Next-Generation Sequencing and Fluorescence in Situ Hybridization Have Comparable Performance Characteristics in the Analysis of Pancreaticobiliary Brushings for Malignancy.

Cytological evaluation of pancreatic or biliary duct brushings is a specific, but insensitive, test for malignancy. We compared adjunctive molecular testing with next-generation sequencing (NGS) relative to fluorescence in situ hybridization (FISH) for detection of high-risk neoplasia or malignancy. Bile duct brushings from 81 specimens were subjected to cytological analysis, FISH using the UroVysion probe set, and targeted NGS. Specimens were placed into negative/atypical (negative) or suspicious/positive (positive) categories depending on cytology and negative or positive categories on the basis of FISH and NGS results. Performance characteristics for each diagnostic modality were calculated on the basis of clinicopathologic follow-up and compared in a receiver operating characteristic analysis. There were 33 high-risk neoplasia/malignant strictures (41%) and 48 benign (59%). NGS revealed driver mutations in 24 cases (30%), including KRAS (21 of 24 cases), TP53 (14 of 24 cases), SMAD4 (6 of 24 cases), and CDKN2A (4 of 24 cases). Cytology had a sensitivity of 67% (95% CI, 48%-82%) and a specificity of 98% (95% CI, 89%-100%). When added to cytology, NGS increased the sensitivity to 85% (95% CI, 68%-95%), leading to a significant increase in the area under the curve in a receiver operating characteristic analysis (P = 0.03). FISH increased the sensitivity to 76% (95% CI, 58%-89%), without significantly increasing the area under the curve. These results suggest that ancillary NGS testing offers advantages over FISH, although studies with larger cohorts are needed to verify these findings.

BRAF V600E mutations are common in pleomorphic xanthoastrocytoma: diagnostic and therapeutic implications.

Pleomorphic xanthoastrocytoma (PXA) is low-grade glial neoplasm principally affecting children and young adults. Approximately 40% of PXA are reported to recur within 10 years of primary resection. Upon recurrence, patients receive radiation therapy and conventional chemotherapeutics designed for high-grade gliomas. Genetic changes that can be targeted by selective therapeutics have not been extensively evaluated in PXA and ancillary diagnostic tests to help discriminate PXA from other pleomorphic and often more aggressive astrocytic malignancies are limited. In this study, we apply the SNaPshot multiplexed targeted sequencing platform in the analysis of brain tumors to interrogate 60 genetic loci that are frequently mutated in 15 cancer genes. In our analysis we detect BRAF V600E mutations in 12 of 20 (60%) WHO grade II PXA, in 1 of 6 (17%) PXA with anaplasia and in 1 glioblastoma arising in a PXA. Phospho-ERK was detected in all tumors independent of the BRAF mutation status. BRAF duplication was not detected in any of the PXA cases. BRAF V600E mutations were identified in only 2 of 71 (2.8%) glioblastoma (GBM) analyzed, including 1 of 9 (11.1%) giant cell GBM (gcGBM). The finding that BRAF V600E mutations are common in the majority of PXA has important therapeutic implications and may help in differentiating less aggressive PXAs from lethal gcGBMs and GBMs.

Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors.

Lung cancers harboring mutations in the epidermal growth factor receptor (EGFR) respond to EGFR tyrosine kinase inhibitors, but drug resistance invariably emerges. To elucidate mechanisms of acquired drug resistance, we performed systematic genetic and histological analyses of tumor biopsies from 37 patients with drug-resistant non-small cell lung cancers (NSCLCs) carrying EGFR mutations. All drug-resistant tumors retained their original activating EGFR mutations, and some acquired known mechanisms of resistance including the EGFR T790M mutation or MET gene amplification. Some resistant cancers showed unexpected genetic changes including EGFR amplification and mutations in the PIK3CA gene, whereas others underwent a pronounced epithelial-to-mesenchymal transition. Surprisingly, five resistant tumors (14%) transformed from NSCLC into small cell lung cancer (SCLC) and were sensitive to standard SCLC treatments. In three patients, serial biopsies revealed that genetic mechanisms of resistance were lost in the absence of the continued selective pressure of EGFR inhibitor treatment, and such cancers were sensitive to a second round of treatment with EGFR inhibitors. Collectively, these results deepen our understanding of resistance to EGFR inhibitors and underscore the importance of repeatedly assessing cancers throughout the course of the disease.

Rapid targeted mutational analysis of human tumours: a clinical platform to guide personalized cancer medicine.

Targeted cancer therapy requires the rapid and accurate identification of genetic abnormalities predictive of therapeutic response. We sought to develop a high-throughput genotyping platform that would allow prospective patient selection to the best available therapies, and that could readily and inexpensively be adopted by most clinical laboratories. We developed a highly sensitive multiplexed clinical assay that performs very well with nucleic acid derived from formalin fixation and paraffin embedding (FFPE) tissue, and tests for 120 previously described mutations in 13 cancer genes. Genetic profiling of 250 primary tumours was consistent with the documented oncogene mutational spectrum and identified rare events in some cancer types. The assay is currently being used for clinical testing of tumour samples and contributing to cancer patient management. This work therefore establishes a platform for real-time targeted genotyping that can be widely adopted. We expect that efforts like this one will play an increasingly important role in cancer management.

Stemless self-quenching reporter molecules identify target sequences in mRNA.

The design of oligonucleotides for gene silencing requires a rational method for identifying hybridization-accessible sequences within the target RNA. To this end, we have developed stem-loop self-quenching reporter molecules (SQRMs) as probes for such sequence. SQRMs have a 5' fluorophore, a quenching moiety on the 3' end, an intervening sequence that forms an approximately 5-basepaired stem, and a loop sequence of approximately 20-30 bases. We have previously described a mapping strategy employing SQRMs to locate stem-loop structures in the target mRNA molecule. We now show that the original design constraint of a basepaired stem is not needed, either in vitro or in vivo. We propose that stemless probes possess sufficient signal-to-noise for use in vivo and that this ratio is an indication of hybridization of the probe to its target. Data showing that these SQRMs can specifically target and reduce c-Myb protein synthesis and can be used for real-time in vivo assays are presented.

Identification of antisense nucleic acid hybridization sites in mRNA molecules with self-quenching fluorescent reporter molecules.

We describe a physical mRNA mapping strategy employing fluorescent self-quenching reporter molecules (SQRMs) that facilitates the identification of mRNA sequence accessible for hybridization with antisense nucleic acids in vitro and in vivo, real time. SQRMs are 20-30 base oligodeoxynucleotides with 5-6 bp complementary ends to which a 5' fluorophore and 3' quenching group are attached. Alone, the SQRM complementary ends form a stem that holds the fluorophore and quencher in contact. When the SQRM forms base pairs with its target, the structure separates the fluorophore from the quencher. This event can be reported by fluorescence emission when the fluorophore is excited. The stem-loop of the SQRM suggests that SQRM be made to target natural stem-loop structures formed during mRNA synthesis. The general utility of this method is demonstrated by SQRM identification of targetable sequence within c-myb and bcl-6 mRNA. Corresponding antisense oligonucleotides reduce these gene products in cells.