Gain Of 12p Research Articles

Chromosomal aberration predict uveal melanoma mutation status

PurposeRationale: In uveal melanoma (UM) non‐random chromosomal aberrations occur and correspond to patients’ prognosis. Mutations in UM specific genes, such as BAP1, SF3B1 and EIF1AX are also used to predict survival. Aim of this study is to identify these mutations corresponding to a specific chromosomal signature in UM.MethodsFor 277 UM patients SNP array data (n = 214) and/or conventional karyotyping (n = 119) of the tumor was available. The mutational status was known in 189 patients. Based on the mutational status, SNP array and conventional karyotyping data was analyzed for recurring copy number variations (CNVs) and structural variants (SVs). Hierarchal clustering of the SNP array data was performed to construct clusters. These clusters were correlated to the mutational status.ResultsBAP1, SF3B1 and EIF1AX‐mutated UMs display specific chromosomal patters with recurring CNVs. Both BAP1‐mutated and SF3B1‐mutated UMs are characterized by specific chromosome anomalies and SF3B1 mutated tumors were characterized by multiple (>3) SVs of the genome. EIF1AX‐mutated UM were characterized by only chromosome 6p gain without additional CNVs. Hierarchal clustering of the SNP array data revealed five clusters of which two clusters predicted BAP1 mutations, one cluster SF3B1 mutations, one cluster EIF1AX mutations or wildtype UM and one cluster predicted SF3B1 mutations, EIF1AX mutations or wildtype.ConclusionUMs with either BAP1, SF3B1 or EIF1AX mutations display a mutation‐specific chromosomal pattern. Based on the chromosomal patterns the genetic mutation in UM can be predicted.

Prognostic impact of the 2016 WHO classification of diffuse gliomas in the French POLA cohort.

The new WHO classification of diffuse gliomas has been refined and now includes the 1p/19q codeletion, IDH1/2 mutation, and histone H3-K27M mutation. Our objective was to assess the prognostic value of the updated 2016 WHO classification in the French POLA cohort. All cases of high-grade oligodendroglial tumors sent for central pathological review and included into the French nationwide POLA cohort were reclassified according to the updated 4th WHO classification. In total, 1041 patients were included, with a median age at diagnosis of 50.4years (range 17.1-84.4). Based on the new histomolecular classification, diagnoses included anaplastic oligodendroglioma IDH mutant and 1p/19q-codeleted (32.5%), anaplastic astrocytoma IDH mutant (IDH (mut)) (11.0%), anaplastic astrocytoma IDH wild type (IDH (wt)) (5.3%), glioblastoma IDH (mut) (17.1%), and glioblastoma IDH (wt) (33.2%). Ten patients presented with a diffuse midline tumor, H3 K27M mutant. The new WHO classification was prognostic for progression-free survival (PFS) and overall survival (OS) (p<0.001). We did not find prognosis differences between grades III and IV for IDH (mut) 1p/19q intact and IDH (wt) gliomas in univariate and multivariate analyses. Among anaplastic astrocytoma IDH (wt), cases with chromosome arm 7p gain and 10q loss (55%) had shorter PFS than the others (p=0.027). In conclusion, the new WHO histomolecular classification of diffuse gliomas presented with high prognostic value. Grading was not discriminant between grade III and IV high-grade gliomas.

Validation of the new glioma WHO classification in the french POLA network: Analysis of 1041 cases.

2015 Background: The upcoming WHO classification of gliomas will be refined taking into account the robust molecular alterations of gliomagenesis: the 1p19q codeletion and IDH1/2mutations. Methods: All cases of high-grade oligodendroglial tumors sent for central pathological review and included into the French nation-wide POLA network were reclassified according the upgrading of the 4th WHO classification that integrates pathological features and molecular data. Immunohistochemical expression of IDH1R132H, and ATRX, 1p19q codeletion, chromosome 7p gain and chromosome 10q loss by genomic-array and IDH1/2and histone K27M mutational statuses by sequencing were evaluated. Results: 1041 patients were included with a median age at diagnosis of 50.4 years (range, 17.1-84.4). Based on the new histo-molecular classification, diagnoses were: anaplastic oligodendroglioma with 1p19q codeletion (32.5%), anaplastic astrocytoma IDH mutated (IDHmt) (11.0%), anaplastic astrocytoma IDHwild-type (IDHwt) (5.3%), glioblastoma IDHmt (17.1%) and glioblastoma IDHwt (33.2%). Ten patients presented a midline tumor with the histone H3-K27M mutation. Both new and oldest WHO classifications were predictive for Progression-Free Survival (PFS) and Overall Survival (OS) but the new histo-molecular classification was more discriminant with higher hazard ratio for PFS (1.603, 1.848, 5.025 and 6.135 versus 1.495, 2.705, 2.799 and 4.933) and for OS (3.588, 3.493, 11.020 and 14.708 versus 2.259, 4.340, 4.214 and 7.534). Grading (III versus IV) was not prognostic for IDHmt non 1p19q codeleted gliomas in univariate analyses (PFS, p=0.5; OS, p=0.8) and multivariate analysis (adjusted by age, type of surgery and first line treatment: PFS, p=0.13; OS, p=0.38). Among anaplastic astrocytoma IDHwt, cases presenting with 7p gain and 10q loss (55%) had a worse prognosis that the others for PFS (p=0.027), suggesting that all anaplastic astrocytoma IDHwt should not be considered as glioblastoma IDHwt. Conclusions: WHO histo-molecular classification of gliomas presented with high predictive and discriminative value, allowing the validation of three main molecular subgroups for the future neuro-oncological trials.

A Meta-Analysis of Retinoblastoma Copy Numbers Refines the List of Possible Driver Genes Involved in Tumor Progression.

