As more clinically relevant cancer genes are identified, comprehensive diagnostic approaches are needed to match patients to therapies, raising the challenge of optimization and analytical validation of assays that interrogate millions of bases of cancer genomes altered by multiple mechanisms. Here we describe a test based on massively parallel DNA sequencing to characterize base substitutions, short insertions and deletions (indels), copy number alterations and selected fusions across 287 cancer-related genes from routine formalin-fixed and paraffin-embedded (FFPE) clinical specimens. We implemented a practical validation strategy with reference samples of pooled cell lines that model key determinants of accuracy, including mutant allele frequency, indel length and amplitude of copy change. Test sensitivity achieved was 95–99% across alteration types, with high specificity (positive predictive value >99%). We confirmed accuracy using 249 FFPE cancer specimens characterized by established assays. Application of the test to 2,221 clinical cases revealed clinically actionable alterations in 76% of tumors, three times the number of actionable alterations detected by current diagnostic tests.

Focal amplifi cation and activating point mutation of the MET gene are well-characterized oncogenic drivers that confer susceptibility to targeted MET inhibitors. Recurrent somatic splice site alterations at MET exon 14 ( MET ex14) that result in exon skipping and MET activation have been characterized, but their full diversity and prevalence across tumor types are unknown. Here, we report analysis of tumor genomic profi les from 38,028 patients to identify 221 cases with MET ex14 mutations (0.6%), including 126 distinct sequence variants. MET ex14 mutations are detected most frequently in lung adenocarcinoma (3%), but also frequently in other lung neoplasms (2.3%), brain glioma (0.4%), and tumors of unknown primary origin (0.4%). Further in vitro studies demonstrate sensitivity to MET inhibitors in cells harboring MET ex14 alterations. We also report three new patient cases with MET ex14 alterations in lung or histiocytic sarcoma tumors that showed durable response to two different MET-targeted therapies. The diversity of MET ex14 mutations indicates that diagnostic testing via comprehensive genomic profi ling is necessary for detection in a clinical setting. SIGNIFICANCE:Here we report the identifi cation of diverse exon 14 splice site alterations in MET that result in constitutive activity of this receptor and oncogenic transformation in vitro . Patients whose tumors harbored these alterations derived meaningful clinical benefi t from MET inhibitors. Collectively, these data support the role of MET ex14 alterations as drivers of tumorigenesis, and identify a unique subset of patients likely to derive benefi t from MET inhibitors. Cancer Discov; 5(8);

We report the development of a biostable methotrexate-immobilized iron oxide nanoparticle drug carrier that may potentially be used for real-time monitoring of drug delivery through magnetic resonance imaging. Methotrexate (MTX) was immobilized on the nanoparticle surface via a poly(ethylene glycol) self-assembled monolayer (PEG SAM). The cytotoxicity of the nanoparticle-drug conjugate (NP-PEG-MTX) to target cells was studied with 9L glioma cells. Cellular uptake experiments showed that the uptake of NP-PEG-MTX conjugates by glioma cells was considerably higher than that of control nanoparticles. Magnetic resonance imaging in 9L cells cultured with NP-PEG-MTX of various concentrations showed significant contrast enhancement. NP-PEG-MTX demonstrated higher cytotoxicity in 9L cells to free MTX in vitro. Leucovorin, an MTX antidote, was used to rescue the cells that had been exposed to NP-PEG-MTX or free MTX, and the experiment verified the biocompatibility of NP-PEG-MTX conjugates and the MTX on NP-PEG-MTX conjugates to be the true source of the cytotoxicity to the target cells. TEM results showed that NP-PEG-MTX conjugates were internalized into the 9L cellular cytoplasm and retained its crystal structure therein for up to 144 h, as identified by electron diffraction. This prolonged particle retention may allow physicians to image tumor cells exposed to the NP-PEG-MTX conjugate over an extended therapeutic time course.

Key Points Novel clinically available comprehensive genomic profiling of both DNA and RNA in hematologic malignancies. Profiling of 3696 clinical hematologic tumors identified somatic alterations that impact diagnosis, prognosis, and therapeutic selection.

Amplification and point mutation in MET are well-characterized oncogenic drivers that confer susceptibility to MET inhibitors, such as crizotinib, particularly in non-small cell lung cancer. Splice site alterations at exon 14 of the MET (METex14) gene result in exon skipping and MET activation through loss of a DpYR motif (Y1003) that recruits the ubiquitin ligase CBL, targeting MET for degradation. METex14 skipping mutations have primarily been detected in lung adenocarcinoma, occurring in ∼3% of cases, but also in neuroblastoma and gastric cancer cell lines. 17 distinct variants have been characterized, however, their full diversity and prevalence across tumor types is unknown. Most importantly, it is unknown whether these mutations confer clinical susceptibility to targeted therapy with multiple MET inhibitors. We report here analysis of 29,714 cancer genomes to identify 168 harboring METex14 splicing mutations, including 101 distinct genomic variants. They are detected most frequently in lung adenocarcinoma (3.1%, 104/3360), but also in other lung neoplasms (2.0%, 40/2022), brain glioma (0.5%, 6/1312), tumors of unknown primary origin (0.4%, 11/2516), and other tumor types (0.03% 7/20504). Lung malignancies with these mutations do not have other characteristic driver mutations, and have a distinct profile of co-occurring mutations, supporting the role of METex14 skipping as the primary drivers of oncogenesis in these specimens. Most importantly, patients with tumors harboring these mutations demonstrate durable response to anti-MET targeted therapy, in several tumor types. These data demonstrate that METex14 splicing mutations define a distinct class of tumors that are responsive to targeted therapy. The diversity of these variants indicates that diagnostic testing via massively parallel sequencing is necessary for detection in a clinical setting. The data also suggest that thousands of cancer genome profiles will be required to discover non-coding cancer drivers with degenerate genomic signatures. Citation Format: Garrett M. Frampton, Siraj Ali, Juliann Chmielecki, Mark Rosenzweig, Timothy Brennan, Zachary Chalmers, Julia Elvin, Alex Fichtenholtz, Kyle Gowan, Joel Greenbowe, Adrienne Johnson, Lily Khaira, Doron Lipson, Caitlin McMahon, Steven Roels, Roman Yelensky, Deborah Morosini, Philip Stephens, Vincent Miller. Activation of MET via diverse exon 14 skipping mutations occurs in multiple tumor types and confers clinical sensitivity to MET inhibitors. [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 1118. doi:10.1158/1538-7445.AM2015-1118