From DNA Copy Number Gains and Tumor Dependencies to Novel Therapeutic Targets for High-Risk Neuroblastoma

Neuroblastoma is a pediatric tumor arising from the sympatho-adrenal lineage and a worldwide leading cause of childhood cancer-related deaths. About half of high-risk patients die from the disease while survivors suffer from multiple therapy-related side-effects. While neuroblastomas present with a low mutational burden, focal and large segmental DNA copy number aberrations are highly recurrent and associated with poor survival. It can be assumed that the affected chromosomal regions contain critical genes implicated in neuroblastoma biology and behavior. More specifically, evidence has emerged that several of these genes are implicated in tumor dependencies thus potentially providing novel therapeutic entry points. In this review, we briefly review the current status of recurrent DNA copy number aberrations in neuroblastoma and provide an overview of the genes affected by these genomic variants for which a direct role in neuroblastoma has been established. Several of these genes are implicated in networks that positively regulate MYCN expression or stability as well as cell cycle control and apoptosis. Finally, we summarize alternative approaches to identify and prioritize candidate copy-number driven dependency genes for neuroblastoma offering novel therapeutic opportunities.

Genomic and Prognostic Heterogeneity Among RAS/BRAFV600E/TP53 Co-Mutated Resectable Colorectal Liver Metastases

Background Hepatic resection is potentially curative for patients with colorectal liver metastases, but the treatment benefit varies. KRAS/NRAS (RAS)/TP53 co-mutations are associated with a poor prognosis after resection, but there is large variation in patient outcome within the mutation groups, and genetic testing is currently not used to determine the likely benefit from surgery. We have investigated the potential for improved prognostic stratification by combined biomarker analysis with DNA copy number aberrations (CNAs) in a tumor heterogeneity setting. Methods We determined the mutation status of KRAS, NRAS, BRAF, and TP53 in 441 liver lesions from 171 patients treated by partial hepatectomy for metastatic colorectal cancer. CNAs were profiled in 232 tumors from 67 of the patients. Results Driver mutations in KRAS, NRAS, BRAFV600E, and TP53, and high-level amplifications affecting cancer-critical genes such as ERBB2 and EGFR, were predominantly homogeneous within patients. RAS/BRAFV600E and TP53 co-mutations were associated with a poor patient outcome (hazard ratio, HR, 3.9, 95% confidence interval, CI, 1.3–11.1, p = 0.012) in multivariable analyses with clinicopathological variables. The genome-wide CNA burden and intra-patient inter-metastatic CNA heterogeneity varied within both the mutated and the wild-type groups. Combined prognostic analyses of RAS/BRAFV600E/TP53 mutations and CNAs, either as a high CNA burden or high inter-metastatic CNA heterogeneity, identified patients with a particularly poor outcome (co-mutation/high CNA burden: HR 2.7, 95% CI 1.2–5.9, p = 0.013; co-mutation/high CNA heterogeneity: HR 2.5, 95% CI 1.1–5.6, p = 0.022). Conclusions DNA copy number profiling identified genomic and prognostic heterogeneity among patients with resectable colorectal liver metastases with co-mutated RAS/BRAFV600E/TP53.

Development of a Genome-Wide Oligonucleotide Microarray Platform for Detection of DNA Copy Number Aberrations in Feline Cancers

The utility of the domestic cat as a model system for biomedical studies was constrained for many years by the absence of a comprehensive feline reference genome sequence assembly. While such a resource now exists, the cat continues to lag behind the domestic dog in terms of integration into the ‘One Health’ era of molecular medicine. Stimulated by the advances being made within the evolving field of comparative cancer genomics, we developed a microarray platform that allows rapid and sensitive detection of DNA copy number aberrations in feline tumors using comparative genomic hybridization analysis. The microarray comprises 110,456 unique oligonucleotide probes anchored at mean intervals of 22.6 kb throughout the feline reference genome sequence assembly, providing ~350-fold higher resolution than was previously possible using this technique. We demonstrate the utility of this resource through genomic profiling of a feline injection-site sarcoma case, revealing a highly disrupted profile of DNA copy number imbalance involving several key cancer-associated genes including KIT, TP53, PTEN, FAS and RB1. These findings were supported by targeted fluorescence in-situ hybridization analysis, which identified major alterations in chromosome structure, including complex intrachromosomal reorganization events typical of those seen in aggressive soft-tissue sarcomas of other species. We then characterized a second mass that was identified at a nearby site in the same patient almost 12 months later. This mass demonstrated a remarkably conserved genomic profile consistent with a recurrence of the original tumor; however the detection of subtle differences reflected evolution of the tumor over time. These findings exemplify the diverse potential of this microarray platform to incorporate domestic cat cancers into comparative and translational research efforts in molecular oncology.

