Next-Generation Sequencing and Applications to the Diagnosis and Treatment of Lung Cancer.

Cancer is a genetic disease characterized by uncontrolled growth of abnormal cells. Over time, somatic mutations accumulate in the cells of an individual due to replication errors, chromosome segregation errors, or DNA damage. When not caught by traditional mechanisms, these somatic mutations can lead to cellular proliferation, the hallmark of cancer. Lung cancer is the leading cause of cancer-related mortality in the United States, accounting for approximately 160,000 deaths annually. Five year survival rates for lung cancer remain low (<50 %) for all stages, with even worse prognosis (<15 %) in late stage cases. Technological advances, including advances in next-generation sequencing (NGS), offer the vision of personalized medicine or precision oncology, wherein an individual's treatment can be based on his or her individual molecular profile, rather than on historical population-based medicine. Towards this end, NGS has already been used to identify new biomarker candidates for the early diagnosis of lung cancer and is increasingly used to guide personalized treatment decisions. In this review we will provide a high-level overview of NGS technology and summarize its application to the diagnosis and treatment of lung cancer. We will also describe how NGS can drive advances that bring us closer to precision oncology and discuss some of the technical challenges that will need to be overcome in order to realize this ultimate goal.

Challenges and opportunities for next-generation sequencing in companion diagnostics.

The rapid decline in sequencing costs has allowed next-generation sequencing (NGS) assays, previously ubiquitous only in research laboratories, to begin making inroads into molecular diagnostics. Genotypic assays - DNA sequencing - include whole genome sequencing, whole exome sequencing, focused assays that target only a handful of genes. Phenotypic assays comprise a broader spectrum of options and can query a variety of epigenetic modifications of DNA (such as ChIP-seq, bisulfite sequencing, DNase-I hypersensitivity site-sequencing, Formaldehyde-Assisted Isolation of Regulatory Elements-sequencing, etc.) that regulate gene expression-related processes or gene expression (RNA-sequencing) itself. To date, the US FDA has only cleared 12 DNA-based companion diagnostic tests, all in cancer. Although challenges exist for NGS in companion diagnostics, the wide-ranging capabilities of NGS offer extraordinary opportunities for the development and implementation of NGS-based companion diagnostics to probe oncogenes, tumor suppressor genes and cancer-enabling genes.

Next-generation sequencing in precision oncology: challenges and opportunities.

High throughput gene sequencing is transforming the utilization of genomics in patient care by providing physicians with a powerful tool to aid the diagnosis and management of disease, particularly in precision oncology. As next-generation sequencing (NGS)-based diagnostic assays are developed, significant hurdles such as assessing tumor heterogeneity, characterizing 'driver' and 'passenger' mutations, typing molecular signatures of individual cancers and determining limits of detection pose significant challenges for clinical laboratories and downstream bioinformatics analyses. Despite these challenges, NGS has the potential to affect all facets of cancer treatment, including early detection and diagnosis through cancer screening in at-risk populations and assessing therapeutic efficacy by detection of circulating tumor DNA via noninvasive blood draws. As the utilization of NGS in precision oncology matures, NGS-based laboratory tests could be used throughout the evolution of cancer in patients and allow for cancers to be monitored and managed as a chronic disease, rather than an acute condition.

Clinical investigational studies for validation of a next-generation sequencing in vitro diagnostic device for cystic fibrosis testing.

PURPOSE: Clinical investigational studies were conducted to demonstrate the accuracy and reproducibility of the Illumina MiSeqDx CF System, a next-generation sequencing (NGS) in vitro diagnostic device for cystic fibrosis testing. METHODS: Two NGS assays - a Clinical Sequencing Assay (Sequencing Assay) and a 139-Variant Assay (Variant Assay) - were evaluated in both an Accuracy Study and a Reproducibility Study, with comparison to bi-directional Sanger sequencing and PCR as reference methods. For each study, positive agreement (PA), negative agreement (NA), and overall agreement (OA) were evaluated. RESULTS: In the Accuracy Study, the Sequencing Assay achieved PA of 99.7% including the polyTG/polyT region and PA of 100% excluding the region. The Variant Assay achieved PA of 100%. NA and OA were >99.99% for both Assays. In the Reproducibility Study, the Sequencing Assay achieved PA of 99.2%; NA and OA were both 99.7%. The Variant Assay achieved PA of 99.8%; NA and OA were both 99.9%. Sample pass rates were 99.7% in both studies for both assays. CONCLUSION: This is the first systematic evaluation of a NGS platform for broad clinical use as an in vitro diagnostic, including accuracy validation with multiple reference methods and reproducibility validation at multiple clinical sites. These NGS-based Assays had accurate and reproducible results which were comparable to or better than other methods currently in clinical use for clinical genetic testing of cystic fibrosis.

Insertion mutations in herpes simplex virus 1 glycoprotein H reduce cell surface expression, slow the rate of cell fusion, or abrogate functions in cell fusion and viral entry.

Of the four required herpes simplex virus (HSV) entry glycoproteins, the precise role of gH-gL in fusion remains the most elusive. The heterodimer gH-gL has been proposed to mediate hemifusion after the interaction of another required glycoprotein, gD, with a receptor. To identify functional domains of HSV-1 gH, we generated 22 randomized linker-insertion mutants. Analyses of 22 gH mutants revealed that gH is relatively tolerant of insertion mutations, as 15 of 22 mutants permitted normal processing and transport of gH-gL to the cell surface. gH mutants that were not expressed well at the cell surface did not function in fusion or viral entry. The screening of gH mutants for function revealed the following: (i) for wild-type gH and some gH mutants, fusion with nectin-1-expressing target cells occurred more rapidly than with herpesvirus entry mediator (HVEM)-expressing target cells; (ii) some gH mutants reduced the rate of cell fusion without abrogating fusion completely, indicating that gH may play a role in governing the kinetics of fusion and may be responsible for a rate-limiting first stage in HSV-1 fusion; and (iii) only one gH mutant, located within the short cytoplasmic tail, completely abrogated function, indicating that the gH cytoplasmic tail is crucial for cell fusion and viral infectivity.

