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.

A modern understanding of the traditional and nontraditional biological functions of angiotensin-converting enzyme.

Angiotensin-converting enzyme (ACE) is a zinc-dependent peptidase responsible for converting angiotensin I into the vasoconstrictor angiotensin II. However, ACE is a relatively nonspecific peptidase that is capable of cleaving a wide range of substrates. Because of this, ACE and its peptide substrates and products affect many physiologic processes, including blood pressure control, hematopoiesis, reproduction, renal development, renal function, and the immune response. The defining feature of ACE is that it is composed of two homologous and independently catalytic domains, the result of an ancient gene duplication, and ACE-like genes are widely distributed in nature. The two ACE catalytic domains contribute to the wide substrate diversity of ACE and, by extension, the physiologic impact of the enzyme. Several studies suggest that the two catalytic domains have different biologic functions. Recently, the X-ray crystal structure of ACE has elucidated some of the structural differences between the two ACE domains. This is important now that ACE domain-specific inhibitors have been synthesized and characterized. Once widely available, these reagents will undoubtedly be powerful tools for probing the physiologic actions of each ACE domain. In turn, this knowledge should allow clinicians to envision new therapies for diseases not currently treated with ACE inhibitors.

Myeloid expression of angiotensin-converting enzyme facilitates myeloid maturation and inhibits the development of myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells which accumulate in cancer, infection and chronic inflammation. These cells suppress T-cell function and the immune response. Angiotensin-converting enzyme (ACE) is a peptidase that is now known to regulate aspects of myelopoiesis. Here, we show that ACE expression correlates with myeloid maturation in vitro. Forced ACE overexpression in monocytic cells reduces the generation of MDSCs. In vivo, mice with a genetic change resulting in myeloid cell ACE overexpression have reduced numbers of blood and splenic MDSCs in a tumor model and in a model of chronic inflammation induced by complete Freund's adjuvant. In contrast, ACE-null mice produce large numbers of MDSCs during chronic inflammation. Macrophages from mice with myeloid ACE overexpressing are more pro-inflammatory and have more tumor-killing activity than cells from wild-type mice. Thus, manipulating myeloid ACE activity can interfere with MDSC development and the maturation of myeloid cells.

Translational utility of next-generation sequencing.

The development of next-generation sequencing (NGS) technology has made DNA sequencing not only rapid and cost-effective, but also highly accurate and reproducible. The translational utility of genomic sequencing is clear, from understanding of human genetic variation and its association with disease risk and individual response to treatment, to the interpretation and translation of the data for clinical decision making. It will be a critical technology for disease characterization and monitoring in molecular pathology and is expected to become a central piece of routine healthcare management which will result in accurate and reliable reporting, a prerequisite for physicians to practice genomic medicine.

Identification of prolyl carboxypeptidase as an alternative enzyme for processing of renal angiotensin II using mass spectrometry.

Angiotensin-converting enzyme 2 (ACE2) catalyzes conversion of ANG II to ANG-(1-7). The present study uses newly established proteomic approaches and genetic mouse models to examine the contribution of alternative renal peptidases to ACE2-independent formation of ANG-(1-7). In situ and in vitro mass spectrometric characterization showed that substrate concentration and pH control renal ANG II processing. At pH ≥6, ANG-(1-7) formation was significantly reduced in ACE2 knockout (KO) mice. However, at pH <6, formation of ANG-(1-7) in ACE2 KO mice was similar to that in wild-type (WT) mice, suggesting alternative peptidases for renal ANG II processing. Furthermore, the dual prolyl carboxypeptidase (PCP)-prolyl endopeptidase (PEP) inhibitor Z-prolyl-prolinal reduced ANG-(1-7) formation in ACE2 KO mice, while the ACE2 inhibitor MLN-4760 had no effect. Unlike the ACE2 KO mice, ANG-(1-7) formation from ANG II in PEP KO mice was not different from that in WT mice at any tested pH. However, at pH 5, this reaction was significantly reduced in kidneys and urine of PCP-depleted mice. In conclusion, results suggest that ACE2 metabolizes ANG II in the kidney at neutral and basic pH, while PCP catalyzes the same reaction at acidic pH. This is the first report demonstrating that renal ANG-(1-7) formation from ANG II is independent of ACE2. Elucidation of ACE2-independent ANG-(1-7) production pathways may have clinically important implications in patients with metabolic and renal disease.

Angiotensin-converting enzyme N-terminal inactivation alleviates bleomycin-induced lung injury.

Bleomycin has potent anti-oncogenic properties for several neoplasms, but drug administration is limited by bleomycin-induced lung fibrosis. Inhibition of the renin-angiotensin system has been suggested to decrease bleomycin toxicity, but the efficacy of such strategies remains uncertain and somewhat contradictory. Our hypothesis is that, besides angiotensin II, other substrates of angiotensin-converting enzyme (ACE), such as the tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), play a significant role in controlling fibrosis. We studied bleomycin-induced lung injury in normotensive mice, termed N-KO and C-KO, which have point mutations inactivating either the N- or C-terminal catalytic sites of ACE, respectively. N-KO, but not C-KO mice, have a marked resistance to bleomycin lung injury as assessed by lung histology and hydroxyproline content. To determine the importance of the ACE N-terminal peptide substrate AcSDKP in the resistance to bleomycin injury, N-KO mice were treated with S-17092, a prolyl-oligopeptidase inhibitor that inhibits the formation of AcSDKP. In response to bleomycin injection, S-17092-treated N-KO mice developed lung fibrosis similar to wild-type mice. In contrast, the administration of AcSDKP to wild-type mice reduced lung fibrosis due to bleomycin administration. This study shows that the inactivation of the N-terminal catalytic site of ACE significantly reduced bleomycin-induced lung fibrosis and implicates AcSDKP in the mechanism of protection. These data suggest a possible means to increase tolerance to bleomycin and to treat fibrosing lung diseases.

