Sequencing of genes of drug response in tumor DNA and implications for precision medicine in cancer patients.

Tumor DNA sequencing is becoming standard-of-care for patient treatment decisions. We evaluated genotype concordance between tumor DNA and genomic DNA from blood and catalogued functional effects of somatic mutations in 21 drug response genes in 752 solid tumor patients. Using a threshold of 10% difference between tumor and blood DNA variant allele fraction (VAF), concordance for heterogenous genotype calls was 78% and increased to 97.5% using a 30% VAF threshold. Somatic mutations were observed in all 21 drug response genes, and 44% of patients had at least one somatic mutation in these genes. In tumor DNA, eight patients had a frameshift mutation in CYP2C8, which metabolizes taxanes. Overall, somatic copy number losses were more frequent than gains, including for CYP2C19 and CYP2D6 which had the most frequent copy number losses. However, copy number gains in TPMT were more than four times as common as losses. Seven % of patients had copy number gains in ABCB1, a multidrug resistance transporter of anti-cancer agents. These results demonstrate tumor-only DNA sequencing might not be reliable to call germline genotypes of drug response variants.

Adopting duplex sequencing technology for genetic toxicity testing: A proof-of-concept mutagenesis experiment with N-ethyl-N-nitrosourea (ENU)-exposed rats.

Duplex sequencing (DS) is an error-corrected next-generation sequencing method in which molecular barcodes informatically link PCR-copies back to their source DNA strands, enabling computational removal of errors in consensus sequences. The resulting background of less than one artifactual mutation per 107 nucleotides allows for direct detection of somatic mutations. TwinStrand Biosciences, Inc. has developed a DS-based mutagenesis assay to sample the rat genome, which can be applied to genetic toxicity testing. To evaluate this assay for early detection of mutagenesis, a time-course study was conducted using male Hsd:Sprague Dawley SD rats (3 per group) administered a single dose of 40 mg/kg N-ethyl-N-nitrosourea (ENU) via gavage, with mutation frequency (MF) and spectrum analyzed in stomach, bone marrow, blood, and liver tissues at 3 h, 24 h, 7 d, and 28 d post-exposure. Significant increases in MF were observed in ENU-exposed rats as early as 24 h for stomach (site of contact) and bone marrow (a highly proliferative tissue) and at 7 d for liver and blood. The canonical, mutational signature of ENU was established by 7 d post-exposure in all four tissues. Interlaboratory analysis of a subset of samples from different tissues and time points demonstrated remarkable reproducibility for both MF and spectrum. These results demonstrate that MF and spectrum can be evaluated successfully by directly sequencing targeted regions of DNA obtained from various tissues⁠, a considerable advancement compared to currently used in vivo gene mutation assays.

Adopting Duplex Sequencing™ Technology for Genetic Toxicity Testing: A Proof-of-Concept Mutagenesis Experiment with N-Ethyl-N-Nitrosourea (ENU)-Exposed Rats.

Duplex sequencing (DuplexSeq) is an error-corrected next-generation sequencing (ecNGS) method in which molecular barcodes informatically link PCR-copies back to their source DNA strands, enabling computational removal of errors by comparing grouped strand sequencing reads. The resulting background of less than one artifactual mutation per 10 7 nucleotides allows for direct detection of somatic mutations. TwinStrand Biosciences, Inc. has developed a DuplexSeq-based mutagenesis assay to sample the rat genome, which can be applied to genetic toxicity testing. To evaluate this assay for early detection of mutagenesis, a time-course study was conducted using male Hsd:Sprague Dawley SD rats (3 per group) administered a single dose of 40 mg/kg N-ethyl-N-nitrosourea (ENU) via gavage, with mutation frequency (MF) and spectrum analyzed in stomach, bone marrow, blood, and liver tissues at 3 h, 24 h, 7 d, and 28 d post-exposure. Significant increases in MF were observed in ENU-exposed rats as early as 24 h for stomach (site of contact) and bone marrow (a highly proliferative tissue) and at 7 d for liver and blood. The canonical, mutational signature of ENU was established by 7 d post-exposure in all four tissues. Interlaboratory analysis of a subset of samples from different tissues and time points demonstrated remarkable reproducibility for both MF and spectrum. These results demonstrate that MF and spectrum can be evaluated successfully by directly sequencing targeted regions of DNA obtained from various tissues, a considerable advancement compared to currently used in vivo gene mutation assays. HIGHLIGHTS: DuplexSeq is an ultra-accurate NGS technology that directly quantifies mutationsENU-dependent mutagenesis was detected 24 h post-exposure in proliferative tissuesMultiple tissues exhibited the canonical ENU mutation spectrum 7 d after exposureResults obtained with DuplexSeq were highly concordant between laboratoriesThe Rat-50 Mutagenesis Assay is promising for applications in genetic toxicology.

