma identified by single nucleotide polymorphism array comparative genome hybridization analysis

Estrogen receptor (ER) remains one of the most important biomarkers for breast cancer subtyping and prognosis, and comparative genome hybridization has greatly contributed to the understanding of global genetic imbalance. The authors used single-nucleotide polymorphism (SNP) arrays to compare overall copy number aberrations (CNAs) as well as loss of heterozygosity (LOH) of the entire human genome in ER-positive and ER-negative breast carcinomas. DNA was extracted from frozen tumor sections of 21 breast carcinoma specimens and analyzed with a proprietary 50K XbaI SNP array. Copy number and LOH probability values were derived for each sample. Data were analyzed using bioinformatics and computational software, and permutation tests were used to estimate the significance of these values. There was a global increase in CNAs and LOH in ER-negative relative to ER-positive cancers. Gain of the long arm of chromosome 1 (1q) and 8q were the most obvious changes common in both subtypes: An increase in the chromosome 1 short arm (1p)/1q ratio was observed in ER-negative samples, and an increased 16p/16q ratio was observed in ER-positive samples. Significant CNAs (adjusted P<.05) in ER-negative relative to ER-positive tumors included 5q deletion, loss of 15q, and gain of 2p and 21q. Copy-neutral LOH (cnLOH) common to both ER-positive and ER-negative samples included 9p21, the p16 tumor suppressor locus, and 4q13, the RCHY1 (ring finger and CHY zinc finger domain-containing 1) oncogene locus. Of particular interest was an enrichment of 17q LOH among the ER-negative tumors, potentially suggesting breast cancer 1 gene (BRCA1) mutations. SNP array detected both genetic imbalances and cnLOH and was capable of discriminating ER-negative breast cancer from ER-positive breast cancer.

Dissecting the Genetic Basis of Marginal Zone Lymphoma Reveals a Predilection for NF-Kb Activation by Clonal MZL Located at Extranodal Sites

AbstractAbstract 4160We propose that the pathophysiology of marginal zone B-cell lymphomas (MZL) could allow for their classification into lymphoid organs located in MZL and extra-nodal MZL of MALT type; the former category encompassing splenic MZL (SMZL) and nodal MZL (NMZL). The rarity of these lymphomas and difficulty in distinguishing them from other low-grade B-cell lymphoma such as lymphoplasmacytic lymphomas hampers further understanding of disease biology. In an attempt to better characterize the genetic basis of these entities and compare acquired genetic changes based on this categorization we performed array-based comparative genomic hybridization (using the 244A array, Agilent) in a cohort of MZL cases. In total we analyzed 101 MZL, including 46 MALT (including 8 cases with t(11;18)(q21;q21)), 35 SMZL and 20 NMZL. Overall, 90.1% cases have copy-number abnormalities (CNA). Genomic complexity was greatest in MALT without t(11;18)(q21;q21)(4 CNA), followed NMZL (3.5 CNA), SMZL (3 CNA) and MALT with t(11;18)(q21;21)(1 CNA). Seventeen minimal deleted regions (MDRs) and 9 minimal amplified regions (MARs) were defined based on the presence in at least one entity with a frequency of 10% or higher. Gains of chromosomes 3, 12 and 18 were found in all entities. Deletion of 6q was the most common abnormality in MALT (21.1%) and, to less extent, SMZL and NMZL. Biallelic and focal monoallelic deletions on 6q23.3-q24.1 identified TNFAIP3 as the sole gene inside the common MDR between entities. Deletion of chromosome 13 was observed in all entities, but NMZL and MALT were characterized by whole chromosome deletions, whereas focal deletions on 13q14.3 (MIR16-1/MIR15A) were identified in SMZL. A subset of abnormalities was shared for more than one entity: deletions 9p21.3 (CDKN2A) and 11q21-q22 (ATM) were found in NMZL and SMZL but not in MALT; deletion 7q32.1-q33 was the most common CNA in of SMZL but was also found in MALT; gain of 6p was found in MALT and NMZL but not in SMZL. Other CNA were only found in one entity such as deletion of 17p13.3-p12 (TP53) in SMZL, deletion of 15q25.3-q26.2 in NMZL. In addition to 17% of cases with the t(11;18)(q21;q21), extra copies of MALT1, focal amplifications of REL and/or monoallelic/biallelic deletions of TNFAIP3 were identified in an additional 46% of MALT lymphomas. These CNA were identified alone or in combination, but all of them were mutually exclusive with the t(11;18)(q21;21), thus totalizing 63% of MALT lymphomas with abnormalities affecting regulator genes from the NF-kB signaling pathway. Moreover, depending on the anatomic site of the MALT lymphoma, the prevalence of CNA affecting the NF-kB pathways varies from 50% in lacrimal glands to 75% in salivary glands. Abnormalities affecting regulator genes of the NF-kB signaling pathways were identified in a significantly lower frequency in NMZL (30%) and SMZL (31%) entities, suggesting its differential role in the pathogenesis of different entities. Using this high-resolution approach we show that while many abnormalities are shared unique patterns of genomic gains and losses characterize subgroups of MZL. Extranodal MZL seems to have a higher propensity of constitutive genetic upregulation of NF-kB pathways. Furthermore, this study remarks the importance of this pathway also in the subgroup of MALT lymphomas without translocations affecting the pathway. Disclosures:Fonseca:Genzyme: Consultancy; Medtronic: Consultancy; BMS: Consultancy; AMGEN: Consultancy; Otsuka: Consultancy; Celgene: Consultancy, Research Funding; Intellikine: Consultancy; Cylene: Research Funding; Onyx: Research Funding; FISH probes prognostication in myeloma: Patents & Royalties.