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.

Personalized Medicine in Ophthalmology: From Pharmacogenetic Biomarkers to Therapeutic and Dosage Optimization.

Rapid progress in genomics and nanotechnology continue to advance our approach to patient care, from diagnosis and prognosis, to targeting and personalization of therapeutics. However, the clinical application of molecular diagnostics in ophthalmology has been limited even though there have been demonstrations of disease risk and pharmacogenetic associations. There is a high clinical need for therapeutic personalization and dosage optimization in ophthalmology and may be the focus of individualized medicine in this specialty. In several retinal conditions, such as age-related macular degeneration, diabetic macular edema, retinal vein occlusion and pre-threshold retinopathy of prematurity, anti-vascular endothelial growth factor therapeutics have resulted in enhanced outcomes. In glaucoma, recent advances in cytoskeletal agents and prostaglandin molecules that affect outflow and remodel the trabecular meshwork have demonstrated improved intraocular pressure control. Application of recent developments in nanoemulsion and polymeric micelle for targeted delivery and drug release are models of dosage optimization, increasing efficacy and improving outcomes in these major eye diseases.

Clinical utility of pharmacogenetic biomarkers in cardiovascular therapeutics: a challenge for clinical implementation.

In the past decade, significant strides have been made in the area of cardiovascular pharmacogenomic research, with the discovery of associations between certain genotypes and drug-response phenotypes. While the motivations for personalized and predictive medicine are promising for patient care and support a model of health system efficiency, the implementation of pharmacogenomics for cardiovascular therapeutics on a population scale faces substantial challenges. The greatest obstacle to clinical implementation of cardiovascular pharmacogenetics may be the lack of both reproducibility and agreement about the validity and utility of the findings. In this review, we present the scientific evidence in the literature for diagnostic variants for the US FDA-labeled cardiovascular therapies, namely CYP2C19 and clopidogrel, CYP2C9/VKORC1 and warfarin, and CYP2D6/ADRB1 and ß-blockers. We also discuss the effect of HMGCR/LDLR in decreasing the effectiveness of low-density lipoprotein cholesterol with statin therapy, the SLCO1B1 genotype and simvastatin myotoxicity, and ADRB1/ADD1 for antihypertensive response.

Increased angiotensin II-induced hypertension and inflammatory cytokines in mice lacking angiotensin-converting enzyme N domain activity.

-Angiotensin-converting enzyme (ACE) is composed of the N- and C-terminal catalytic domains. To study the role of the ACE domains in the inflammatory response, N-knockout (KO) and C-KO mice, models lacking 1 of the 2 ACE domains, were analyzed during angiotensin II-induced hypertension. At 2 weeks, N-KO mice have systolic blood pressures that averaged 173±4.6 mm Hg, which is more than 25 mm Hg higher than the blood pressures observed in wild-type or C-KO mice (146±3.2 and 147±4.2 mm Hg). After 3 weeks, blood pressure differences between N-KO, C-KO, and wild-type were even more pronounced. Macrophages from N-KO mice have increased expression of tumor necrosis factor α after stimulation with either lipopolysaccharide (about 4-fold) or angiotensin II (about 2-fold), as compared with C-KO or wild-type mice. Inhibition of the enzyme prolyl oligopeptidase, responsible for the formation of acetyl-SerAspLysPro and other peptides, eliminated the blood pressure difference and the difference in tumor necrosis factor α expression between angiotensin II-treated N-KO and wild-type mice. However, this appears independent of acetyl-SerAspLysPro. These data establish significant differences in the inflammatory response as a function of ACE N- or C-domain catalytic activity. They also indicate a novel role of prolyl oligopeptidase in the cytokine regulation and in the blood pressure response to experimental hypertension.

Personalized medicine and pharmacogenetic biomarkers: progress in molecular oncology testing.

In the field of oncology, clinical molecular diagnostics and biomarker discoveries are constantly advancing as the intricate molecular mechanisms that transform a normal cell into an aberrant state in concert with the dysregulation of alternative complementary pathways are increasingly understood. Progress in biomarker technology, coupled with the companion clinical diagnostic laboratory tests, continue to advance this field, where individualized and customized treatment appropriate for each individual patient define the standard of care. Here, we discuss the current commonly used predictive pharmacogenetic biomarkers in clinical oncology molecular testing: BRAF V600E for vemurafenib in melanoma; EML4-ALK for crizotinib and EGFR for erlotinib and gefitinib in non-small-cell lung cancer; KRAS against the use of cetuximab and panitumumab in colorectal cancer; ERBB2 (HER2/neu) for trastuzumab in breast cancer; BCR-ABL for tyrosine kinase inhibitors in chronic myeloid leukemia; and PML/RARα for all-trans-retinoic acid and arsenic trioxide treatment for acute promyelocytic leukemia.

Different in vivo functions of the two catalytic domains of angiotensin-converting enzyme (ACE).

Angiotensin-converting enzyme (ACE) can cleave angiotensin I, bradykinin, neurotensin and many other peptide substrates in vitro. In part, this is due to the structure of ACE, a protein composed of two independent catalytic domains. Until very recently, little was known regarding the specific in vivo role of each ACE domain, and they were commonly regarded as equivalent. This is not true, as shown by mouse models with a genetic inactivation of either the ACE N- or C-domain. In vivo, most angiotensin II is produced by the ACE C-domain. Some peptides, such as the anti-fibrotic peptide AcSDKP, are substrates only of the ACE N-domain. Knowing the in vivo role of each ACE domain has great significance for developing ACE domain-specific inhibitors and for understanding the full effects of the anti-ACE pharmaceuticals in widespread clinical use.