High Resolution aCGH Analysis Research Articles

Characterization of FGFR1 Locus in sqNSCLC Reveals a Broad and Heterogeneous Amplicon.

FGFR1 amplification occurs in ~20% of sqNSCLC and trials with FGFR inhibitors have selected FGFR1 amplified patients by FISH. Lung cancer cell lines were profiled for sensitivity to AZD4547, a potent, selective inhibitor of FGFRs 1–3. Sensitivity to FGFR inhibition was associated with but not wholly predicted by increased FGFR1 gene copy number. Additional biomarker assays evaluating expression of FGFRs and correlation between amplification and expression in clinical tissues are therefore warranted. We validated nanoString for mRNA expression analysis of 194 genes, including FGFRs, from clinical tumour tissue. In a panel of sqNSCLC tumours 14.4% (13/90) were FGFR1 amplified by FISH. Although mean FGFR1 expression was significantly higher in amplified samples, there was significant overlap in the range of expression levels between the amplified and non-amplified cohorts with several non-amplified samples expressing FGFR1 to levels equivalent to amplified samples. Statistical analysis revealed increased expression of FGFR1 neighboring genes on the 8p12 amplicon (BAG4, LSM1 and WHSC1L1) in FGFR1 amplified tumours, suggesting a broad rather than focal amplicon and raises the potential for codependencies. High resolution aCGH analysis of pre-clinical and clinical samples supported the presence of a broad and heterogeneous amplicon around the FGFR1 locus. In conclusion, the range of FGFR1 expression levels in both FGFR1 amplified and non-amplified NSCLC tissues, together with the breadth and intra-patient heterogeneity of the 8p amplicon highlights the need for gene expression analysis of clinical samples to inform the understanding of determinants of response to FGFR inhibitors. In this respect the nanoString platform provides an attractive option for RNA analysis of FFPE clinical samples.

Abstract IA22: An oncogenic enhancer drives NOTCH1-induced leukemia.

Abstract Most genetic abnormalities found in cancer are located in intergenic regions and their role in cancer development remains poorly understood. Here we report the identification of a long range enhancer driving NOTCH1-induced transformation in T-ALL. Using genome wide ChIPseq mapping of NOTCH1 binding and functional epigenetic marks in T-ALL we identified a NOTCH1-occupied enhancer located in a gene desert island in 8q24, +1,427 kb 3' from the MYC locus. This regulatory element named N-Me for NOTCH-bound MYC enhancer activates MYC reporter constructs in T-ALL cells and directly interacts with MYC promoter sequences via long range chromatin loops. Notably, high resolution aCGH analysis revealed recurrent focal duplications at chromosome 8q24 encompassing this enhancer in 8/160 (5%) T-ALL cases. Functional characterization of N-Me in T-cell development and transformation using N-Me knockout and conditional knockout mice revealed a major role for this enhancer during thymocyte development and in T-ALL. Thus N-Me null animals showed a marked and selective reduction in thymus size and cellularity and were completely resistant to NOTCH1-induced transformation. Moreover, secondary deletion of N-Me in established NOTCH1 induced leukemias from tamoxifen-inducible N-Me conditional knockout mice induced profound antileukemic effects with extended survival and almost complete suppression of leukemia initiating cell activity. Altogether, these results identify N-Me as a major downstream effector NOTCH1 critically required for MYC expression in T-cell development and leukemia. Citation Format: Adolfo Ferrando. An oncogenic enhancer drives NOTCH1-induced leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA22.