A genome-wide association analysis of temozolomide response using lymphoblastoid cell lines shows a clinically relevant association with MGMT.

OBJECTIVE: Recently, lymphoblastoid cell lines (LCLs) have emerged as an innovative model system for mapping gene variants that predict the dose response to chemotherapy drugs. METHODS: In the current study, this strategy was expanded to the in-vitro genome-wide association approach, using 516 LCLs derived from a White cohort to assess the cytotoxic response to temozolomide. RESULTS: Genome-wide association analysis using ∼2.1 million quality-controlled single-nucleotide polymorphisms (SNPs) identified a statistically significant association (P<10(-8)) with SNPs in the O(6)-methylguanine-DNA methyltransferase (MGMT) gene. We also show that the primary SNP in this region is significantly associated with the differential gene expression of MGMT (P<10(-26)) in LCLs and differential methylation in glioblastoma samples from The Cancer Genome Atlas. CONCLUSION: The previously documented clinical and functional relationships between MGMT and temozolomide response highlight the potential of well-powered genome-wide association studies of the LCL model system to identify meaningful genetic associations.

Cancer pharmacogenomics: DNA genotyping and gene expression profiling to identify molecular determinants of chemosensitivity.

Cancer patients exhibit a wide heterogeneity in their responses to chemotherapy. Improvement in chemotherapeutic responses could be achieved by gaining more detailed information on the molecular determinants (i.e., DNA, RNA or protein) underlying this heterogeneity. Pharmacogenomics approaches can be used to integrate information on drug responsiveness with alterations in molecular entities, often on a genome-wide scale. By using information gleaned from pharmacogenomics studies, it is anticipated that cancer chemotherapy can be tailored to the individual patient or tumor phenotype. This review focuses on pharmacogenomics studies conducted to gain insight into the molecular determinants of chemosensitivity to cancer chemotherapeutics.

Applications of genomic tools to colorectal cancer therapeutics.

Clinically and histopathologically similar colorectal cancers exhibit considerable variability in their responses to chemotherapeutics. The advent of genomic technologies has enabled the unbiased determination of changes in DNA and RNA, alterations that may be responsible for, or predictive of the variability in response to chemotherapy. This review highlights several advances made in applying genomic tools toward colorectal cancer therapeutics. Progress has been made using gene expression profiling to identify which colorectal cancer patients would benefit most from adjuvant chemotherapy. In addition, advances have been made in colorectal cancer pharmacogenomics by identifying gene expression patterns associated with sensitivity to specific chemotherapeutic agents. Lastly, the use of genome-wide mutation screening of individual tumor samples to identify the profiles of mutated genes is explored. Future research toward integrating genomic information with clinical and histopathological data is expected to lead to improved therapeutic management of colorectal cancer.

Genetic variation determines VEGF-A plasma levels in cancer patients.

