Somatic inactivating PTPRJ mutations and dysregulated pathways identified in canine malignant melanoma by integrated comparative genomic analysis.

Canine malignant melanoma, a significant cause of mortality in domestic dogs, is a powerful comparative model for human melanoma, but little is known about its genetic etiology. We mapped the genomic landscape of canine melanoma through multi-platform analysis of 37 tumors (31 mucosal, 3 acral, 2 cutaneous, and 1 uveal) and 17 matching constitutional samples including long- and short-insert whole genome sequencing, RNA sequencing, array comparative genomic hybridization, single nucleotide polymorphism array, and targeted Sanger sequencing analyses. We identified novel predominantly truncating mutations in the putative tumor suppressor gene PTPRJ in 19% of cases. No BRAF mutations were detected, but activating RAS mutations (24% of cases) occurred in conserved hotspots in all cutaneous and acral and 13% of mucosal subtypes. MDM2 amplifications (24%) and TP53 mutations (19%) were mutually exclusive. Additional low-frequency recurrent alterations were observed amidst low point mutation rates, an absence of ultraviolet light mutational signatures, and an abundance of copy number and structural alterations. Mutations that modulate cell proliferation and cell cycle control were common and highlight therapeutic axes such as MEK and MDM2 inhibition. This mutational landscape resembles that seen in BRAF wild-type and sun-shielded human melanoma subtypes. Overall, these data inform biological comparisons between canine and human melanoma while suggesting actionable targets in both species.

Integrated genomic analyses reveal frequent TERT aberrations in acral melanoma.

Genomic analyses of cutaneous melanoma (CM) have yielded biological and therapeutic insights, but understanding of non-ultraviolet (UV)-derived CMs remains limited. Deeper analysis of acral lentiginous melanoma (ALM), a rare sun-shielded melanoma subtype associated with worse survival than CM, is needed to delineate non-UV oncogenic mechanisms. We thus performed comprehensive genomic and transcriptomic analysis of 34 ALM patients. Unlike CM, somatic alterations were dominated by structural variation and absence of UV-derived mutation signatures. Only 38% of patients demonstrated driver BRAF/NRAS/NF1 mutations. In contrast with CM, we observed PAK1 copy gains in 15% of patients, and somatic TERT translocations, copy gains, and missense and promoter mutations, or germline events, in 41% of patients. We further show that in vitro TERT inhibition has cytotoxic effects on primary ALM cells. These findings provide insight into the role of TERT in ALM tumorigenesis and reveal preliminary evidence that TERT inhibition represents a potential therapeutic strategy in ALM.

microRNA changes in liver tissue associated with fibrosis progression in patients with hepatitis C.

BACKGROUND & AIMS: Accumulating evidence indicates that microRNAs play a role in a number of disease processes including the pathogenesis of liver fibrosis in hepatitis C infection. Our goal is to add to the accruing information regarding microRNA deregulation in liver fibrosis to increase our understanding of the underlying mechanisms of pathology and progression. METHODS: We used next generation sequencing to profile all detectable microRNAs in liver tissue and serum from patients with hepatitis C, stages F1-F4 of fibrosis. RESULTS: We found altered expression of several microRNAs, in particular, miR-182, miR199a-5p, miR-200a-5p and miR-183 were found to be significantly upregulated in tissue from liver biopsies of hepatitis C patients with advanced fibrosis, stage F3 and F4, when compared with liver biopsies from patients with early fibrosis, stages F1 and F2. We also found miR-148-5p, miR-1260b, miR-122-3p and miR-378i among the microRNAs most significantly down-regulated from early to advanced fibrosis of the liver. We also sequenced the serum microRNAs; however, we were not able to detect significant changes in circulating microRNAs associated with fibrosis stage after adjusting for multiple tests. CONCLUSIONS: Adding measurements of tissue microRNAs acquired during routine biopsies will continue to increase our knowledge of underlying mechanisms of fibrosis. Our goal is that these data, in combination with studies from other researchers and future long-term studies, could be used to enhance the staging accuracy of liver biopsies and expand the surveillance of patients at increased risk for cancer and progression to advanced fibrosis.