A20 Gene Deregulation In Waldenstrom's Macroglobulinemia.

AbstractAbstract 3628 Background.Waldenstrom's macroglobulinemia (WM) is a rare lymphoproliferative disorder characterized by bone marrow (BM) infiltration of lymphoplasmacytic cells and monoclonal IgM gammopathy. The deletion 6q is the most frequent cytogenetic aberration in WM and it has been suspected that this region harbours a tumour suppressor gene of pathogenic significance for WM. Comparative genomic hybridization (CGH) array delineated several minimal deleted regions (MDR) on 6q and pointed out key regulator genes of the NFKB pathway involved in these deletions, including A20 gene. The zinc finger protein A20 has been characterized as dual inhibitor of NFKB activation and was recently described as a tumor suppressor gene in lymphoma. However, the mechanisms of A20 deregulation are not fully understood in WM. We aimed to study the mechanisms of A20 deregulation in WM using gene expression profiling (GEP) and single nucleotide polymorphism (SNP) based arrays, a powerful high resolution method allowing both the detection of loss of heterozygosity (LOH) and copy number alteration (CNA) analysis in the same experiment. Methods.We have studied BM samples from 42 patients (pts) with WM (31 males, mean age: 67 years). Conventional karyotype study was analyzed following stimulation with IL2 and DSP30 of BM cells. FISH analysis was performed to detect deletion 6q21 (MYB probe). DNA and RNA were extracted following BM CD19 B cell negative selection. Quantification of copy number of A20 gene was performed using real time PCR, RNAse P gene was used as reference gene. Genome-Wide Human SNP Array 6.0 (Affymetrix chips) was used to detect both LOH and CNA in 31 pts. Paired samples (B cells/normal T lymphocytes) were used as an intra-individual reference to distinguish germline polymorphisms from somatic abnormalities in 29 patients. Size, position and location of genes were referenced using UCSC HG18 assembly, LOH and CNA were identified using genotyping console 3.02 software (Affymetrix) and Partek genomic suite. Gene expression profiling (GEP) was performed in 11 pts using Affymetrix U133A arrays. Gene-expression intensity values were log transformed, normalized and analyzed using GeneSpring GX software. Results.SNP array identified deletion 6q in 9/31 (29%) patients, confirmed by FISH analysis. All cases were monoallelic deletion. In 90% of cases, deletion 6q was associated with others genetic abnormalities. In one case, we observed a loss of 6q combined with a gain of 6p. Deletion 6q was associated with gain of chromosome 4 in 2 cases, and with deletion of 13q in 3 cases. The MDR was located on chromosome 6q21-6q25. This MDR contained several candidate genes included always deletion of A20 gene, located on 6q23, and was confirmed by real time DNA PCR quantification in all cases. Overall, the frequency of A20 gene deletion in our entire WM cohort (N=42 pts) was 29.2% (12/42), determined by real time DNA PCR of the copy number of A20 gene.In patient without deletion 6q, we looked at potential deregulated mechanisms of A20 by LOH with no CNA, e.g. UPD (uniparental disomy). We have not seen any UPD targeting the A20 gene.We then looked at A20 gene expression by GEP, and found no significant variation of A20 gene expression related to A20 monoallelic deletion as compared to pts with two copies, reflecting probably the existence of other mechanisms of A20 gene deregulation. This result was consistent with the absence of clear difference in genome-wide expression profiling in pts with 6q deletion and those without the deletion. As A20 is a key player in the negative feedback loop regulation of NFKB, we also looked at the gene expression of a set of genes involved in NFKB pathway. The presence or absence of A20 deletion did not influence the gene expression profiling of this NFKB pathway gene set. Conclusion.We described a high frequency of deletion of A20 gene. In most cases, deletion of A20 was associated with other genetic abnormalities. A20 deletion was not associated with a significant signature by GEP. Additional studies are needed to understand the cellular consequences of A20 deletions in the pathogenesis of WM. Disclosures:Leleu:Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