Angiogenesis is essential in tumor biology and is regulated by vascular endothelial growth factor (VEGF) ligands and receptors. Here we aimed to discover genetic variants associated with levels of circulating angiogenic proteins in cancer patients. Plasma was collected at baseline in 216 pancreatic and 114 colorectal cancer patients. Thirty-one angiogenic proteins were measured by ELISA. 484,523 Single Nucleotide Polymorphisms (SNP) were tested for association with plasma levels for each protein in pancreatic cancer patients. Three top-ranked hits were then genotyped in colorectal cancer patients, where associations with the same proteins were measured. The results demonstrated rs2284284 and MCP1 (P-value = 6.7e-08), rs7504372 and VEGF-C (P-value = 9.8e-09), and rs7767396 and VEGF-A (P-value = 5.8e-09) were SNP-protein pairs identified in pancreatic cancer patients. In colorectal cancer patients, only rs7767396 (A > G) and VEGF-A was validated (P-value = 5.18e-05). The AA genotype of rs7767396 exhibited 2.04-2.3 and 2.7-3.4-fold higher VEGF-A levels than those with AG and GG genotypes. The G allele of rs7767396 reduces binding of the NF-AT1 transcription factor. In conclusion, a common genetic variant predicts the plasma levels of VEGF-A in cancer patients through altered binding of NF-AT1.

VHL substrate transcription factor ZHX2 as an oncogenic driver in clear cell renal cell carcinoma.

Inactivation of the von Hippel-Lindau (VHL) E3 ubiquitin ligase protein is a hallmark of clear cell renal cell carcinoma (ccRCC). Identifying how pathways affected by VHL loss contribute to ccRCC remains challenging. We used a genome-wide in vitro expression strategy to identify proteins that bind VHL when hydroxylated. Zinc fingers and homeoboxes 2 (ZHX2) was found as a VHL target, and its hydroxylation allowed VHL to regulate its protein stability. Tumor cells from ccRCC patients with VHL loss-of-function mutations usually had increased abundance and nuclear localization of ZHX2. Functionally, depletion of ZHX2 inhibited VHL-deficient ccRCC cell growth in vitro and in vivo. Mechanistically, integrated chromatin immunoprecipitation sequencing and microarray analysis showed that ZHX2 promoted nuclear factor κB activation. These studies reveal ZHX2 as a potential therapeutic target for ccRCC.

Heat map visualization of high-density clinical chemistry data.

Clinical chemistry data are routinely generated as part of preclinical animal toxicity studies and human clinical studies. With large-scale studies involving hundreds or even thousands of samples in multiple treatment groups, it is currently difficult to interpret the resulting complex, high-density clinical chemistry data. Accordingly, we conducted this study to investigate methods for easy visualization of complex, high-density data. Clinical chemistry data were obtained from male rats each treated with one of eight different acute hepatotoxicants from a large-scale toxicogenomics study. The raw data underwent a Z-score transformation comparing each individual animal's clinical chemistry values to that of reference controls from all eight studies and then were visualized in a single graphic using a heat map. The utility of using a heat map to visualize high-density clinical chemistry data was explored by clustering changes in clinical chemistry values for >400 animals. A clear distinction was observed in animals displaying hepatotoxicity from those that did not. Additionally, while animals experiencing hepatotoxicity showed many similarities in the observed clinical chemistry alterations, distinct differences were noted in the heat map profile for the different compounds. Using a heat map to visualize complex, high-density clinical chemistry data in a single graphic facilitates the identification of previously unrecognized trends. This method is simple to implement and maintains the biological integrity of the data. The value of this clinical chemistry data transformation and visualization will manifest itself through integration with other high-density data, such as genomics data, to study physiology at the systems level.

Identification of genes implicated in methapyrilene-induced hepatotoxicity by comparing differential gene expression in target and nontarget tissue.

BACKGROUND: Toxicogenomics experiments often reveal thousands of transcript alterations that are related to multiple processes, making it difficult to identify key gene changes that are related to the toxicity of interest. OBJECTIVES: The objective of this study was to compare gene expression changes in a nontarget tissue to the target tissue for toxicity to help identify toxicity-related genes. METHODS: Male rats were given the hepatotoxicant methapyrilene at two dose levels, with livers and kidneys removed 24 hr after one, three, and seven doses for gene expression analysis. To identify gene changes likely to be related to toxicity, we analyzed genes on the basis of their temporal pattern of change using a program developed at the National Institute of Environmental Health Sciences, termed "EPIG" (extracting gene expression patterns and identifying co-expressed genes). RESULTS: High-dose methapyrilene elicited hepatic damage that increased in severity with the number of doses, whereas no treatment-related lesions were observed in the kidney. High-dose methapyrilene elicited thousands of gene changes in the liver at each time point, whereas many fewer gene changes were observed in the kidney. EPIG analysis identified patterns of gene expression correlated to the observed toxicity, including genes associated with endoplasmic reticulum stress and the unfolded protein response. CONCLUSIONS: By factoring in dose level, number of doses, and tissue into the analysis of gene expression elicited by methapyrilene, we were able to identify genes likely to not be implicated in toxicity, thereby allowing us to focus on a subset of genes to identify toxicity-related processes.