Differences in Host Innate Responses among Coccidioides Isolates in a Murine Model of Pulmonary Coccidioidomycosis.

Coccidioides immitis and Coccidioides posadasii are soil-dwelling fungi and the causative agents of coccidioidomycosis, a mycosis endemic to certain semiarid regions in the Americas. The most common route of infection is by inhalation of airborne Coccidioides arthroconidia. Once a susceptible host inhales the conidia, a transition to mature endosporulated spherules can occur within the first 5 days of infection. For this study, we examined the host response in a murine model of coccidioidomycosis during a time period of infection that has not been well characterized. We collected lung tissue and bronchoalveolar lavage fluid (BALF) from BALB/c mice that were infected with a C. immitis pure strain, a C. immitis hybrid strain, or a C. posadasii strain as well as uninfected mice. We compared the host responses to the Coccidioides strains used in this study by assessing the level of transcription of selected cytokine genes in lung tissues and characterized host and fungal proteins present in BALF. Host response varied depending on the Coccidioides strain that was used and did not appear to be overly robust. This study provides a foundation to begin to dissect the host immune response early in infection, to detect abundant Coccidioides proteins, and to develop diagnostics that target these early time points of infection.

Advancing a clinically relevant perspective of the clonal nature of cancer.

Cancers frequently arise as a result of an acquired genomic instability and the subsequent clonal evolution of neoplastic cells with variable patterns of genetic aberrations. Thus, the presence and behaviors of distinct clonal populations in each patient's tumor may underlie multiple clinical phenotypes in cancers. We applied DNA content-based flow sorting to identify and isolate the nuclei of clonal populations from tumor biopsies, which was coupled with array CGH and targeted resequencing. The results produced high-definition genomic profiles of clonal populations from 40 pancreatic adenocarcinomas and a set of prostate adenocarcinomas, including serial biopsies from a patient who progressed to androgen-independent metastatic disease. The genomes of clonal populations were found to have patient-specific aberrations of clinical relevance. Furthermore, we identified genomic aberrations specific to therapeutically responsive and resistant clones arising during the evolution of androgen-independent metastatic prostate adenocarcinoma. We also distinguished divergent clonal populations within single biopsies and mapped aberrations in multiple aneuploid populations arising in primary and metastatic pancreatic adenocarcinoma. We propose that our high-definition analyses of the genomes of distinct clonal populations of cancer cells in patients in vivo can help guide diagnoses and tailor approaches to personalized treatment.

Genomic analysis and selected molecular pathways in rare cancers.

It is widely accepted that many cancers arise as a result of an acquired genomic instability and the subsequent evolution of tumor cells with variable patterns of selected and background aberrations. The presence and behaviors of distinct neoplastic cell populations within a patient's tumor may underlie multiple clinical phenotypes in cancers. A goal of many current cancer genome studies is the identification of recurring selected driver events that can be advanced for the development of personalized therapies. Unfortunately, in the majority of rare tumors, this type of analysis can be particularly challenging. Large series of specimens for analysis are simply not available, allowing recurring patterns to remain hidden. In this paper, we highlight the use of DNA content-based flow sorting to identify and isolate DNA-diploid and DNA-aneuploid populations from tumor biopsies as a strategy to comprehensively study the genomic composition and behaviors of individual cancers in a series of rare solid tumors: intrahepatic cholangiocarcinoma, anal carcinoma, adrenal leiomyosarcoma, and pancreatic neuroendocrine tumors. We propose that the identification of highly selected genomic events in distinct tumor populations within each tumor can identify candidate driver events that can facilitate the development of novel, personalized treatment strategies for patients with cancer.

Amplification and overexpression of vinculin are associated with increased tumour cell proliferation and progression in advanced prostate cancer.