High-Resolution SNP-Array Profiling of Chronic Lymphocytic Leukemia

AbstractAbstract 50Genomic aberrations are important prognostic factors in chronic lymphocytic leukemia (CLL) [Döhner et al., 2000]. However, known genomic aberrations fail to fully explain the biologic and clinical heterogeneity of the disease. We sought to precisely map copy number alterations (CNA) and copy number neutral losses of heterozygocity (LOH) to better characterize known recurrent aberrations and to identify new genetic lesions.We used Affymetrix 6.0 single nucleotide polymorphism (SNP) array analyses on CD19 sorted CLL cells. Data were analyzed using dChipSNP, a modified array normalization algorithm guided by cytogenetic abnormalities and a circular binary segmentation. We studied samples from 346 patients enrolled on the CLL8 trial of the German CLL Study Group. Data of 145 samples were analyzed against intraindividual reference DNA (paired), data of 201 samples against a pool of reference DNA (unpaired). FISH data were available for all samples, the distribution of genomic aberrations was as follows: del(13q14) in 59.8%, del(11q23) in 26.3%, trisomy 12 in 11.6%, and del(17p13) in 8.4%. IGHV was mutated in 32.9%, unmutated in 63.3%, and unknown in 3.8%.In total, 261 tumor-specific CNA could be discovered among the 145 paired samples. Genomic aberrations were found in 85.5% of these cases. The average number of aberrations per case was 1.8; according to the hierarchical model of genomic aberrations, it was 3.5 in del(17p), 2.4 in del(11q23), 1.7 in del(13q14) single, and 0.5 in normal karyotype CLL. The minimally deleted region (MDR) on 13q14 was 277.25 kb in size and contained mir15a and mir16, DLEU1 and DLEU2; RFP2 was not part of the MDR. Deletions on 13q were highly heterogeneous in size, ranging from 294 kb to 68 Mb. On 11q23 the MDR only contained ATM, the smallest lesion of 78.5 kb being intragenic; in two of theses cases, the deletion size was too small to be detected by FISH analysis. TP53 was affected in all del(17p13) cases except two; one tumor-specific deletion of 635.7 kb was detected in cytoband 17p13.2 harboring 30 genes and a second deletion of 780 kb in 17p13.3 containing – among 15 other genes – MNT, a tumor suppressor acting as an antagonist of MYC. A partial trisomy on chromosome 12 was not detected. Of the 261 CNA, 95 were located in genomic regions that are not evaluated by our routine FISH probe panel; 17 regions were affected recurrently: del(1p35.3) [2/145], del(1q23.3) [2/145], del(1q42.12) [2/145], +2p [5/145], del(3p21.31) [2/145], del(6p25.3) [3/145], +(6p25.3) [2/145], del(6q) [11/145], del(7q23.1) [2/145], +(8q24.21) [3/145], del(9q13-q21.13) [2/145], del(10q24) [2/145], del(14q24.3) [2/145], del(14q12.3) [2/145], del(15q15.1) [2/145], +18 [3/145] and +19 [7/145]. The frequency of these CNA was subsequently evaluated within the cohort of 201 (unpaired) samples. Five of 17 regions were affected in more than 2% in the whole cohort: +2p, del(6q), +8q24.21, del(15q15.1), and +19. Gain of 2p was found in 6.9% of cases, the minimally amplified region was 1.9 Mb in size and contained e.g. BCL11A and REL. Del(6q) was detected in 6,4%, the deletions were heterogeneous, an MDR could not be identified. 16 cases had 8q24.21 gains, the minimally amplified region was delineated by three intragenic gains in MYC. 14 cases had loss in 15q15.1 focussing on MGA, a potential suppressor of transcriptional activation by MYC. 8 cases had total or partial gains of chromosome 19, among those two overlapping partial gains with a minimally amplified region of 2.17 Mb in 19p13.2. Tumor-specific LOH were identified in 6.0% (9/145) located on 13q in three cases and in one case each on 17p, 12q, 11p, 1p, 3 and 22q. The LOH on chromosomes one and three overlapped with recurrent losses in 1p35.3 and 3p21.31. Essential members of the ATR-pathway were located in these regions: ATRIP and RPA2. However, mutational analyses of the two candidate genes in 48 cases revealed no mutations.SNP array analysis is a reliable tool to identify and further characterize genomic aberrations in CLL. MDR on 13q14 was delineated to a 277.25 kb segment affecting mir15a, mir16, DLEU1 and DLEU2 but not RFP2; the MDR on 11q23 to a segment only containing ATM. Cases with del(11q23) and del(17p) showed a higher genomic complexity than those with normal karyotype or del(13q14) as single abnormality. Relatively few novel genetic lesions were identified. Although occurring at low frequency, they may lead to the discovery of new genes involved in CLL pathogenesis. Disclosures:Stilgenbauer:Amgen: Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Sanofi Aventis: Research Funding.