­­­­­­N-Me, a Long Range T-Cell Specific Oncogenic Enhancer in T-ALL

Over the last years, numerous studies have dissected the mutational landscape of T-cell acute lymphoblastic leukemia (T-ALL) resulting in the identification of numerous oncogenes and tumor suppressors implicated in T-cell transformation. However, most genetic abnormalities found in cancer are located in intergenic regions, whose role in cancer development, if any, remains poorly understood. Here we hypothesized that recurrent cancer-associated intergenic mutations, amplifications and deletions may implicate strong transcriptional regulatory sequences responsible for the activation of key oncogenic factors in the pathogenesis of T-ALL. To address this question, we first used ChIPseq analysis to map the genomic landscape of enhancer sequences controlled by NOTCH1, a critical T-ALL oncogene activated by mutations in over 60% of human T-ALLs. In addition, we performed high resolution aCGH analysis of somatic chromosomal amplifications and deletions in a comprehensive series of 160 T-ALL samples. These analyses revealed recurrent focal duplications at chromosome 8q24 in 8/160 (5%) T-ALL cases in an area devoid of protein-coding genes containing a prominent 1 kb NOTCH1-binding peak. Strikingly, this putative oncogenic element is located +1,427 kb 3’ from the MYC locus and chromatin configuration 3C analysis demonstrated its direct association with the MYC proximal promoter. Multispecies DNA sequence alignment revealed remarkable conservation of this region in mammals, birds and reptiles and local ChIP analysis revealed bona fide active enhancer features including P300 occupancy and high levels of H3K4me1 with low levels of H3K4me3. In addition, detailed analysis of epigenetic marks across 64 hematopoietic and non-hematopoietic cell lines and tissues revealed that his regulatory element is located within a major superenhancer specifically active in T-cells. Based on these results, we proposed that this regulatory sequence, hereby named N-Me for NOTCH-bound MYCenhancer, could function as an important regulatory element driving the activation of MYC downstream of NOTCH1 in T-ALL. Consistently, luciferase reporter assays showed strong, orientation-independent and NOTCH-dependent activation of reporter constructs containing N-Me in association with a -2.5 kb MYC proximal promoter in JURKAT T-ALL cells.Next and to formally test the functional relevance of this enhancer in T-cell development and transformation we generated and characterized N-Me knockout and conditional knockout mice. N-Me null animals were viable and showed a marked and selective reduction in thymus size and cellularity as their only developmental alteration. Detailed immunophenotypic analysis of N-Me knockout thymocytes demonstrated accumulation of double negative 3 (DN3) T cells and marked reductions in double positive and CD4+ and CD8+ single positive cells. Mechanistically, this phenotype was associated with a marked reduction in Myc expression in DN3, DN4 and ISP cells. Moreover, transplantation of N-Me knockout bone marrow hematopoietic progenitors demonstrated that this phenotype is cell autonomous, and can be rescued upon retroviral expression of MYC. Next, we analyzed the role of N-Me in the induction of NOTCH-driven leukemias by transplanting mice with N-Me wild-type and knockout hematopoietic progenitors infected with retroviruses expressing a mutant constitutively active form of NOTCH1 (ΔE-NOTCH1). In this context, mice transplanted with ΔE-NOTCH1-infected N-Me wild type cells developed T-ALL with 100% penetrance 6 weeks after transplant. In contrast, mice transplanted with ΔE-NOTCH1-N-Me knockout cells showed complete resistance to NOTCH1-induced transformation remaining 100% leukemia-free at 15 weeks post-transplant. In addition, secondary deletion of N-Me in established NOTCH1 induced leukemias from tamoxifen-inducible N-Me conditional knockout mice (Rosa26TMCre N-Meflox/flox) induced profound antileukemic effects with extended survival and almost complete suppression of leukemia initiating cell activity. Altogether, these results identify N-Me as the first long range oncogenic enhancer directly implicated in the pathogenesis of human leukemia. DisclosuresNo relevant conflicts of interest to declare.

Modulation of chemotherapeutic drug resistance in neuroblastoma SK-N-AS cells by the neural apoptosis inhibitory protein and miR-520f.

The acquisition of multidrug resistance is a major impediment to the successful treatment of neuroblastoma, a clinically heterogeneous cancer accounting for ∼15% of all pediatric cancer deaths. The MYCN transcription factor, whose gene is amplified in ∼30% of high-risk neuroblastoma cases, influences drug resistance by regulating a cadre of genes, including those involved with drug efflux, however, other high-risk subtypes of neuroblastoma lacking MYCN amplification, such as those with chromosome 11q deletions, also acquire multidrug resistance. To elucidate additional mechanisms involved with drug resistance in non-MYCN amplified tumour cells, an SK-N-AS subline (SK-N-AsCis24) that is significantly resistant to cisplatin and cross resistant to etoposide was developed through a pulse-selection process. High resolution aCGH analysis of SK-N-AsCis24 revealed a focal gain on chromosome 5 containing the coding sequence for the neural apoptosis inhibitory protein (NAIP). Significant overexpression of NAIP mRNA and protein was documented, while experimental modulation of NAIP levels in both SK-N-AsCis24 and in parental SK-N-AS cells confirmed that NAIP was responsible for the drug resistant phenotype by apoptosis inhibition. Furthermore, a decrease in the NAIP targeting microRNA, miR-520f, was also demonstrated to be partially responsible for increased NAIP levels in SK-N-AsCis24. Interestingly, miR-520f levels were determined to be significantly lower in postchemotherapy treatment tumours relative to matched prechemotherapy samples, consistent with a role for this miRNA in the acquisition of drug resistance in vivo, potentially through decreased NAIP targeting. Our findings provide biological novel insight into neuroblastoma drug-resistance and have implications for future therapeutic research.