Androgen withdrawal is the standard treatment for advanced prostate cancer. Although this therapy is initially effective, nearly all prostate cancers become refractory to it. Approximately 15% of these castration-resistant prostate cancers harbour a genomic amplification at 10q22. The aim of this study was to explore the structure of the 10q22 amplicon and to determine the major driving genes. Application of high-resolution array-CGH using the 244k Agilent microarrays to cell lines with 10q22 amplification allowed us to narrow down the common amplified region to a region of 5.8 megabases. We silenced each of the genes of this region by an RNAi screen in the prostate cancer cell lines PC-3 and 22Rv1. We selected genes with a significant growth reduction in the 10q22 amplified cell line PC-3, but not in the non-amplified 22Rv1 cells, as putative target genes of this amplicon. Immunohistochemical analysis of the protein expression of these candidate genes on a tissue microarray enriched for 10q22 amplified prostate cancers revealed vinculin as the most promising target of this amplicon. We found a strong association between vinculin gene amplification and overexpression (p < 0.001). Further analysis of 443 specimens from across all stages of prostate cancer progression showed that vinculin expression was highest in castration-resistant prostate cancers, but negative or very low in benign prostatic hyperplasia (p < 0.0001). Additionally, high tumour cell proliferation measured by Ki67 expression was significantly associated with high vinculin expression in prostate cancer (p < 0.0001). Our data suggest that vinculin is a major driving gene of the 10q22 amplification in prostate cancer and that vinculin overexpression might contribute to prostate cancer progression by enhancing tumour cell proliferation.

Genomic signatures of cancer: basis for individualized risk assessment, selective staging and therapy.

The development of DNA microarray and quantitative real-time polymerase chain reaction technologies has allowed for precise quantitation of RNA expression of hundreds to thousands of genes. These technologies have significantly impacted the study and understanding of cancer in terms of its molecular and genetic characteristics. In this review article, breast cancer, colon cancer, and adrenal carcinoma have been chosen to illustrate the principle and techniques of genomic profiling and to illustrate how such methods may be used to develop genomic signatures for personalized risk assessment and to individualize patient treatment.

A small-molecule activator of the unfolded protein response eradicates human breast tumors in mice.

Metastatic estrogen receptor α (ERα)-positive breast cancer is presently incurable. Seeking to target these drug-resistant cancers, we report the discovery of a compound, called ErSO, that activates the anticipatory unfolded protein response (a-UPR) and induces rapid and selective necrosis of ERα-positive breast cancer cell lines in vitro. We then tested ErSO in vivo in several preclinical orthotopic and metastasis mouse models carrying different xenografts of human breast cancer lines or patient-derived breast tumors. In multiple orthotopic models, ErSO treatment given either orally or intraperitoneally for 14 to 21 days induced tumor regression without recurrence. In a cell line tail vein metastasis model, ErSO was also effective at inducing regression of most lung, bone, and liver metastases. ErSO treatment induced almost complete regression of brain metastases in mice carrying intracranial human breast cancer cell line xenografts. Tumors that did not undergo complete regression and regrew remained sensitive to retreatment with ErSO. ErSO was well tolerated in mice, rats, and dogs at doses above those needed for therapeutic responses and had little or no effect on normal ERα-expressing murine tissues. ErSO mediated its anticancer effects through activation of the a-UPR, suggesting that activation of a tumor protective pathway could induce tumor regression.

Identifying treatment options for BRAFV600 wild-type metastatic melanoma: A SU2C/MRA genomics-enabled clinical trial.

Although combination BRAF and MEK inhibitors are highly effective for the 40-50% of cutaneous metastatic melanomas harboring BRAFV600 mutations, targeted agents have been ineffective for BRAFV600wild-type (wt) metastatic melanomas. The SU2C Genomics-Enabled Medicine for Melanoma Trial utilized a Simon two-stage optimal design to assess whether comprehensive genomic profiling improves selection of molecular-based therapies for BRAFV600wt metastatic melanoma patients who had progressed on standard-of-care therapy, which may include immunotherapy. Of the response-evaluable patients, binimetinib was selected for 20 patients randomized to the genomics-enabled arm, and nine were treated on the alternate treatment arm. Response rates for 27 patients treated with targeted recommendations included one (4%) partial response, 18 (67%) with stable disease, and eight (30%) with progressive disease. Post-trial genomic and protein pathway activation mapping identified additional drug classes that may be considered for future studies. Our results highlight the complexity and heterogeneity of metastatic melanomas, as well as how the lack of response in this trial may be associated with limitations including monotherapy drug selection and the dearth of available single and combination molecularly-driven therapies to treat BRAFV600wt metastatic melanomas.