BackgroundWhile RB1 loss initiates retinoblastoma development, additional somatic copy number alterations (SCNAs) can drive tumor progression. Although SCNAs have been identified with good concordance between studies at a cytoband resolution, accurate identification of single genes for all recurrent SCNAs is still challenging. This study presents a comprehensive meta-analysis of genome-wide SCNAs integrated with gene expression profiling data, narrowing down the list of plausible retinoblastoma driver genes.MethodsWe performed SCNA profiling of 45 primary retinoblastoma samples and eight retinoblastoma cell lines by high-resolution microarrays. We combined our data with genomic, clinical and histopathological data of ten published genome-wide SCNA studies, which strongly enhanced the power of our analyses (N = 310).ResultsComprehensive recurrence analysis of SCNAs in all studies integrated with gene expression data allowed us to reduce candidate gene lists for 1q, 2p, 6p, 7q and 13q to a limited gene set. Besides the well-established driver genes RB1 (13q-loss) and MYCN (2p-gain) we identified CRB1 and NEK7 (1q-gain), SOX4 (6p-gain) and NUP205 (7q-gain) as novel retinoblastoma driver candidates. Depending on the sample subset and algorithms used, alternative candidates were identified including MIR181 (1q-gain) and DEK (6p gain). Remarkably, our study showed that copy number gains rarely exceeded change of one copy, even in pure tumor samples with 100% homozygosity at the RB1 locus (N = 34), which is indicative for intra-tumor heterogeneity. In addition, profound between-tumor variability was observed that was associated with age at diagnosis and differentiation grades.InterpretationSince focal alterations at commonly altered chromosome regions were rare except for 2p24.3 (MYCN), further functional validation of the oncogenic potential of the described candidate genes is now required. For further investigations, our study provides a refined and revised set of candidate retinoblastoma driver genes.

MP49-01 NEXT GENERATION SEQUENCING OF CELL FREE DNA REVEALS GENOMIC ABERRATIONS IN METASTATIC UROTHELIAL CARCINOMA

You have accessJournal of UrologyBladder Cancer: Invasive III1 Apr 2016MP49-01 NEXT GENERATION SEQUENCING OF CELL FREE DNA REVEALS GENOMIC ABERRATIONS IN METASTATIC UROTHELIAL CARCINOMA Tilman Todenhöfer, Stanislav Volik, Bernie Eigl, Scott North, Sonal Brahmbhatt, Anne Haegert, Johannes Mischinger, Arnulf Stenzl, Stephane LeBihan, Alexander Wyatt, Colin Collins, and Peter Black Tilman TodenhöferTilman Todenhöfer More articles by this author , Stanislav VolikStanislav Volik More articles by this author , Bernie EiglBernie Eigl More articles by this author , Scott NorthScott North More articles by this author , Sonal BrahmbhattSonal Brahmbhatt More articles by this author , Anne HaegertAnne Haegert More articles by this author , Johannes MischingerJohannes Mischinger More articles by this author , Arnulf StenzlArnulf Stenzl More articles by this author , Stephane LeBihanStephane LeBihan More articles by this author , Alexander WyattAlexander Wyatt More articles by this author , Colin CollinsColin Collins More articles by this author , and Peter BlackPeter Black More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.414AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES To date, only limited data is available on genomic alterations present in metastatic lesions of patients with metastatic urothelial carcinoma (mUC). The constant release of circulating cell free DNA (cfDNA) from tumor cells into the peripheral blood stream may provide a valuable source for detection of cancer associated somatic copy number variations and mutations. The value of next-generation sequencing (NGS) of cfDNA to identify unknown genomic mutations in patients with bladder cancer has not been addressed. The aim of the present pilot study was to evaluate the feasibility of analysis of somatic mutations and copy number variations in plasma cfDNA of patients with mUC. METHODS Peripheral blood (20ml) was collected from 8 patients with mUC before chemotherapy. Plasma cfDNA was isolated using the QIAAmp Circulating Nucleic Acid Kit. DNA was extracted from peripheral blood mononuclear cells (PBMCs) using the DNEasy Kit (Qiagen) for assessment of germ-line DNA alterations. Library construction was performed using the Ampliseq Comprehensive Cancer Panel (ThermoFisher). NGS was done on an ION Proton platform (ThermoFisher). Copy number variations and mutations were compared to publically available datasets from UC tissue. RESULTS The median amount of cfDNA extracted was 202.2 ng (range 42.3-530.4). We detected strong mutation candidates in 6/8 patients (Table 1) and copy number changes consistent with previously reported alterations in UC. Common copy number variations included gain of 1p (5/8 patients), 7p (5/8 patients), and 17q (3/8 patients including one with high-level focal amplification of ERBB2) and loss of 6q (6/8 patients). CONCLUSIONS Non-invasive characterization of genomic changes in peripheral blood of patients with mUC using NGS of plasma cfDNA is feasible and detects copy number changes and mutations in a high proportion of patients. Analysis of cfDNA might therefore provide an improved understanding of genomic changes in mUC that could enable the personalized use of targeted therapies. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e664 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Tilman Todenhöfer More articles by this author Stanislav Volik More articles by this author Bernie Eigl More articles by this author Scott North More articles by this author Sonal Brahmbhatt More articles by this author Anne Haegert More articles by this author Johannes Mischinger More articles by this author Arnulf Stenzl More articles by this author Stephane LeBihan More articles by this author Alexander Wyatt More articles by this author Colin Collins More articles by this author Peter Black More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

PRAME as an Independent Biomarker for Metastasis in Uveal Melanoma.