Integrative Genomic Analysis of Cholangiocarcinoma Identifies Distinct IDH-Mutant Molecular Profiles.

Cholangiocarcinoma (CCA) is an aggressive malignancy of the bile ducts, with poor prognosis and limited treatment options. Here, we describe the integrated analysis of somatic mutations, RNA expression, copy number, and DNA methylation by The Cancer Genome Atlas of a set of predominantly intrahepatic CCA cases and propose a molecular classification scheme. We identified an IDH mutant-enriched subtype with distinct molecular features including low expression of chromatin modifiers, elevated expression of mitochondrial genes, and increased mitochondrial DNA copy number. Leveraging the multi-platform data, we observed that ARID1A exhibited DNA hypermethylation and decreased expression in the IDH mutant subtype. More broadly, we found that IDH mutations are associated with an expanded histological spectrum of liver tumors with molecular features that stratify with CCA. Our studies reveal insights into the molecular pathogenesis and heterogeneity of cholangiocarcinoma and provide classification information of potential therapeutic significance.

Differential gene expression profiling in whole blood during acute systemic inflammation in lipopolysaccharide-treated rats.

Microarrays have been used to evaluate the expression of thousands of genes in various tissues. However, few studies have investigated the change in gene expression profiles in one of the most easily accessible tissues, whole blood. We utilized an acute inflammation model to investigate the possibility of using a cDNA microarray to measure the gene expression profile in the cells of whole blood. Blood was collected from male Sprague-Dawley rats at 2 and 6 h after treatment with 5 mg/kg (ip) LPS. Hematology showed marked neutrophilia accompanied by lymphopenia at both time points. TNF-alpha and IL-6 levels were markedly elevated at 2 h, indicating acute inflammation, but by 6 h the levels had declined. Total RNA was isolated from whole blood and hybridized to the National Institute of Environmental Health Sciences Rat Chip v.3.0. LPS treatment caused 226 and 180 genes to be differentially expressed at 2 and 6 h, respectively. Many of the differentially expressed genes are involved in inflammation and the acute phase response, but differential expression was also noted in genes involved in the cytoskeleton, cell adhesion, oxidative respiration, and transcription. Real-time RT-PCR confirmed the differential regulation of a representative subset of genes. Principal component analysis of gene expression discriminated between the acute inflammatory response apparent at 2 h and the observed recovery underway at 6 h. These studies indicate that, in whole blood, changes in gene expression profiles can be detected that are reflective of inflammation, despite the adaptive shifts in leukocyte populations that accompany such inflammatory processes.

A randomized trial of etoposide + cisplatin versus vinblastine + bleomycin + cisplatin + cyclophosphamide + dactinomycin in patients with good-prognosis germ cell tumors.

Standard chemotherapy for disseminated germ cell tumors (GCT) cures most patients but causes considerable acute toxicity, including treatment-related death due to septicemia during neutropenia and pulmonary fibrosis. In addition, chronic and delayed toxicities, particularly Raynaud's phenomenon, have been reported in 6% to 37% of treated patients. In an attempt to minimize the acute and chronic effects of treatment which are related primarily to vinblastine and bleomycin, a randomized trial comparing the efficacy and toxicity of vinblastine + bleomycin + cisplatin + cyclophosphamide + dactinomycin (VAB-6) and etoposide + cisplatin (EP) was conducted on 164 eligible patients with good-prognosis GCT. Seventy-nine of 82 (96%) patients receiving VAB-6 and 76/82 (93%) receiving EP achieved a complete remission (CR) with or without adjunctive surgery. Similar proportions of patients in both arms were found at surgery to have necrosis/fibrosis or mature teratoma. With a median follow-up of 24.4 months in the VAB-6 arm and 25.9 months in the EP arm, the total, relapse-free, and event-free survival distributions were similar in the two arms. Patients receiving EP experienced less emesis (P = .05), higher nadir WBC (P = .06) and platelet counts (P = .01), less magnesium wasting (P = .0001), less mucositis (P = .09), and no pulmonary toxicity. No treatment-related mortality was observed. EP is an efficacious and less toxic regimen and is recommended for good-prognosis patients with disseminated GCT.