Synthetic lethal RNAi screening identifies sensitizing targets for gemcitabine therapy in pancreatic cancer.

BACKGROUND: Pancreatic cancer retains a poor prognosis among the gastrointestinal cancers. It affects 230,000 individuals worldwide, has a very high mortality rate, and remains one of the most challenging malignancies to treat successfully. Treatment with gemcitabine, the most widely used chemotherapeutic against pancreatic cancer, is not curative and resistance may occur. Combinations of gemcitabine with other chemotherapeutic drugs or biological agents have resulted in limited improvement. METHODS: In order to improve gemcitabine response in pancreatic cancer cells, we utilized a synthetic lethal RNAi screen targeting 572 known kinases to identify genes that when silenced would sensitize pancreatic cancer cells to gemcitabine. RESULTS: Results from the RNAi screens identified several genes that, when silenced, potentiated the growth inhibitory effects of gemcitabine in pancreatic cancer cells. The greatest potentiation was shown by siRNA targeting checkpoint kinase 1 (CHK1). Validation of the screening results was performed in MIA PaCa-2 and BxPC3 pancreatic cancer cells by examining the dose response of gemcitabine treatment in the presence of either CHK1 or CHK2 siRNA. These results showed a three to ten-fold decrease in the EC50 for CHK1 siRNA-treated cells versus control siRNA-treated cells while treatment with CHK2 siRNA resulted in no change compared to controls. CHK1 was further targeted with specific small molecule inhibitors SB 218078 and PD 407824 in combination with gemcitabine. Results showed that treatment of MIA PaCa-2 cells with either of the CHK1 inhibitors SB 218078 or PD 407824 led to sensitization of the pancreatic cancer cells to gemcitabine. CONCLUSION: These findings demonstrate the effectiveness of synthetic lethal RNAi screening as a tool for identifying sensitizing targets to chemotherapeutic agents. These results also indicate that CHK1 could serve as a putative therapeutic target for sensitizing pancreatic cancer cells to gemcitabine.

Multiscale, multimodal analysis of tumor heterogeneity in IDH1 mutant vs wild-type diffuse gliomas.

Glioma is recognized to be a highly heterogeneous CNS malignancy, whose diverse cellular composition and cellular interactions have not been well characterized. To gain new clinical- and biological-insights into the genetically-bifurcated IDH1 mutant (mt) vs wildtype (wt) forms of glioma, we integrated data from protein, genomic and MR imaging from 20 treatment-naïve glioma cases and 16 recurrent GBM cases. Multiplexed immunofluorescence (MxIF) was used to generate single cell data for 43 protein markers representing all cancer hallmarks, Genomic sequencing (exome and RNA (normal and tumor) and magnetic resonance imaging (MRI) quantitative features (protocols were T1-post, FLAIR and ADC) from whole tumor, peritumoral edema and enhancing core vs equivalent normal region were also collected from patients. Based on MxIF analysis, 85,767 cells (glioma cases) and 56,304 cells (GBM cases) were used to generate cell-level data for 24 biomarkers. K-means clustering was used to generate 7 distinct groups of cells with divergent biomarker profiles and deconvolution was used to assign RNA data into three classes. Spatial and molecular heterogeneity metrics were generated for the cell data. All features were compared between IDH mt and IDHwt patients and were finally combined to provide a holistic/integrated comparison. Protein expression by hallmark was generally lower in the IDHmt vs wt patients. Molecular and spatial heterogeneity scores for angiogenesis and cell invasion also differed between IDHmt and wt gliomas irrespective of prior treatment and tumor grade; these differences also persisted in the MR imaging features of peritumoral edema and contrast enhancement volumes. A coherent picture of enhanced angiogenesis in IDHwt tumors was derived from multiple platforms (genomic, proteomic and imaging) and scales from individual proteins to cell clusters and heterogeneity, as well as bulk tumor RNA and imaging features. Longer overall survival for IDH1mt glioma patients may reflect mutation-driven alterations in cellular, molecular, and spatial heterogeneity which manifest in discernable radiological manifestations.