Uveal melanoma (UM) can be classified by gene expression profiling (GEP) into Class 1 (low metastatic risk) and Class 2 (high metastatic risk), the latter being strongly associated with mutational inactivation of the tumor suppressor BAP1. Nevertheless, a small percentage of Class 1 tumors give rise to metastatic disease. The purpose of this study was to identify biomarkers of metastasis in Class 1 tumors. A total of 389 consecutive patients with UM were assigned to Class 1 or Class 2 using a prospectively validated 12-gene prognostic classifier. Selected tumors were further analyzed using global GEP and single nucleotide polymorphism microarrays. PRAME (preferentially expressed antigen in melanoma) mRNA expression was analyzed in 64 Class 1 tumors by qPCR. Among Class 1 UMs, the most significant predictor of metastasis was PRAME mRNA expression (P = 0.0006). The 5-year actuarial rate of metastasis was 0% for Class1(PRAME-), 38% for Class1(PRAME+), and 71% for Class 2 tumors. Median metastasis-free survival for Class1(PRAME+) patients was 88 months, compared to 32 months for Class 2 patients. Findings were validated using three independent datasets, including one using disomy 3 to identify low-risk UM. Chromosome copy number changes associated with Class1(PRAME+) tumors included gain of 1q, 6p, 8q, and 9q and loss of 6q and 11q. PRAME expression was associated with larger tumor diameter (P = 0.05) and SF3B1 mutations (P = 0.003). PRAME is an independent prognostic biomarker in UM, which identifies increased metastatic risk in patients with Class 1 or disomy 3 tumors. This finding may further enhance the accuracy of prognostic testing and precision medicine for UM.

Cytogenetic Findings of an Undifferentiated Testicular Sarcoma

Primary testicular sarcomas are extremely rare. In contrast, germ cell tumors (GCTs) with sarcomatous components (SCs) that can have similar pathologic features are more frequently reported. We report a primary undifferentiated testicular sarcoma with cytogenetic analysis for amplification of 12p. A 36-year-old male initially presented with 9.4 cm right testicular tumor and no metastatic disease. He was lost to follow-up but re-presented 2 years later with tumor growth to 21 cm and multiple pulmonary and bone metastases. Histologically, the testicular tumor revealed an undifferentiated sarcoma. Fluorescence in situ hybridization (FISH) of paraffin-embedded tissue showed no amplification of 12p. Gain of isochromosome 12p (i(12p)) is a specific chromosomal anomaly present in most GCTs. The cytogenetic analysis indicated the possibility that the tumor was not of GCT origin. This is the first report of primary testicular undifferentiated sarcoma with cytogenetic analysis for i(12p). J Med Cases. 2016;7(1):7-12 doi: http://dx.doi.org/10.14740/jmc2356w

Genomic Alterations Important for the Prognosis in Patients with Follicular Lymphoma Treated on SWOG Study S0016

Background: FL is a common, indolent, yet typically incurable disease characterized by clinical and genetic heterogeneity. Recent studies suggested that TNFRSF14 mutation and 1p36 deletion were associated with worse outcome in FL and that TNFAIP3 mutation on 6q occurred at FL transformation. However, no genomic biomarkers are currently in clinical use for prognostic and therapeutic decisions. We aimed to identify novel genomic aberrations associated with prognosis of patients with FL in the context of a randomized cooperative group trial (Press 2013 JCO).Patients and Methods: We employed a comprehensive genomic array testing strategy to assess the common genomic abnormalities, including copy number aberrations (CNAs), copy-neutral loss-of-heterozygosity (cnLOH), and common cancer gene mutations. Our study set includes all patients enrolled in the SWOG FL study S0016 with available pre-treatment tissue specimens (n=250). To date, chromosome genomic array testing (CGAT) has been performed on archived formalin-fixed, paraffin embedded (FFPE) lymphoma tissue specimens from 158 patients. A SWOG pathologist reviewed all tissues prior to CGAT to ensure that each sample met the diagnostic criteria for FL and had at least 30% tumor content. The OncoScan platform was used for CGAT with data analysis performed by Nexus Express. Statistical analysis was performed using the Cox-proportional hazard regression model. Hazard ratio (HR) and 95% confidence interval (CI) were estimated from the multivariate model. For this exploratory analysis, statistical significance was determined at an alpha-level of 0.05 without adjustment for multiple comparisons.Results: Most samples (>90%) showed multiple chromosome abnormalities (median 10, range 1-65). The most frequently affected chromosome arms were: 1p (49.3%), 6q (41.7%), 6p (40.5%), 1q (38.6%), 16p or 22q (36%), and 18q (35.4%). Specific common CNAs were deletions of 1p, 6q, and 10q, gains of X, 1q, 2p, 7, 8q, 12q, 17q, and 18, and cnLOH of 1p, 6p, and 16p. Point mutations were not identified. Univariate analysis for progression free survival (PFS) identified several aberrations (of the 94 aberrations assessed) significantly associated with prognosis at unadjusted .05 level; these include gain of 12p, 17q, 18q, and Yp, deletions of 2q, 6q, 8q, 9p, and Yq, and deletion or cnLOH of 9p and 10q. Multivariate analysis adjusting for FLIPI risk, bulky disease, and combined serum β2M and LDH levels demonstrated that selected markers, including deletion or cnLOH of 9p and 10q and gain of 12p and 17q, remained significant for PFS (HR 2.0 for 9p, 0.5 for 10q, 2.8 for 12p, and 2.0 for 17q), while FLIPI risk and serum β2M/LDH levels were no longer significant. In particular, estimated two-year PFS was lower in patients with 9p deletion or cnLOH (60% vs 77%), gain of 12p (62% vs 78%), and gain of 17q (59% vs 80%) compared with those without.Conclusion: We confirmed the frequent 1p and 6q deletions in FL reported in literature. In addition, we identified several genomic aberrations that may be prognostic of FL patients. Among these, deletions and cnLOH of CDKN2A (9p), PTEN (10q) and CREBBP (16p) are of significant interest for their known tumor suppressor functions. Gain of 12p, 17q, and 18q may also help identify oncogenes and other activating tumorigenic processes relevant to disease progression and survival. Multivariate analysis suggested that the genomic aberrations might add to currently utilized clinical risk stratification as a means to identify high risk patients at diagnosis of follicular lymphoma.Support: This work was supported by Affymetrix Inc., NIH/NCI National Clinical Trials Network (NCTN) grants CA180888 and CA180819, and in part by GlaxoSmithKline. DisclosuresHsi:Abbvie: Research Funding; Cellerent Therapeutics: Research Funding; Eli Lilly: Research Funding; Onyx: Honoraria; Seattle Genetics: Honoraria. Smith:Pharmacyclics: Consultancy; Celgene: Consultancy. Fang:Affymetrix: Research Funding.