DOCK4 deletion at 7q31.1 in a de novo acute myeloid leukemia with a normal karyotype

Acute myeloid leukemia with a normal karyotype (NK-AML) has been assigned to an intermediate prognostic risk group. However, there is a marked variability in outcome within this group of AML, suggesting a significant biological and molecular heterogeneity. Chromosomal abnormalities may be cryptic by metaphase cytogenetics, but still have an impact on the process of leukemogenesis and/or the clinical outcome of NK-AML. Therefore, we analyzed the genomic complement of NK-AML cases to search for the presence of submicroscopic genomic imbalances. We applied high-resolution oligo-based aCGH analysis to a cohort of AML patients with a normal karyotype, and FISH to validate the aCGH findings. Relative gene expression levels were examined by qPCR. High-resolution aCGH analysis of 21 NK-AML patients revealed one patient with a rare submicroscopic deletion at 7q31.1. This female patient exhibited a rapid disease progression and a dismal outcome. The deletion was mono-allelic, approximately 189,1 kb in size, and encompassed the major 3' part of the DOCK4 gene. The expression level of the DOCK4 gene in her leukemic cells was decreased when compared to CD34+ cells from healthy controls. When compared to AML patients with -7/del(7q) as the sole chromosomal anomaly, the DOCK4 expression level was found to be similarly low. This is the first report of an acquired partial deletion of the DOCK4 gene in a patient with de novo NK-AML. DOCK4 is a strong tumor suppressor candidate, required for GTPase activation in signal transduction pathways controlling cellular proliferation, adhesion, migration and phagocytosis. Our findings may be relevant for understanding of the process of leukemogenesis and the response to therapy in a subset of NK-AML patients.

Abstract 3133: Identification and functional validation of novel genetically-linked breast cancer targets through pooled gain-of-function screening.

Abstract Breast cancer is one of the most common cancer types, with greater than 450,000 deaths reported per year worldwide. Through genome wide sequencing efforts, multiple genetic alterations have been identified, including mutations and amplifications in genes such as v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2), GATA binding protein 3 (GATA3), phosphatidylinositol 3-kinase alpha catalytic subunit (PIK3CA) as well as novel genomic rearrangements such as the recently identified MAGI3-AKT3 fusion. Now that breast cancer can be characterized to an unprecedented level, one of the key challenges remaining is to identify and distinguish critical ‘driver’ events responsible for tumor progression, from neutral ‘passenger’ lesions. In order to achieve this, we utilized high resolution aCGH analysis of 50 purified breast cancer samples (made up of Her2+, estrogen receptor positive (ER+) and triple negative tumors with variable responses to SOC regimens), in combination with a Gain-of-Function transformation screen to identify and validate novel breast targets. 158 genomic regions were found to be recurrently amplified, consisting of 759 genes in total. The top 32 focally amplified genes, along with 12 cancer-relevant mutant alleles were prioritized and a library generated utilizing the pTRIPZ-tetracycline regulated inducible lentiviral vector system. These 44 genes were subsequently combined into 16 different target pools (5-13 targets per pool, co-expressing genes that were co-amplified in the same clinical specimen) and evaluated for their ability to transform immortalized breast epithelial MCF10A cells (both wild-type and p53 -/- cells). Through this screening approach, p21-activated kinase 1 (PAK1) was identified, whose kinase activity was required to robustly transform MCF10A cells through regulating multiple signalling pathways including MAPK. Several other putative oncogenes were also identified and will be presented here, including the glycosyltransferse asparagine-linked glycosylation 8 (ALG8). Interestingly, PAK1 and ALG8 are co-amplified in both breast (8%) and ovarian cancers (11%). Our target validation studies have suggested that ALG8 can support PAK1-induced transformation, as dramatic suppression of soft-agar colony growth was seen in co-amplified breast cancer cell lines upon combined siRNA treatment to both targets. Thus, this combined high resolution aCGH profiling and functional screening approach has enabled the successful identification of novel oncogenic targets in breast cancer. Citation Format: Krishna Vasudevan, Axel Hernandez, Zhongwu Lai, Yonghong Xiao, Nin Guan, Carolyn Hardy, Robert Godin, Christopher Denz, Minwei Ye, Elizabeth Lenkiewicz, Stephanie Savage, Michael T. Barrett, Donna Prunkard, Peter Rabinovitch, Mark Basik, Ewa Przybytkowski, Kevin Webster, Michael Zinda, Emma-Louise Jenkins. Identification and functional validation of novel genetically-linked breast cancer targets through pooled gain-of-function screening. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3133. doi:10.1158/1538-7445.AM2013-3133