Discovery of genetic profiles impacting response to chemotherapy: application to gemcitabine.

Chemotherapy is a major treatment modality for individuals affected by cancer. Currently, a number of genome-based technologies are being adopted to identify genes associated with drug response; however, large-scale genetic association applications are still limited. Here we describe a novel strategy based on the genetic and drug response data of the NCI60 cell lines to discover potential candidate genetic variants associated with variable response to chemotherapy. As an example we have applied this strategy to discover single genetic markers and haplotypes from candidate genes previously implicated in the pharmacobiology of gemcitabine. Single-marker association analyses have implicated the association of four SNPs within the gene loci of CDC5L, EPC2, POLS, and PARP1. We have also investigated the combined effect of SNPs using haplotype-based analysis. Accordingly, we have shown modest association of haplotypes in six genes, whereas the most significant associations included a haplotype of the POLS gene. The hypothesis-generating tool presented in this study can be applied to drugs profiled in the NCI60 cell line screen and provides an effective means for the identification of genes associated with drug response. The results obtained using this novel methodology can be used to better design the clinical trials for effective study of the chemotherapeutic agents and thus provide a basis for individualized chemotherapy.

Functional evidence implicating S100P in prostate cancer progression.

S100P protein regulates calcium signal transduction and mediates cytoskeletal interaction, protein phosphorylation and transcriptional control. We have previously shown how elevated S100P levels in prostate cancer strongly correlate with progression to metastatic disease. In our study, we evaluated the functional significance of S100P expression on prostate tumor growth in vitro and in vivo. S100P levels were modulated by overexpressing S100P in PC3 prostate cancer cells and by silencing S100P levels in 22Rv1 prostate cancer cells. Overexpression of S100P in PC3 cells promoted cell growth, increased the percentage of S-phase cells, decreased basal apoptosis rate and promoted anchorage independent growth in soft agar. Furthermore, prostate cancer cells overexpressing S100P were protected against camptothecin-induced apoptosis. Conversely, silencing of S100P in 22Rv1 cells using siRNA resulted in a prominent cytostatic effect. The influence of S100P on tumor growth and metastases were assessed in vivo. S100P-overexpressing PC3 cells had a dramatically increased tumor formation compared to controls. Microarray analysis showed the involvement of growth pathways including increased androgen receptor expression in S100P-overexpressing cells. These results provide the first functional proof that S100P overexpression can upregulate androgen receptor expression and thereby promote prostate cancer progression by increasing cell growth. Moreover, the results confirm the oncogenic nature of S100P in prostate cancer and suggest that the protein may directly confer resistance to chemotherapy. Hence, S100P could be considered a potential drug target or a chemosensitization target, and could also serve as a biomarker for aggressive, hormone-refractory and metastatic prostate cancer.

Network Rewiring in Cancer: Applications to Melanoma Cell Lines and the Cancer Genome Atlas Patients.

Genes do not work in isolation, but rather as part of networks that have many feedback and redundancy mechanisms. Studying the properties of genetic networks and how individual genes contribute to overall network functions can provide insight into genetically-mediated disease processes. Most analytical techniques assume a network topology based on normal state networks. However, gene perturbations often lead to the rewiring of relevant networks and impact relationships among other genes. We apply a suite of analysis methodologies to assess the degree of transcriptional network rewiring observed in different sets of melanoma cell lines using whole genome gene expression microarray profiles. We assess evidence for network rewiring in melanoma patient tumor samples using RNA-sequence data available from The Cancer Genome Atlas. We make a distinction between "unsupervised" and "supervised" network-based methods and contrast their use in identifying consistent differences in networks between subsets of cell lines and tumor samples. We find that different genes play more central roles within subsets of genes within a broader network and hence are likely to be better drug targets in a disease state. Ultimately, we argue that our results have important implications for understanding the molecular pathology of melanoma as well as the choice of treatments to combat that pathology.