The Gain of the Short Arm of Chromosome 2 (2p+) Induces XPO1 Overexpression and Drug Resistance in Chronic Lymphocytic Leukemia

Introduction: CLL is a heterogeneous disease in terms of response to treatment, with some patients reaching complete and prolonged remissions, while others relapsing early and requiring several lines of treatments. This highly variable course is partly explained by the existence of a heterogenic panel of genetic alterations (mutations, chromosomal abnormalities) that allow the development of drug-resistant aggressive CLL subclones. Therefore, a functional characterization of the cytogenetic alterations associated to CLL drug resistance may provide new means of improving the current therapeutic strategies. We and others have already reported that the gain of 2p (2p+) is recurrent in CLL. However, the candidate gained gene(s) on the 2p remain to be identified. Previously data: we have observed that the 2p gain is frequent in previously untreated CLL Binet stages B/C (21/132, 15.9%), and is associated with bad prognostic factors, such as 11q deletion (p=0.0008) and unmutated IGHV (p=0.02). Using a SNP-array approach, we have identified a minimally gained region of 1.28Mb on 2p16.1-15. This region included the gene CRM1/XPO1 (Chromosome Region Maintenance 1/Exportin-1), a gene also recurrently mutated in CLL. A qPCR assessment confirmed that XPO1 was overexpressed in the 2p+/CLL patients (1.4-fold increase compared to 2p-/CLL; p=0.02). The objective of our work was to identify the potential role of XPO1 in CLL drug resistance by using the selective XPO1 inhibitor Selinexor (KPT-330, provided by Karyopharm Therapeutics), which is currently in Phase II human clinical trials in hematological and solid cancers.Methods: We have analyzed 36 2p+/CLL and we have searched for XPO1 mutations in 436 CLL samples. CLL drug resistance associated to XPO1 overexpression/mutation was assessed by measuring the rate of programmed cell death (PCD) on cells from 2p- and wildtype (wt) XPO1/CLL (n=20), 2p+/XPO1 wt/CLL (n=8) and on XPO1 mut/CLL (n=6). After 24 hours treatment with Fludarabin + Cyclophosphamid + Rituximab (FCR), Ibrutinib (Ibru), Idelalisib + Rituximab (Ide+R) and Selinexor, cells were stained with Annexin-V and propidium iodide and PCD was assessed by flow cytometry. KPT-301 was used as a negative control. For the inhibition assay, the inhibitor Q-VD-Oph was added 30 min before inducing cell death. Mitochondrial membrane depolarisation was assessed using tetramethyllrhodamine ethyl ester probe and flow cytometry analysis.Results: (i) Using a FISH approach, we fully confirmed the gain of XPO1 in 2p+/CLL samples. Additionally, we found that the XPO1 gain was often subclonal, suggesting that it tends to arise late in leukemic development. Longitudinal FISH analyses, performed on 8 2p+/CLL-treated patients, showed a similar or increasing percentage of cells carrying XPO1 gain at relapse, when compared to diagnosis; (ii) XPO1 was mutated in 23/436 (5.3%) CLL and in 2/30 (6.7%) 2p+/CLL; (iii) Selinexor induced PCD in 2p-/XPO1 wt/CLL (35% of PCD). The results were similar in all tested CLL, independently of prognostic factors (del13q, tri12, del11q, del17p, IGHV status), while sparing the non leukemic cells from patients or B cells from healthy donors; (iv) Selinexor induced CLL PCD through a caspase-dependant apoptotic pathway, as evidenced by inhibition of cell death by Q-VD-Oph, and cleavage of the caspase-3. Selinexor also induced mitochondrial depolarization and was associated with upregulation and activation of the pro-apopototic Bax protein; (v) XPO1 mut/CLL were significantly resistant to PCD induced by Selinexor (p=0.003). In contrast, the mutations in XPO1 had no effect in FCR and Ibru PCD induction; (vi) 2p+/CLL cells were resistant to PCD induced by all tested drugs: FCR (p=0.01), Ibru (p=0.003), Ide+R (p=0.004) and Selinexor (p=0.0001).Conclusion: Our data show that 2p+/CLL is associated to FCR, Ibru and Ide+R drug resistance. Strikingly, Selinexor, a new XPO1 inhibitor, is unable to induce PCD in 2p+ and/or XPO1 mut CLL, which strongly suggests a key role for XPO1 in the CLL drug resistance associated to the 2p gain. Altogether, our work provide substantial progress in the understanding of the role of XPO1 in CLL drug resistance and suggests that the assessment of the 2p gain and the mutations in XPO1 will be considered before to decide a CLL therapy. As 2p gain could be observed in other B malignancies, it is tempting to extend these recommendations to all Selinexor treatments. DisclosuresChoquet:Janssen: Consultancy; Roche: Consultancy. Leblond:Janssen: Consultancy, Honoraria, Speakers Bureau; GSK: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Mundipharma: Honoraria.