Insertional mutations in herpes simplex virus type 1 gL identify functional domains for association with gH and for membrane fusion.

Glycoprotein L (gL) is one of four glycoproteins required for the entry of herpes simplex virus (HSV) into cells and for virus-induced cell fusion. This glycoprotein oligomerizes with gH to form a membrane-bound heterodimer but can be secreted when expressed without gH. Twelve unique gL linker-insertion mutants were generated to identify regions critical for gH binding and gH/gL processing and regions essential for cell fusion and viral entry. All gL mutants were detected on the cell surface in the absence of gH, suggesting incomplete cleavage of the signal peptide or the presence of a cell surface receptor for secreted gL. Coexpression with gH enhanced the levels of cell surface gL detected by antibodies for all gL mutants except those that were defective in their interactions with gH. Two insertions into a conserved region of gL abrogated the binding of gL to gH and prevented gH expression on the cell surface. Three other insertions reduced the cell surface expression of gH and/or altered the properties of gH/gL heterodimers. Altered or absent interaction of gL with gH was correlated with reduced or absent cell fusion activity and impaired complementation of virion infectivity. These results identify a conserved domain of gL that is critical for its binding to gH and two noncontiguous regions of gL, one of which contains the conserved domain, that are critical for the gH/gL complex to perform its role in membrane fusion.

Differential effects on cell fusion activity of mutations in herpes simplex virus 1 glycoprotein B (gB) dependent on whether a gD receptor or a gB receptor is overexpressed.

Glycoprotein B (gB) of herpes simplex virus (HSV) is one of four glycoproteins essential for viral entry and cell fusion. Recently, paired immunoglobulin-like type 2 receptor (PILRalpha) was identified as a receptor for HSV type 1 (HSV-1) gB. Both PILRalpha and a gD receptor were shown to participate in HSV-1 entry into certain cell types. The purpose of this study was to determine whether insertional mutations in gB had differential effects on its function with PILRalpha and the gD receptor, nectin-1. Previously described gB mutants and additional newly characterized mutants were used in this study. We found that insertional mutations near the N terminus and C terminus of gB and especially in the central region of the ectodomain reduced cell fusion activity when PILRalpha was overexpressed much more than when nectin-1 was overexpressed. Most of the insertions reduced the binding of gB to PILRalpha, for at least some forms of gB, but this reduction did not necessarily correlate with the selective reduction in cell fusion activity with PILRalpha. These results suggest that the regions targeted by the relevant mutations are critical for functional activity with PILRalpha. They also suggest that, although both the binding of gB to a gB receptor and the binding of gD to a gD receptor may be required for HSV-induced cell fusion, the two receptor-binding activities may have unequal weights in triggering fusogenic activity, depending on the ratios of gB and gD receptors or other factors.

Alternative entry receptors for herpes simplex virus and their roles in disease.

Either herpesvirus entry mediator (HVEM, TNFRSF14) or nectin-1 (PVRL1) is sufficient for herpes simplex virus (HSV) infection of cultured cells. The contribution of individual receptors to infection in vivo and to disease is less clear. To assess this, Tnfrsf14(-/-) and/or Pvrl1(-/-) mice were challenged intravaginally with HSV-2. Infection of the vaginal epithelium occurred in the absence of either HVEM or nectin-1 but was virtually undetectable when both receptors were absent, indicating that either HVEM or nectin-1 was necessary. Absence of nectin-1 (but not HVEM) reduced efficiency of infection of the vaginal epithelium and viral spread to the nervous system, attenuating neurological disease and preventing external lesion development. While nectin-1 proved not to be essential for infection of the nervous system, it is required for the full manifestations of disease. This study illustrates the value of mutant mice for understanding receptor contributions to disease caused by a human virus.

Random linker-insertion mutagenesis to identify functional domains of herpes simplex virus type 1 glycoprotein B.

Herpes simplex virus glycoprotein B (gB) is one of four glycoproteins essential for viral entry and cell fusion. Recently, an x-ray structure of the nearly full-length trimeric gB ectodomain was determined. Five structural domains and two linker regions were identified in what is probably a postfusion conformation. To identify functional domains of gB, we performed random linker-insertion mutagenesis. Analyses of 81 mutants revealed that only 27 could fold to permit processing and transport of gB to the cell surface. These 27 mutants fell into three categories. Insertions into two regions excluded from the solved structure (the N terminus and the C-terminal cytoplasmic tail) had no negative effect on cell fusion and viral entry activity, identifying regions that can tolerate altered structure without loss of function. Insertions into a disordered region in domain II and the adjacent linker region also permitted partial cell fusion and viral entry activity. Insertions at 16 other positions resulted in loss of cell fusion and viral entry activity, despite detectable levels of cell surface expression. Four of these insertion sites were not included in the solved structure. Two were between residues exposed to a cavity that is too small to accommodate the 5-amino acid insertions, consistent with the solved structure being different from the native prefusion structure. Ten were between residues exposed to the surface of the trimer, identifying regions that may be critical for interactions with other viral proteins or cellular components or for transitions from the prefusion to postfusion state.