Nonlinear mixed effects dose response modeling in high throughput drug screens: application to melanoma cell line analysis.

Cancer cell lines are often used in high throughput drug screens (HTS) to explore the relationship between cell line characteristics and responsiveness to different therapies. Many current analysis methods infer relationships by focusing on one aspect of cell line drug-specific dose-response curves (DRCs), the concentration causing 50% inhibition of a phenotypic endpoint (IC50). Such methods may overlook DRC features and do not simultaneously leverage information about drug response patterns across cell lines, potentially increasing false positive and negative rates in drug response associations. We consider the application of two methods, each rooted in nonlinear mixed effects (NLME) models, that test the relationship relationships between estimated cell line DRCs and factors that might mitigate response. Both methods leverage estimation and testing techniques that consider the simultaneous analysis of different cell lines to draw inferences about any one cell line. One of the methods is designed to provide an omnibus test of the differences between cell line DRCs that is not focused on any one aspect of the DRC (such as the IC50 value). We simulated different settings and compared the different methods on the simulated data. We also compared the proposed methods against traditional IC50-based methods using 40 melanoma cell lines whose transcriptomes, proteomes, and, importantly, BRAF and related mutation profiles were available. Ultimately, we find that the NLME-based methods are more robust, powerful and, for the omnibus test, more flexible, than traditional methods. Their application to the melanoma cell lines reveals insights into factors that may be clinically useful.

Ponatinib Shows Potent Antitumor Activity in Small Cell Carcinoma of the Ovary Hypercalcemic Type (SCCOHT) through Multikinase Inhibition.

Purpose: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, aggressive ovarian cancer in young women that is universally driven by loss of the SWI/SNF ATPase subunits SMARCA4 and SMARCA2. A great need exists for effective targeted therapies for SCCOHT.Experimental Design: To identify underlying therapeutic vulnerabilities in SCCOHT, we conducted high-throughput siRNA and drug screens. Complementary proteomics approaches profiled kinases inhibited by ponatinib. Ponatinib was tested for efficacy in two patient-derived xenograft (PDX) models and one cell-line xenograft model of SCCOHT.Results: The receptor tyrosine kinase (RTK) family was enriched in siRNA screen hits, with FGFRs and PDGFRs being overlapping hits between drug and siRNA screens. Of multiple potent drug classes in SCCOHT cell lines, RTK inhibitors were only one of two classes with selectivity in SCCOHT relative to three SWI/SNF wild-type ovarian cancer cell lines. We further identified ponatinib as the most effective clinically approved RTK inhibitor. Reexpression of SMARCA4 was shown to confer a 1.7-fold increase in resistance to ponatinib. Subsequent proteomic assessment of ponatinib target modulation in SCCOHT cell models confirmed inhibition of nine known ponatinib target kinases alongside 77 noncanonical ponatinib targets in SCCOHT. Finally, ponatinib delayed tumor doubling time 4-fold in SCCOHT-1 xenografts while reducing final tumor volumes in SCCOHT PDX models by 58.6% and 42.5%.Conclusions: Ponatinib is an effective agent for SMARCA4-mutant SCCOHT in both in vitro and in vivo preclinical models through its inhibition of multiple kinases. Clinical investigation of this FDA-approved oncology drug in SCCOHT is warranted. Clin Cancer Res; 24(8); 1932-43. ©2018 AACR.

Osteoprotegerin in prostate cancer bone metastasis.