Intratumor heterogeneity of DNA copy number aberrations in gastric and oesophageal adenocarcinomas.

78 Background: DNA copy number aberrations (CNAs) are common in oesophageal and gastric adenocarcinomas (OGCs) and display extensive inter-tumour heterogeneity. CNA patterns define gastric cancer molecular subtypes and ERBB2 amplifications, present in a small fraction of patients with OGC, are predictive for ERBB2-targeted drug sensitivity. Together, this suggests a critical role of CNAs determining OGC tumour biology and clinical outcomes. Despite this, predictive and prognostic CNA biomarkers have not been identified for the majority of OGCs, precluding the development of effective personalized therapy approaches for these aggressive tumours. Intra-tumour heterogeneity, characterized by the presence of multiple subclones with distinct genetic profiles within an individual cancer, can hinder the accurate molecular analysis and classification of tumours. The aim of this pilot study was to assess whether chemotherapy-naïve localized OGCs display intra-tumour macroheterogeneity of CNA profiles. Methods: Tissue specimens from four tumour regions representing the macroscopic spatial extent of each of five OGCs were systematically collected after surgical resection. DNA extracted from these FFPE specimens was analysed by molecular inversion probe SNP arrays for high resolution CNA detection. Results: Comparison of genome wide copy number and B-allele frequency profiles suggested highly concordant CNA profiles across the regions from individual primary tumours. Eight driver CNAs leading to amplification of the MET, KRAS, ERBB2, PIK3CA or FGFR2 oncogenes were identified in 4/5 tumours. Only one out of these eight driver CNA’s, harbouring the KRAS oncogene, was heterogeneous within a tumour. Conclusions: Although chromosomal instability is thought to be common in this tumour type, this pilot study suggests that macroheterogeneity is limited and that CNA profiles assessed from a single tumour biopsy are likely to be representative of the dominant CNA profile of localized OGCs. Thus, clinical correlative CNA analyses may be possible from single biopsies of localized OGCs. Mutational heterogeneity and microheterogeneity in microdissected and single cells are currently being investigated.

Identification of Novel Breast Cancer Subtype-Specific Biomarkers by Integrating Genomics Analysis of DNA Copy Number Aberrations and miRNA-mRNA Dual Expression Profiling

Breast cancer is a heterogeneous disease with well-defined molecular subtypes. Currently, comparative genomic hybridization arrays (aCGH) techniques have been developed rapidly, and recent evidences in studies of breast cancer suggest that tumors within gene expression subtypes share similar DNA copy number aberrations (CNA) which can be used to further subdivide subtypes. Moreover, subtype-specific miRNA expression profiles are also proposed as novel signatures for breast cancer classification. The identification of mRNA or miRNA expression-based breast cancer subtypes is considered an instructive means of prognosis. Here, we conducted an integrated analysis based on copy number aberrations data and miRNA-mRNA dual expression profiling data to identify breast cancer subtype-specific biomarkers. Interestingly, we found a group of genes residing in subtype-specific CNA regions that also display the corresponding changes in mRNAs levels and their target miRNAs’ expression. Among them, the predicted direct correlation of BRCA1-miR-143-miR-145 pairs was selected for experimental validation. The study results indicated that BRCA1 positively regulates miR-143-miR-145 expression and miR-143-miR-145 can serve as promising novel biomarkers for breast cancer subtyping. In our integrated genomics analysis and experimental validation, a new frame to predict candidate biomarkers of breast cancer subtype is provided and offers assistance in order to understand the potential disease etiology of the breast cancer subtypes.