The genetic and clinical significance of MYCN gain as detected by FISH in neuroblastoma

MYCN amplification (MYCN-A) is a strong prognostic factor in neuroblastoma (NB). MYCN gain which is a low level of MYCN-A as determined by FISH. It is unclear whether the MYCN gain is the pre-status of MYCN-A. This study assessed the status of MYCN gene and chromosome 2p of MYCN-A, MYCN gain and no MYCN amplification using a single nucleotide polymorphism (SNP) array, and the clinical implication of MYCN gain in NB. The status of the MYCN gene was determined by FISH in 47 primary NB samples and the status of chromosome 2p in all cases was analyzed using an SNP array. 8 of the 47 cases analyzed using FISH showed MYCN-A, 7 cases showed MYCN gain and 32 cases showed no MYCN amplification. An SNP array analysis showed that only 2 of 8 cases with MYCN-A by FISH had both amplification of MYCN region and distal 2p gain and other 6 cases had amplification of the MYCN region without distal 2p gain. All 7 cases with MYCN gain by FISH had distal 2p gain without amplification of the MYCN region, and all 32 cases with no MYCN amplification by FISH demonstrated neither the amplification of the MYCN region nor the 2p gain. 5-year overall survival rate of patients with MYCN gain (n=7, 71.4%) was not significant different from that of patients with no MYCN amplification (n=32, 90.6%) by FISH (p=0.11). These results suggested that the MYCN gain detected by FISH represents the 2p gain, and the MYCN gain is not considered to represent the pre-status of MYCN amplification.