Secretory Breast Carcinoma: A Histopathologic and Genomic Spectrum Characterized by a Joint Specific ETV6-NTRK3 Gene Fusion.

Secretory breast carcinoma (SBC) is a rare breast carcinoma with distinctive morphologic features and a recurrent specific chromosomal translocation t(12;15)(p13;q25), usually of low histologic grade and favorable prognosis. We describe the morphologic and genetic characteristics of 11 cases of SBC from 10 patients. Histologic and immunohistochemical analyses, fluorescence in situ hybridization using break-apart probes specific to ETV6 on 12p13, reverse transcription polymerase chain reaction with in-house probes specific to the ETV6-NTRK3 gene fusion, and DNA copy number variation by array comparative genomic hybridization analyses were performed on all cases. Seven cases were of low histologic grade, 3 were intermediate, and 1 had high-grade nuclear atypia, necrosis, and numerous mitoses. This patient had a fatal outcome. Five cases displayed low hormonal receptor expression, whereas the rest had basal-type immunoprofiles. All interpretable cases harbored an ETV6-NTRK3 gene fusion by reverse transcription polymerase chain reaction and/or an ETV6 rearrangement by fluorescence in situ hybridization, with duplication of the oncogenic derivative in 2 cases. Array comparative genomic hybridization analysis showed simplex genomic profiles. The 2 cases with ETV6-NTRK3 duplication included a gain of 12p starting from the ETV6 locus to the telomere, associated with a gain of the 15q from the centromere to NTRK3 in 1 case, and in the other a normal profile up to NTRK3 on 15q, and then a loss up to the telomere, suggesting loss of corresponding normal chromosome 15. These findings provide a novel insight into the morphologic and genetic spectrum of SBC, ranging from low-grade to high-grade histology, with occasional low hormonal receptor expression, simplex genomic profiles, and possible unfavorable course.

Heterogeneity revealed by integrated genomic analysis uncovers a molecular switch in malignant uveal melanoma.

Gene expression profiles as well as genomic imbalances are correlated with disease progression in uveal melanoma (UM). We integrated expression and genomic profiles to obtain insight into the oncogenic mechanisms in development and progression of UM. We used tumor tissue from 64 enucleated eyes of UM patients for profiling. Mutations and genomic imbalances were quantified with digital PCR to study tumor heterogeneity and molecular pathogenesis. Gene expression analysis divided the UM panel into three classes. Class I presented tumors with a good prognosis and a distinct genomic make up that is characterized by 6p gain. The UM with a bad prognosis were subdivided into class IIa and class IIb. These classes presented similar survival risks but could be distinguished by tumor heterogeneity. Class IIa presented homogeneous tumors while class IIb tumors, on average, contained 30% of non-mutant cells. Tumor heterogeneity coincided with expression of a set of immune genes revealing an extensive immune infiltrate in class IIb tumors. Molecularly, class IIa and IIb presented the same genomic configuration and could only be distinguished by 8q copy number. Moreover, UM establish in the void of the immune privileged eye indicating that in IIb tumors the infiltrate is attracted by the UM. Combined our data show that chromosome 8q contains the locus that causes the immune phentotype of UM. UM thereby provides an unique opportunity to study immune attraction by tumors.

Abstract 4262: PRAME as a biomarker for a new molecular subclass of uveal melanoma

Abstract Introduction: Uveal melanoma is the most common primary intraocular tumor of the eye and has a propensity for fatal hematogenous metastasis. Gene expression profiling is routinely used in clinic to classify uveal melanoma into prognostic subgroups: Class 1 (low metastatic risk) and Class 2 (high metastatic risk). Approximately 40% of Class 2 patients metastasize, which accounts for 85% of metastasis. The remaining 15% of patients that metastasize are Class 1 based on gene expression profiling, however this study demonstrates that the Class 1 patients that metastasize (Class1met+) can be defined as a new molecular subclass, distinct from Class 1 patients that do not metastasize (Class1met-) and Class 2 patients. Methods: Tumor samples from Class1met+ and Class1met- patients with at least 1 year of follow-up underwent histopathologic, cytogenetic and transcriptomic analyses. Results: In a comparison of Class1met+ and Class1met- tumors, the only significant difference was a larger mean basal tumor diameter in Class1met+ tumors. Cytogenetic analyses revealed more extensive copy number gains and losses in Class1met+ tumors. The most significant differentiating features in Class1met+ tumors included 1q gain, 6p gain, 6q loss, and 8q gain. Using principal component analysis, the Class1met+ tumors clustered separately from the Class1met- tumors, indicating the existence of significant transcriptomic differences between the two subgroups. Strikingly, the most highly differentially expressed gene in Class1met+ tumors was preferentially expressed antigen in melanoma (PRAME); mean mRNA expression of PRAME in Class1met+ tumors was 61-fold higher than in Class1met- tumors. Discussion: We define a new subtype of uveal melanoma and demonstrate that gene expression of PRAME can distinguish between Class1met+ patients and Class1met- patients. Citation Format: Matthew G. Field, Christina L. Decatur, J. William Harbour. PRAME as a biomarker for a new molecular subclass of uveal melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4262. doi:10.1158/1538-7445.AM2015-4262

Genetic abnormalities in adolescents and young adults with neuroblastoma: A report from the Italian Neuroblastoma group.

