High-throughput oncogene mutation profiling in human cancer.

Systematic efforts are underway to decipher the genetic changes associated with tumor initiation and progression. However, widespread clinical application of this information is hampered by an inability to identify critical genetic events across the spectrum of human tumors with adequate sensitivity and scalability. Here, we have adapted high-throughput genotyping to query 238 known oncogene mutations across 1,000 human tumor samples. This approach established robust mutation distributions spanning 17 cancer types. Of 17 oncogenes analyzed, we found 14 to be mutated at least once, and 298 (30%) samples carried at least one mutation. Moreover, we identified previously unrecognized oncogene mutations in several tumor types and observed an unexpectedly high number of co-occurring mutations. These results offer a new dimension in tumor genetics, where mutations involving multiple cancer genes may be interrogated simultaneously and in 'real time' to guide cancer classification and rational therapeutic intervention.

An oncogenic role for ETV1 in melanoma.

Copy gains involving chromosome 7p represent one of the most common genomic alterations found in melanomas, suggesting the presence of "driver" cancer genes. We identified several tumor samples that harbored focal amplifications situated at the peak of common chromosome 7p gains, in which the minimal common overlapping region spanned the ETV1 oncogene. Fluorescence in situ hybridization analysis revealed copy gains spanning the ETV1 locus in >40% of cases, with ETV1 amplification (>6 copies/cell) present in 13% of primary and 18% of metastatic melanomas. Melanoma cell lines, including those with ETV1 amplification, exhibited dependency on ETV1 expression for proliferation and anchorage-independent growth. Moreover, overexpression of ETV1 in combination with oncogenic NRAS(G12D) transformed primary melanocytes and promoted tumor formation in mice. ETV1 overexpression elevated microphthalmia-associated transcription factor expression in immortalized melanocytes, which was necessary for ETV1-dependent oncogenicity. These observations implicate deregulated ETV1 in melanoma genesis and suggest a pivotal lineage dependency mediated by oncogenic ETS transcription factors in this malignancy.

Profiling critical cancer gene mutations in clinical tumor samples.

BACKGROUND: Detection of critical cancer gene mutations in clinical tumor specimens may predict patient outcomes and inform treatment options; however, high-throughput mutation profiling remains underdeveloped as a diagnostic approach. We report the implementation of a genotyping and validation algorithm that enables robust tumor mutation profiling in the clinical setting. METHODOLOGY: We developed and implemented an optimized mutation profiling platform ("OncoMap") to interrogate approximately 400 mutations in 33 known oncogenes and tumor suppressors, many of which are known to predict response or resistance to targeted therapies. The performance of OncoMap was analyzed using DNA derived from both frozen and FFPE clinical material in a diverse set of cancer types. A subsequent in-depth analysis was conducted on histologically and clinically annotated pediatric gliomas. The sensitivity and specificity of OncoMap were 93.8% and 100% in fresh frozen tissue; and 89.3% and 99.4% in FFPE-derived DNA. We detected known mutations at the expected frequencies in common cancers, as well as novel mutations in adult and pediatric cancers that are likely to predict heightened response or resistance to existing or developmental cancer therapies. OncoMap profiles also support a new molecular stratification of pediatric low-grade gliomas based on BRAF mutations that may have immediate clinical impact. CONCLUSIONS: Our results demonstrate the clinical feasibility of high-throughput mutation profiling to query a large panel of "actionable" cancer gene mutations. In the future, this type of approach may be incorporated into both cancer epidemiologic studies and clinical decision making to specify the use of many targeted anticancer agents.

Modeling genomic diversity and tumor dependency in malignant melanoma.

The classification of human tumors based on molecular criteria offers tremendous clinical potential; however, discerning critical and "druggable" effectors on a large scale will also require robust experimental models reflective of tumor genomic diversity. Here, we describe a comprehensive genomic analysis of 101 melanoma short-term cultures and cell lines. Using an analytic approach designed to enrich for putative "driver" events, we show that cultured melanoma cells encompass the spectrum of significant genomic alterations present in primary tumors. When annotated according to these lesions, melanomas cluster into subgroups suggestive of distinct oncogenic mechanisms. Integrating gene expression data suggests novel candidate effector genes linked to recurrent copy gains and losses, including both phosphatase and tensin homologue (PTEN)-dependent and PTEN-independent tumor suppressor mechanisms associated with chromosome 10 deletions. Finally, sample-matched pharmacologic data show that FGFR1 mutations and extracellular signal-regulated kinase (ERK) activation may modulate sensitivity to mitogen-activated protein kinase/ERK kinase inhibitors. Genetically defined cell culture collections therefore offer a rich framework for systematic functional studies in melanoma and other tumors.