Alternating cycles of etoposide plus cisplatin and VAB-6 in the treatment of poor-risk patients with germ cell tumors.

A phase II trial of 6 months of alternating etoposide plus cisplatin (EP) and cyclophosphamide, vinblastine, actinomycin D, bleomycin, cisplatin (VAB-6) was conducted in 41 evaluable patients in an attempt to improve the treatment results in those patients considered to have "poor-risk" germ cell tumors (GCT). Eight of 14 (57%) patients with mediastinal and retroperitoneal GCTs achieved complete remission (CR), and five (36%) remain alive and free of disease. Fourteen of 27 patients (52%) with poor-risk testicular cancer achieved CR, and ten (37%) remain alive and free of disease. Two patients with seminoma, one each with a testicular and extragonadal primary tumor, achieved durable CRs. Toxicity was tolerable, but greater than that of VAB-6 alone. The response and survival of the 39 patients with nonseminomatous tumors were found to be identical to the results of 29 patients with nonseminomatous GCTs and poor-risk characteristics who were treated with VAB-6 alone. Thus, this 6-month schedule of alternating months of chemotherapy is not recommended for patients with poor-risk GCTs. Patients with such tumors should be referred to centers conducting prospective trials, so that research seeking better therapy may continue.

Working memory performance and cholinergic effects in the ventral tegmental area and substantia nigra.

The nicotinic antagonist mecamylamine has been found to impair working memory performance in the radial-arm maze (RAM) after s.c. or i.c.v. administration. Mecamylamine has important interactions with dopaminergic (DA) systems. Mecamylamine-induced memory deficits in the RAM are potentiated by the D2 antagonist raclopride and reversed by the D2 agonist quinpirole. The nicotinic agonist nicotine has been found to improve working memory performance in the RAM after s.c. or i.c.v. administration. Nicotine-induced memory improvement in the RAM is potentiated by the D2 agonist quinpirole. The midbrain DA nuclei, the substantia nigra (SN) and the ventral tegmental area (VTA) have relatively dense concentrations of nicotinic receptors which may be critical sites of action for mecamylamine and nicotine. In the current study, the effects of mecamylamine (1, 3.3 and 10 micrograms/side) infusions into the SN (n = 12) and VTA (n = 13) on working memory in the radial-arm maze were examined in adult female Sprague-Dawley rats. The 10-micrograms/side dose of mecamylamine significantly impaired radial-arm maze working memory performance when infused into either the SN or VTA. No significant effects of mecamylamine on response latency were seen. The nicotinic agonists cytisine (0.1, 0.33 and 1.0 microgram/side) and nicotine (0.3, 1.0 and 3.3 micrograms/side) were administered in a counterbalanced order. The high dose of cytisine (1 microgram/side) nearly caused a significant deficit in choice accuracy. Nicotine slightly depressed choice accuracy but not significantly in this study. The interaction of nicotine and mecamylamine was then studied. A dose of 1.0 microgram/side of nicotine caused a significant decrease in choice accuracy. Interestingly, this was significantly reversed by a 3.3-micrograms/side dose of mecamylamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Perinatal exposure to environmental tobacco smoke induces adenylyl cyclase and alters receptor-mediated cell signaling in brain and heart of neonatal rats.