Studies of genomic imbalances and the MYB-NFIB gene fusion in polymorphous low-grade adenocarcinoma of the head and neck

Polymorphous low-grade adenocarcinoma (PLGA) is a malignancy predominantly originating from the minor salivary glands. The molecular events underlying the pathogenesis of PLGA is poorly understood and no recurrent genetic aberrations have so far been identified. We used genome-wide, high-resolution aCGH analysis to explore genomic imbalances in 9 cases of PLGA. Because of the well-known morphologic similarities between PLGA and adenoid cystic carcinoma (ACC) we also analyzed all tumors for expression of the recently identified ACC-associated MYB-NFIB gene fusion. aCGH analysis revealed that the PLGA genome contains comparatively few copy number alterations (CNAs). Gains/losses of whole chromosomes or chromosome arms were more than twice as common as partial CNAs. Two cases showed gain of chromosome 8 and one case each gain of chromosome 9, loss of chromosome 22 and loss of the Y chromosome. One case showed loss of the entire 6q arm and one case an interstitial deletion of a 33-Mb segment within 6q22.1-q24.3. This region contains the MYB oncogene and the candidate tumor suppressor gene PLAGL1. RT-PCR analysis revealed that one of the 9 PLGAs expressed the ACC-associated MYB-NFIB gene fusion, illustrating the diagnostic difficulties associated with the diagnosis of these morphologically partly overlapping entities. Taken together, our findings indicate that the PLGA genome is genetically stable and contains comparatively few CNAs which is in line with the clinical observation that PLGA is a slow-growing, low-grade carcinoma with low metastatic potential.

Chromosomal Alterations in Gene Expression-Defined Pediatric Aggressive B-Cell Non-Hodgkin Lymphoma (B-NHL).

Abstract 2922Poster Board II-898 Introduction:Lymphomas derived from mature lymphocytes in children and adolescents are predominantly aggressive B-cell non-Hodgkin lymphomas (B-NHL), including Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). Differences in the clinical course between pediatric and adult aggressive B-NHL suggest distinct pathogenetic mechanisms. This study sought to identify both shared and unique genetic alterations between gene expression-defined pediatric and adult cases of BL and DLBCL. Patients and Methods:Gene expression profiling (GEP) was done on 45 BL and 18 DLBCL specimens from patients 18 years of age or younger, and 38 BL and 106 DLBCL from adult patients. Pediatric specimens were collected from the Cooperative Human Tissue Network (CHTN) pediatric NHL repository through the Children's Oncology Group (COG) and adult specimens were collected from the Nebraska Lymphoma Study Group Registry and Tissue Bank through the Lymphoma/Leukemia Molecular Profiling Project (LLMPP). Previously-published gene signatures were used to classify lymphomas molecularly into mBL and mDLBCL groups (Dave et al., NEJM, 2006). The mDLBCL tumors were further classified into activated B-cell-like (ABC), germinal center B-cell-like (GCB), and primary mediastinal B-cell lymphoma (PMBL) subtypes (Rosenwald et al., JEM, 2003). High resolution array comparative genomic hybridization (aCGH) was done on a subset of the pediatric cases using the 250K NspI Human Mapping Array (Affymetrix) to detect DNA copy number alterations (CNA). Results:Molecular classification of the pediatric cases resulted in a 20% reclassification rate for cases with a morphologic diagnosis of BL or DLBCL. Among the 63 pediatric cases, there were 38 mBL (3 of which were DLBCL by morphology), 23 mDLBCL (9 of which were BL by morphology) and two cases which were unclassifiable by the molecular gene signatures. Comparison of the GEP profiles for adult and pediatric mBL failed to identify pathways that differed significantly; however high resolution aCGH analysis revealed a number of abnormalities in the pediatric cases not previously reported in BL, including gains of 3q21, 11q13 and 16p11. A predominance of the GCB to ABC subtype (3:1) was found among pediatric mDLBCL patients. Two cases with mediastinal tumors were classified as PMBL molecularly. Both PMBL cases were female and carried copy number gains of the Rel/BCL11A locus. Comparison of adult and pediatric GCB mDLBCL gene expression revealed enrichment in B-cell surface molecules and markers of antigen-dependent B-cell activation in the adult cases. aCGH analysis identified abnormalities that were both shared (+12q15, +19q13, -6q) between adult and pediatric mDLBCL and unique (-4p14, -19q13.32, +16p11.2) to the pediatric cases. Correlation of DNA copy number and gene expression revealed potential candidate genes for these loci. Conclusions:Pediatric BL and DLBCL classified by morphology were reclassified molecularly in a significant fraction of cases. Although pediatric BL and DLBCL are treated similarly, defining homogeneous molecular entities will be relevant for developing new therapies and future clinical trials. In general, pediatric cases have a more favorable outcome relative to adult patients. However, it is unclear whether this is due to the ability of children to tolerate very intensive therapies or whether distinct pathogenetic mechanisms modulate the disease course. Higher B-cell receptor signaling in adult relative to pediatric GCB DLBCL may be relevant to the outcome. The identification of previously undetected chromosomal alterations unique to the pediatric cases also suggests distinct pathogenetic mechanisms. Elucidation of the underlying genes may provide insight into factors which modulate outcome and could provide novel therapeutic targets with reduced toxicity. Disclosures:Gascoyne:Roche Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Molecular cytogenetic characterization of two cases with constitutional distal 11q duplication/triplication

