Integrated genomic DNA/RNA profiling of hematologic malignancies in the clinical setting.

The spectrum of somatic alterations in hematologic malignancies includes substitutions, insertions/deletions (indels), copy number alterations (CNAs), and a wide range of gene fusions; no current clinically available single assay captures the different types of alterations. We developed a novel next-generation sequencing-based assay to identify all classes of genomic alterations using archived formalin-fixed paraffin-embedded blood and bone marrow samples with high accuracy in a clinically relevant time frame, which is performed in our Clinical Laboratory Improvement Amendments-certified College of American Pathologists-accredited laboratory. Targeted capture of DNA/RNA and next-generation sequencing reliably identifies substitutions, indels, CNAs, and gene fusions, with similar accuracy to lower-throughput assays that focus on specific genes and types of genomic alterations. Profiling of 3696 samples identified recurrent somatic alterations that impact diagnosis, prognosis, and therapy selection. This comprehensive genomic profiling approach has proved effective in detecting all types of genomic alterations, including fusion transcripts, which increases the ability to identify clinically relevant genomic alterations with therapeutic relevance.

PREDICTING SIGNIFICANCE OF UNKNOWN VARIANTS IN GLIAL TUMORS THROUGH SUB-CLASS ENRICHMENT.

Glial tumors have been heavily studied and sequenced, leading to scores of findings about altered genes. This explosion in knowledge has not been matched with clinical success, but efforts to understand the synergies between drivers of glial tumors may alleviate the situation. We present a novel molecular classification system that captures the combinatorial nature of relationships between alterations in these diseases. We use this classification to mine for enrichment of variants of unknown significance, and demonstrate a method for segregating unknown variants with functional importance from passengers and SNPs.

Activation of MET via diverse exon 14 splicing alterations occurs in multiple tumor types and confers clinical sensitivity to MET inhibitors.

UNLABELLED: Focal amplification 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 (METex14) 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 profiles from 38,028 patients to identify 221 cases with METex14 mutations (0.6%), including 126 distinct sequence variants. METex14 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 METex14 alterations. We also report three new patient cases with METex14 alterations in lung or histiocytic sarcoma tumors that showed durable response to two different MET-targeted therapies. The diversity of METex14 mutations indicates that diagnostic testing via comprehensive genomic profiling is necessary for detection in a clinical setting. SIGNIFICANCE: Here we report the identification 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 benefit from MET inhibitors. Collectively, these data support the role of METex14 alterations as drivers of tumorigenesis, and identify a unique subset of patients likely to derive benefit from MET inhibitors.

Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing.

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.

Methotrexate-immobilized poly(ethylene glycol) magnetic nanoparticles for MR imaging and drug delivery.

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.