Less than 5% of neuroblastomas (NB) occur in adolescents and young adults (AYA), in whom the disease has an indolent and fatal course. We studied the genomic profile and histological characteristics of 34 NBs from AYA patients enrolled in the Italian Neuroblastoma Registry (INBR) between 1979 and 2009. Disease was disseminated in 20 patients and localized in 14; 30/34 tumors were classified as NB and 4/34 as nodular ganglioneuroblastoma (nGNB). Segmental Chromosome Aberrations (SCAs) were observed in 29 tumors (85%) namely 1p imbalance (58%), 17q gain (52%), 9p loss (32%), 11q loss (30%), 1q gain (17%), 7q gain (17%), 2p gain (14%), 3p loss (14%), and 4p loss (7%). MYCN amplification and MYCN gain were detected in 3 (10%) and 2 cases (7%) respectively. An anaplastic lymphoma receptor tyrosine kinase (ALK) gene mutation study on the available cases from this cohort revealed 4/25 (16%) mutated cases. In parallel, alpha thalassaemia/mental retardation syndrome X linked (ATRX) gene mutations were also sought, a novel mutation being detected in 1/21 (4,7%) cases. This study confirmed the low incidence of MYCN amplification in AYA and recorded a high frequency of 17q gain and 9p and 11q loss independently from the stage of the disease. The presence of 1q gain, which identifies patients with particularly aggressive disease, relapse and poor survival, was also detected. Furthermore, the frequency of ALK mutations suggests that a target-based therapy with ALK inhibitors might be effective in this subset of patients.

Patterns of chromosomal copy-number alterations in intrahepatic cholangiocarcinoma.

BackgroundIntrahepatic cholangiocarcinomas (ICC) are relatively rare malignant tumors associated with a poor prognosis. Recent studies using genome-wide sequencing technologies have mainly focused on identifying new driver mutations. There is nevertheless a need to investigate the spectrum of copy number aberrations in order to identify potential target genes in the altered chromosomal regions. The aim of this study was to characterize the patterns of chromosomal copy-number alterations (CNAs) in ICC.Methods53 patients having ICC with frozen material were selected. In 47 cases, DNA hybridization has been performed on a genomewide SNP array. A procedure with a segmentation step and a calling step classified genomic regions into copy-number aberration states. We identified the exclusively amplified and deleted recurrent genomic areas. These areas are those showing the highest estimated propensity level for copy loss (resp. copy gain) together with the lowest level for copy gain (resp. copy loss). We investigated ICC clustering. We analyzed the relationships between CNAs and clinico-pathological characteristics.ResultsThe overall genomic profile of ICC showed many alterations with higher rates for the deletions. Exclusively deleted genomic areas were 1p, 3p and 14q. The main exclusively amplified genomic areas were 1q, 7p, 7q and 8q. Based on the exclusively deleted/amplified genomic areas, a clustering analysis identified three tumors groups: the first group characterized by copy loss of 1p and copy gain of 7p, the second group characterized by 1p and 3p copy losses without 7p copy gain, the last group characterized mainly by very few CNAs. From univariate analyses, the number of tumors, the size of the largest tumor and the stage were significantly associated with shorter time recurrence. We found no relationship between the number of altered cytobands or tumor groups and time to recurrence.ConclusionThis study describes the spectrum of chromosomal aberrations across the whole genome. Some of the recurrent exclusive CNAs harbor candidate target genes. Despite the absence of correlation between CNAs and clinico-pathological characteristics, the co-occurence of 7p gain and 1p loss in a subgroup of patients may suggest a differential activation of EGFR and its downstream pathways, which may have a potential effect on targeted therapies.

Whole-exome sequencing reveals the mutational spectrum of testicular germ cell tumours.

Testicular germ cell tumours (TGCTs) are the most common cancer in young men. Here we perform whole-exome sequencing (WES) of 42 TGCTs to comprehensively study the cancer's mutational profile. The mutation rate is uniformly low in all of the tumours (mean 0.5 mutations per Mb) as compared with common cancers, consistent with the embryological origin of TGCT. In addition to expected copy number gain of chromosome 12p and mutation of KIT, we identify recurrent mutations in the tumour suppressor gene CDC27 (11.9%). Copy number analysis reveals recurring amplification of the spermatocyte development gene FSIP2 (15.3%) and a 0.4 Mb region at Xq28 (15.3%). Two treatment-refractory patients are shown to harbour XRCC2 mutations, a gene strongly implicated in defining cisplatin resistance. Our findings provide further insights into genes involved in the development and progression of TGCT.

Non-invasive detection of genomic imbalances in Hodgkin/Reed-Sternberg cells in early and advanced stage Hodgkin's lymphoma by sequencing of circulating cell-free DNA: a technical proof-of-principle study

Hodgkin's lymphoma is one of the most common lymphoid neoplasms in young adults, but the low abundance of neoplastic Hodgkin/Reed-Sternberg cells in the tumour hampers the elucidation of its pathogenesis, biology, and diversity. After an incidental observation that genomic aberrations known to occur in Hodgkin's lymphoma were detectable in circulating cell-free DNA, this study was undertaken to investigate whether circulating cell-free DNA can be informative about genomic imbalances in Hodgkin's lymphoma. We applied massive parallel sequencing to circulating cell-free DNA in a prospective study of patients with biopsy proven nodular sclerosis Hodgkin's lymphoma. Genomic imbalances in Hodgkin/Reed-Sternberg cells were investigated by fluorescence in-situ hybridisation (FISH) on tumour specimens. By non-invasive prenatal testing, we observed several genomic imbalances in circulating cell-free DNA of a pregnant woman, who was subsequently diagnosed with early-stage nodular sclerosis Hodgkin's lymphoma stage IIA during gestation. FISH on tumour tissue confirmed corresponding genomic imbalances in Hodgkin/Reed-Sternberg cells. We prospectively studied circulating cell-free DNA of nine nodular sclerosis Hodgkin's lymphoma cases: eight at first diagnosis and one at first relapse. Seven patients had stage IIA disease and two had stage IVB disease. In eight, genomic imbalances were detected, including, among others, gain of chromosomes 2p and 9p, known to occur in Hodgkin's lymphoma. These gains and losses in circulating cell-free DNA were extensively validated by FISH on Hodgkin/Reed-Sternberg cells in biopsy samples. Initiation of chemotherapy induced normalisation of circulating cell-free DNA profiles within 2-6 weeks. The cell cycle indicator Ki67 and cleaved caspase-3 were detected in Hodgkin/Reed-Sternberg cells by immunohistochemistry, suggesting high turnover of Hodgkin/Reed-Sternberg cells. In early and advanced stage nodular sclerosis Hodgkin's lymphoma, genomic imbalances in Hodgkin/Reed-Sternberg cells can be identified by massive parallel sequencing of circulating cell-free DNA at diagnosis. The rapid normalisation of circulating cell-free DNA profiles on therapy initiation suggests a potential role for circulating cell-free DNA profiling in early response monitoring. This finding creates several new possibilities for exploring the diversity of Hodgkin's lymphoma, and has potential implications for the future clinical development of biomarkers and precision therapy for this malignancy. KU Leuven-University of Leuven and University Hospitals Leuven.