Perinatal exposure to environmental tobacco smoke (ETS) has adverse effects on neurobehavioral development. In the current study, rats were exposed to ETS during gestation, during the early neonatal period, or both. Brains and hearts were examined for alterations in adenylyl cyclase (AC) activity and for changes in beta-adrenergic and m2-muscarinic cholinergic receptors and their linkage to AC. ETS exposure elicited induction of total AC activity as monitored with the direct enzymatic stimulant, forskolin. In the brain, the specific coupling of beta-adrenergic receptors to AC was inhibited in the ETS groups, despite a normal complement of beta-receptor binding sites. In the heart, ETS evoked a decrease in m2-receptor expression. In both tissues, the effects of postnatal ETS, mimicking passive smoking, were equivalent to (AC) or greater than (m2-receptors) those seen with prenatal ETS mimicking active smoking; the effects of combined prenatal and postnatal exposure were equivalent to those seen with postnatal exposure alone. These data indicate that ETS exposure evokes changes in cell signaling that recapitulate those caused by developmental nicotine treatment. Since alterations in AC signaling are known to affect cardiorespiratory function, the present results provide a mechanistic link reinforcing the participation of ETS exposure, including postnatal ETS, in disturbances culminating in events like Sudden Infant Death Syndrome.

Neonatal chlorpyrifos exposure targets multiple proteins governing the hepatic adenylyl cyclase signaling cascade: implications for neurotoxicity.

Chlorpyrifos has been hypothesized to interact with receptors and transduction proteins involved in the production of cyclic AMP, contributing to adverse effects on cell replication and differentiation. We studied the effects of neonatal chlorpyrifos exposure on hepatic adenylyl cyclase (AC) activity, as the liver accumulates the highest concentrations of chlorpyrifos and is the site for generation of its active metabolite, chlorpyrifos oxon. Newborn rats were given 1 mg/kg of chlorpyrifos s.c. on PN1-4. On PN5, 24 h after the last dose, AC catalytic activity was induced as assessed by the response to the direct AC stimulant, Mn(2+). In contrast, AC activation dependent upon interaction of the enzyme with G-proteins (forskolin) did not show any enhancement, suggesting impairment of G-protein function. This conclusion was confirmed by impaired responsiveness to fluoride, which directly activates G-proteins. In addition, the response of AC to hormonal signals was altered in a receptor-selective manner, with an enhanced response to glucagon but not to the beta-adrenoceptor agonist, isoproterenol. The effects of chlorpyrifos on AC signaling displayed a critical developmental period of vulnerability, as treatment of older rats (PN11-14) failed to cause substantial induction of AC or interference with G-protein signaling, although it did still enhance the glucagon response. In all cases, the effects of chlorpyrifos disappeared within a few days of discontinuing treatment. These results stand in contrast to the delayed deterioration of AC signaling seen in the brain after the same chlorpyrifos treatment. The temporal and organ selectivity of chlorpyrifos' effects on the AC cascade suggest that disruption of membrane signaling occurs consequent to selective effects on cell development, rather than representing a direct interaction between chlorpyrifos and signaling proteins.

Perinatal exposure to environmental tobacco smoke upregulates nicotinic cholinergic receptors in monkey brain.

In humans, perinatal exposure to environmental tobacco smoke (ETS) is associated with neurobehavioral deficits. In the current study, we exposed Rhesus monkeys to ETS in late gestation and in the early neonatal period, and examined changes in neurotransmitter receptors in the brainstem and caudal portion of the cerebral cortex. Nicotinic acetylcholine receptors were markedly upregulated and the effect was selective in that there were no changes in m(2)-muscarinic acetylcholine receptors or in beta-adrenergic receptors. Nicotinic receptor upregulation is indicative of chronic cell stimulation by nicotine, and is a hallmark of nicotine-induced neuroteratogenesis. These results indicate that perinatal ETS exposes the fetus and neonate to quantities of nicotine that are sufficient to alter brain development.

Results of intravenous digital subtraction angiography (IVDSA) as a screening method for renovascular hypertension.

Since January 1982 we have combined urography in hypertensive patients with intravenous angiography of the renal arteries. In more than 80% of 163 patients the examination was of good diagnostic quality and both renal arteries were well visualised. IVDSA has a lower morbidity and cost than conventional angiography. It is more accurate than urography and therefore offers better screening of a selected group of hypertensive patients.