Here, we report two cases with isolated distal 11q rearrangement and multiple congenital anomalies. The first patient is a two-and-a-half year old male referred to our genetics clinic due to dysmorphic features and developmental delay including speech delay. Using conventional and molecular cytogenetic techniques, we demonstrate that he carries a recombinant chromosome with duplication of the 11q23.3q24.2 region resulting from an intrachromosomal insertion in the father. The second patient was originally reported by Partida-Perez, et al. [Partida-Perez et al., 2006] as having a tandem duplication of the 11q23.3 region. We performed array comparative genomic hybridization (aCGH) on this patient in order to map the exact region of the duplication, and demonstrated that the patient actually had a triplication within 11q23.3. We compare the clinical features of our two patients with those previously reported to further delineate the phenotype of isolated distal 11q duplication. Our study also demonstrates the clinical usefulness of whole genome high resolution aCGH analysis as a powerful molecular cytogenetic tool capable of detecting genomic imbalances due to cytogenetically visible but uncertain rearrangements.

A familial inverted duplication/deletion of 2p25.1–25.3 provides new clues on the genesis of inverted duplications

We studied a family in which the same 10 Mb inverted duplication of 2p25.3-p25.1 segregates in two children and their father, all showing a trisomy phenotype. As FISH analysis demonstrated that the duplication was inverted, we suspected that a contiguous terminal deletion was also present, according to the classical inv dup del type of rearrangements. Although FISH with 2p and 2q subtelomeric probes gave normal results, 100 kb resolution array-C/GH (aCGH) showed that, beside the duplication, a 273 kb deletion was also present. The presence of a single-copy region between the deleted and duplicated regions was further suspected through high-resolution aCGH analysis (approximately 20 kb), although only one informative spot having a normal log ratio was detected. The precise structure of the rearrangement was re-defined by real-time PCR and breakpoint cloning, demonstrating the presence of a 2680 bp single-copy sequence between deleted and duplicated regions and the involvement of a simple repeat with the potential for forming a non-B DNA structure. The rearrangement was not mediated by segmental duplications or short inverted repeats, and the double-strand break might have been repaired by non-homologous end joining or microhomology-mediated intrastrand repair. These data highlight the fact that concomitant deletions associated with inverted duplications are very likely to be more frequent than classical cytogenetic methods alone have been able to demonstrate. The phenotypic effects of the trisomy and of the terminal 2p deletion are discussed.

C6-04: Genomic profiling of tumour recurrence in non-small cell lung carcinoma

C6-04: Genomic profiling of tumour recurrence in non-small cell lung carcinoma