BIRC3 disruption and Copy Number Aberrations in Chronic Lymphocytic Leukemia (CLL) Patients with 11q Deletion

Introduction. Chronic lymphocytic leukemia (CLL) with 11q deletion has been associated to a poor prognosis, but the clinical course of patients carrying this lesion is variable. This aberration, most often monoallelic, is present in 10-17% of newly diagnosed CLL and in 20-30% of patients with progressive or chemorefractory disease. The minimal deleted region (MDR) (2-3 Mbp) is located on the 11q22.3-q23.1 region and includes ATM. Moreover, 30-40% of 11q- CLL have also an inactivating ATM mutation on the other allele. The deleted region on 11q can also include BIRC3, a gene that is often deleted or mutated in advanced/chemorefractory stages of the disease. Although BIRC3 disruption has been associated to a poor prognosis, its prognostic implications in addition to ATM deletion are not well defined. The aim of this study was to perform a copy number aberration (CNA) and gene sequencing analyses on a cohort of CLL patients with 11q- in order to identify subgroups with potential prognostic relevance based on: i) the inclusion of BIRC3 in the deleted region; ii) the presence of BIRC3 mutation; iii) the presence of other CNAs.Methods. The study has included 55 untreated CLL patients followed at our Institution or enrolled in GIMEMA clinical trials (2003-2013). Genomic DNA was extracted from peripheral blood samples. CNA analysis was performed by genomic hybridization on the CytoScan HD array (Affymetrix), which contains more than 2.6 x 106 markers for copy number analysis and 750.000 SNPs. Data were analyzed using both Partek Genomics Suite and ChAS (Chromosome Analysis Suite, Affymetrix) software. The resulting CNAs were verified by visual examination of the plotted copy number profiles. BIRC3 mutations (exons 6-9) were evaluated by Sanger sequencing. Time to first treatment (TFT) was calculated from the date of diagnosis to the date of first therapy or last follow-up; progression-free survival (PFS) from the date of first therapy to the date of progression, death or last follow-up.Results. Baseline characteristics of the 55 cases were as follows: median age at diagnosis 59 years (range 39-84), male gender in 81.8% of patients, progressive disease in 62%. All patients showed 11q- by FISH (median 80%, range 25-99% of nuclei); germline IGHV were present in 96.4% of cases; TP53 deletion in 1 case and TP53 mutation in none; NOTCH1 mutation in 4/40 cases; SF3B1 mutation in 5/40 (all mutually exclusive with only 1 case having both SF3B1 and BIRC3 mutations). By CytoScan HD array, the size of 11q- was very variable, ranging from 0.36 Mbp to 65.14 Mbp; the MDR was located on 11q22.3 region, encompassing 4 genes (ACAT1, ATM, CUL5, NPAT). BIRC3 was included in the deleted region in 45/55 cases (81.8%) and was mutated in 4/54 (7.5%), being always deleted on the other allele. Beside 11q-, 51 cases (92.7%) showed several additional CNAs (average 4.9, range 1-14 per patient), with 5 recurrent lesions: 2p gain in 11 cases, del4(p15.2) in 6, del19(p13.3) in 6, 8q gain in 5 and del4(q22.1) in 4. BIRC3 deletion was not associated to the number of additional CNAs nor to specific CNA. After a follow-up of 59.6 months (range 7.4-229.7), 40 of 47 evaluable patients have received treatment (median TFT 15.8 months, range 0-167). BIRC3 deleted cases (n=37) showed a TFT not significantly different from WT cases (n=10). Conversely, BIRC3 mutation was associated to a shorter TFT (p <0.0001), 0.5 vs 19.3 months of WT cases. Notably, 3/4 cases with the biallelic BIRC3 disruption had a marked hyperleukocytosis at diagnosis (212.4-302.8 x 109L). The presence of additional CNAs was associated to a shorter, though not significant, TFT considering either >3 CNAs larger than 5 Mb (n=14) or >10 CNAs (n=5), or the presence of 2p gain, del4(p15.2), del19(p13.3) or 8q gain. So far, 22 patients have been evaluated for PFS after first-line therapy (median 28.7 months). BIRC3 deleted cases (n=17) were not associated to a shorter PFS compared to WT cases (n=5), in line with the results from Rose-Zerilli et al (Haematologica 2014).Conclusions. Among CLL with 11q-: 1) BIRC3 deletion involves more than 80% of cases, whilst the mutation is rare (7.5%); 2) BIRC3 deletion is not associated to a higher genomic complexity; 3) BIRC3 deletion does not seem to influence TFT or PFS of 11q- CLL; 4) BIRC3 mutation is strongly associated to a short TFT; 5) BIRC3 biallelic lesions can be associated to a marked hyperleucocytosis at diagnosis and immediate need of treatment. DisclosuresNo relevant conflicts of interest to declare.