Multiple metastases from cutaneous malignant melanoma patients may display heterogeneous genomic and epigenomic patterns

Disseminated melanoma is an aggressive disease with fatal outcome. Better understanding of the underlying biology is needed to find effective treatment. We applied microarray-based comparative genomic hybridization, gene expression and CpG island methylation analysis of primary tumors and multiple metastases from five melanoma patients with the aim of analyzing the molecular patterns of melanoma progression. Epigenetic profiling showed that the multiple metastases after a single primary melanoma share similar methylation patterns for many genes, although differences in methylation between the lesions were evident for several genes, example, PTEN, TFAP2C, and RARB. In addition, DNA copy number and global gene expression profiles of tumors from individual patients were highly similar, confirming common origin of metastases. Some of the identified genomic aberrations, for example, gain of chromosome 6p and loss of chromosomes 6q and 10, persisted during progression, indicating early changes highly important for melanoma development. Homozygous deletions at 3p26.1 and 6q23.2-q23.3 appeared in two consecutive metastases originating from the same primary tumor, respectively, in a mutually exclusive manner that provides evidence for two genetically different subclones. However, in another case, the similarity of the copy number aberrations in subsequent metastatic lesions suggests sequential metastatic development through the clonal evolution. These data are further corroborated by a switch in CDH1 and CDH2 expression between metastases from the same patient. In conclusion, our results provide evidence for different models of metastatic progression in melanoma.

Identification of Candidate Tumor Oncogenes by Integrative Molecular Analysis of Choroidal Melanoma Fine-Needle Aspiration Biopsy Specimens

To report integrative molecular analysis of choroidal melanoma fine-needle aspiration biopsy specimens to identify candidate tumor oncogenes. Thirty-one choroidal melanoma fine-needle aspiration biopsy specimens were analyzed using cytopathologic diagnosis of melanoma, fluorescence in situ hybridization for chromosome 3, cytogenetic characterization (GeneChip Human 250K NSPI Mapping Arrays; Affymetrix, Santa Clara, California), and gene expression profiles (GeneChip Human Genome U133 Plus 2.0 Arrays, Affymetrix). These analyses were performed by clustering of cytogenetic aberrations, sorting by chromosome 3 loss and chromosome 6p gain, and comparing gene expression profiles in chromosome 3 loss- and chromosome 6p-gain tumors to identify genes with differential expression based on cytogenetic characteristics. Of 31 choroidal melanoma biopsy specimens included in this study, 19 tumors had chromosome 3 loss, and 12 tumors without chromosome 3 loss had chromosome 6p gain. Comparative RNA analysis for these 2 groups revealed 49 genes with greater than 4-fold higher expression and 31 genes with greater than 4-fold lower expression in chromosome 3-loss tumors relative to chromosome 6p-gain tumors. Molecular analysis of choroidal melanoma fine-needle aspiration biopsy specimens demonstrated 2 cytogenetically distinct groups characterized by chromosome 3 loss or chromosome 6p gain. In chromosome 3-loss melanomas relative to chromosome 6p-gain melanomas, integrative RNA analysis revealed genes with higher expression and lower expression and identified several genes that have not been reported in previous studies. Genes differentially expressed between chromosome 3-loss and chromosome 6p-gain melanomas may provide new knowledge about the biologic nature of choroidal melanoma and may contribute to the development of targeted therapies.

2p24 Gain Region Harboring MYCN Gene Compared with MYCN Amplified and Nonamplified Neuroblastoma : Biological and Clinical Characteristics

Although the role of MYCN amplification in neuroblastoma is well established, the biological and clinical characteristics of the 2p gain region harboring the MYCN gene remain unclear. The aim of this study was to compare the biological and clinical characteristics of these tumors with MYCN amplified and nonamplified neuroblastoma and to determine their impact on disease outcome. Samples from 177 patients were analyzed by fluorescence in situ hybridization, including MYCN, 1p, 17q, and 11q regions; 2p gain was identified in 25 patients, MYCN amplification in 31, and no amplification in 121 patients. Patients with 2p gain had a significantly worse 5-year event-free survival rate than patients with no MYCN amplified (P < 0.001), and an intermediate 5-year overall survival rate difference existed between the MYCN amplified tumors (P = 0.025) and nonamplified (P = 0.003) groups. All of the 2p gain samples were associated with segmental and/or numerical alterations in the other tested regions. The presence of segmental alterations with or without MYCN amplification was recently found to be the strongest predictor of relapse in a multivariate analysis. The results of the present study suggest that the determination of MYCN gene copy number relative to chromosome 2, when evaluating MYCN status at diagnosis, may help to reveal the underlying genetic pattern of these tumors and better understand their clinical behavior.