Osteoprotegerin (OPG), a critical regulator of osteoclastogenesis, is expressed by prostate cancer cells, and OPG levels are increased in patients with prostate cancer bone metastases. The objective of this study was to investigate the effects of OPG overexpression on prostate cancer cells and prostate cancer/bone cell interactions in vitro and in vivo. OPG-transfected C4-2 cells expressed 8.0 ng OPG per mL per 10(6) cells, whereas no OPG was detected in the media of C4-2 cells transfected with a control plasmid. OPG overexpressed by C4-2 cells protected these cells from tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis and decreased osteoclast formation. Subcutaneous OPG-C4-2 and pcDNA-C4-2 tumors exhibited similar growth and take-rate characteristics. However, when grown in bone, tumor volume was decreased in OPG-C4-2 versus pcDNA-C4-2 (P=0.0017). OPG expressed by C4-2 cells caused increases in bone mineral density (P=0.0074) and percentage of trabecular bone volume (P=0.007), and decreases in numbers of osteoblasts and osteoclasts when compared with intratibial pcDNA-C4-2 tumors (P=0.003 and P=0.019, respectively). In summary, our data show that increased expression of OPG in C4-2 cells does not directly affect proliferation of prostate cancer cells but indirectly decreases growth of C4-2 tumors in the bone environment. Our data also show that OPG expressed by C4-2 cells inhibits bone lysis associated with C4-2 bone metastasis, which results in net increases in bone volume. We therefore hypothesize that OPG expressed in prostate cancer patient bone metastases may be at least partially responsible for the osteoblastic character of most prostate cancer bone lesions.

Analysis of variability in high throughput screening data: applications to melanoma cell lines and drug responses.

High-throughput screening (HTS) strategies and protocols have undergone significant development in the last decade. It is now possible to screen hundreds of thousands of compounds, each exploring multiple biological phenotypes and parameters, against various cell lines or model systems in a single setting. However, given the vast amount of data such studies generate, the fact that they use multiple reagents, and are often technician-intensive, questions have been raised about the variability, reliability and reproducibility of HTS results. Assessments of the impact of the multiple factors in HTS studies could arguably lead to more compelling insights into the robustness of the results of a particular screen, as well as the overall quality of the study. We leveraged classical, yet highly flexible, analysis of variance (ANOVA)-based linear models to explore how different factors contribute to the variation observed in a screening study of four different melanoma cell lines and 120 drugs over nine dosages studied in two independent academic laboratories. We find that factors such as plate effects, appropriate dosing ranges, and to a lesser extent, the laboratory performing the screen, are significant predictors of variation in drug responses across the cell lines. Further, we show that when sources of variation are quantified and controlled for, they contextualize claims of inconsistencies and reveal the overall quality of the HTS studies performed at each participating laboratory. In the context of the broader screening study, we show that our analysis can also elucidate the robust effects of drugs, even those within specific cell lines.

A prospective pilot study of genome-wide exome and transcriptome profiling in patients with small cell lung cancer progressing after first-line therapy.

BACKGROUND: Small cell lung cancer (SCLC) that has progressed after first-line therapy is an aggressive disease with few effective therapeutic strategies. In this prospective study, we employed next-generation sequencing (NGS) to identify therapeutically actionable alterations to guide treatment for advanced SCLC patients. METHODS: Twelve patients with SCLC were enrolled after failing platinum-based chemotherapy. Following informed consent, genome-wide exome and RNA-sequencing was performed in a CLIA-certified, CAP-accredited environment. Actionable targets were identified and therapeutic recommendations made from a pharmacopeia of FDA-approved drugs. Clinical response to genomically-guided treatment was evaluated by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1. RESULTS: The study completed its accrual goal of 12 evaluable patients. The minimum tumor content for successful NGS was 20%, with a median turnaround time from sample collection to genomics-based treatment recommendation of 27 days. At least two clinically actionable targets were identified in each patient, and six patients (50%) received treatment identified by NGS. Two had partial responses by RECIST 1.1 on a clinical trial involving a PD-1 inhibitor + irinotecan (indicated by MLH1 alteration). The remaining patients had clinical deterioration before NGS recommended therapy could be initiated. CONCLUSIONS: Comprehensive genomic profiling using NGS identified clinically-actionable alterations in SCLC patients who progressed on initial therapy. Recommended PD-1 therapy generated partial responses in two patients. Earlier access to NGS guided therapy, along with improved understanding of those SCLC patients likely to respond to immune-based therapies, should help to extend survival in these cases with poor outcomes.