Intragenic anaplastic lymphoma kinase (ALK) rearrangements: translocations as a novel mechanism of ALK activation in neuroblastoma tumors.

Anaplastic lymphoma kinase (ALK) has been demonstrated to be deregulated in sporadic as well as in familiar cases of neuroblastoma (NB). Whereas ALK-fusion proteins are common in lymphoma and lung cancer, there are few reports of ALK rearrangements in NB indicating that ALK mainly exerts its oncogenic capacity via activating mutations and/or overexpression in this tumor type. In this study, 332 NB tumors and 13 cell lines were screened by high resolution single nucleotide polymorphism microarray. Gain of 2p was detected in 23% (60/332) of primary tumors and 46% (6/13) of cell lines, while breakpoints at the ALK locus were detected in four primary tumors and two cell lines. These were further analyzed by next generation sequencing and a targeted enrichment approach. Samples with both ALK and MYCN amplification displayed complex genomic rearrangements with multiple breakpoints within the amplicon. None of the translocations characterized in primary NB tumors are likely to result in a chimeric protein. However, immunohistochemical analysis reveals high levels of phosphorylated ALK in these samples despite lack of initial exons, possibly due to alternative transcription initiation sites. Both ALK proteins predicted to arise from such alterations and from the abnormal ALK exon 4-11 deletion observed in the CLB-BAR cell line show strong activation of downstream targets STAT3 and extracellular signal-regulated kinase (ERK) when expressed in PC12 cells. Taken together, our data indicate a novel, although rare, mechanism of ALK activation with implications for NB tumorigenesis.

ASTROCYTIC TUMORS WITH GAIN ON 7Q AND THOSE WITH GAIN OF WHOLE CHROMOSOME 7 BELONG TO DISTINCT SUBGROUPS CHARACTERIZED BY VARIOUS GENETIC FACTORS INCLUDING IDH1 AND TP53

BACKGROUND: Many genetic studies on gliomas have reported the utility of DNA copy number aberrations (CNAs) in tumor classification, and we have previously shown that grade II–III adult supratentorial gliomas could be classified into clinically relevant subgroups based on the CNAs. The major CNAs in astrocytic tumors of these grades were gain of whole chromosome 7 (+7) and that on chromosomal arm 7q (+7q), and tumors with +7 had a worse outcome than tumors with 7q. However, it was not confirmed that the former could develop from the latter; that is, it is not clear that a stepwise DNA copy number gain on chromosome 7, beginning as partial gain of 7q, develops along with glioma progression. We then aimed to establish glioma development pathway model by combining information on CNAs and other genetic information. METHODS: We analyzed the chromosomal DNA copy number aberrations of 160 adult supratentorial gliomas using comparative genomic hybridization as well as mutational status of IDH1 and TP53 by PCR-based direct sequencing. We also performed immunohistochemistry for c-Met whose gene is located in 7q34, that is included in the region with copy number gain in both +7 and +7q tumors. RESULTS: The most frequent aberration in IDH1 mutant tumors was -1p/19q followed by +7q, and, in contrast, IDH1 wild-type tumors showed +7 and, less frequently in grade II-III tumors, -10q. Kaplan-Meier estimates for progression free survival showed that the tumors with mutant IDH1, -1p/19q, or +7q (in the absence of +7p) survived longer than tumors with wildtype IDH1, +7, or -10q. Furthermore, +7q tumors showed TP53 mutation and c-Met overexpression whereas +7 tumors did not show these features. Paired analysis of astrocytic tumors for primary and recurrent disease showed that the majority of +7q tumors progressed without additional gain of 7p. In contrast to tumors with gain on chromosome 7, there is a lack of evidence to indicate that tumors with 1p/19q loss are composed of multiple lineages. CONCLUSIONS: We found +7q tumors were distinct tumor lineage from tumors with whole 7 gain, and, although associated with CNA in common region of the chromosome, tumors with a gain on chromosome 7 (mostly astrocytic) comprise multiple lineages, and such differences in their biological nature should be taken into consideration during their clinical management. SECONDARY CATEGORY: n/a.

Genomic characterization of ependymomas reveals 6q loss as the most common aberration.

Ependymomas are rare tumors of the central nervous system (CNS). They are classified based on tumor histology and grade, but the prognostic value of the WHO grading system remains controversial. Treatment is mainly surgical and by radiation. An improved knowledge of ependymoma biology is important to elucidate the pathogenesis, to improve classification schemes, and to identify novel potential treatment targets. Only 113 ependymoma karyotypes with chromosome aberrations are registered in the Mitelman database. We present the first study of ependymoma genomes combining karyotyping and high resolution comparative genomic hybridization (HR-CGH). Nineteen tumor samples were collected from three pediatric and 15 adult patients treated at Oslo University Hospital between 2005 and 2012. Histological diagnoses included subependymoma and myxopapillary ependymoma (WHO grade I), ependymoma (WHO grade II) and anaplastic ependymoma (WHO grade III). Four tumors were intraspinal and 15 were intracranial. Seventeen samples were successfully karyotyped, HR-CGH analysis was undertaken on 17 samples, and 15 of 19 tumors were analyzed using both methods. Twelve tumors had karyotypic abnormalities, mostly gains or losses of whole chromosomes. Structural rearrangements were found in four tumors, in two of which 2p23 was identified as a breakpoint region. Twelve tumors displayed genomic imbalances by HR-CGH analysis with loss of material at 6q as the most common. 6q loss, which was detected by one or both methods in seven of 12 (58%) abnormal tumors, and 5p gain (observed in five tumors; 42%) were the most common genomic aberrations in this series.