BPG: Seamless, automated and interactive visualization of scientific data.

BACKGROUND: We introduce BPG, a framework for generating publication-quality, highly-customizable plots in the R statistical environment. RESULTS: This open-source package includes multiple methods of displaying high-dimensional datasets and facilitates generation of complex multi-panel figures, making it suitable for complex datasets. A web-based interactive tool allows online figure customization, from which R code can be downloaded for integration with computational pipelines. CONCLUSION: BPG provides a new approach for linking interactive and scripted data visualization and is available at http://labs.oicr.on.ca/boutros-lab/software/bpg or via CRAN at https://cran.r-project.org/web/packages/BoutrosLab.plotting.general.

Pathway-based subnetworks enable cross-disease biomarker discovery.

Biomarkers lie at the heart of precision medicine. Surprisingly, while rapid genomic profiling is becoming ubiquitous, the development of biomarkers usually involves the application of bespoke techniques that cannot be directly applied to other datasets. There is an urgent need for a systematic methodology to create biologically-interpretable molecular models that robustly predict key phenotypes. Here we present SIMMS (Subnetwork Integration for Multi-Modal Signatures): an algorithm that fragments pathways into functional modules and uses these to predict phenotypes. We apply SIMMS to multiple data types across five diseases, and in each it reproducibly identifies known and novel subtypes, and makes superior predictions to the best bespoke approaches. To demonstrate its ability on a new dataset, we profile 33 genes/nodes of the PI3K pathway in 1734 FFPE breast tumors and create a four-subnetwork prediction model. This model out-performs a clinically-validated molecular test in an independent cohort of 1742 patients. SIMMS is generic and enables systematic data integration for robust biomarker discovery.

Promoter hypomethylation of NY-ESO-1, association with clinicopathological features and PD-L1 expression in non-small cell lung cancer.

Cancer-Testis antigens (CTA) are immunogenic molecules with normal tissue expression restricted to testes but with aberrant expression in up to 30% of non-small cell lung cancers (NSCLCs). Regulation of CTA expression is mediated in part through promoter DNA methylation. Recently, immunotherapy has altered treatment paradigms in NSCLC. Given its immunogenicity and ability to be re-expressed through demethylation, NY-ESO-1 promoter methylation, protein expression and its association with programmed death receptor ligand-1 (PD-L1) expression and clinicopathological features were investigated. Lung cancer cell line demethylation resulting from 5-Aza-2'-deoxycytidine treatment was associated with both NY-ESO-1 and PD-L1 re-expression in vitro but not increased chemosensitivity. NY-ESO-1 hypomethylation was observed in 15/94 (16%) of patient samples and associated with positive protein expression (P < 0.0001). In contrast, PD-L1 expression was observed in 50/91 (55%) but strong expression in only 12/91 (13%) cases. There was no association between NY-ESO-1 and PD-L1 expression, despite resultant re-expression of both by 5-Aza-2'-deoxycytidine. Importantly, NY-ESO-1 hypomethylation was found to be an independent marker of poor prognosis in patients not treated with chemotherapy (HR 3.59, P = 0.003) in multivariate analysis. In patients treated with chemotherapy there were no differences in survival associated with NY-ESO-1 hypomethylation. Collectively, these results provided supporting evidence for the potential use of NY-ESO-1 hypomethylation as a prognostic biomarker in stage 3 NSCLCs. In addition, these data highlight the potential to incorporate demethylating agents to enhance immune activation, in tumours currently devoid of immune infiltrates and expression of immune checkpoint genes.

Molecular heterogeneity of non-small cell lung carcinoma patient-derived xenografts closely reflect their primary tumors.

Availability of lung cancer models that closely mimic human tumors remains a significant gap in cancer research, as tumor cell lines and mouse models may not recapitulate the spectrum of lung cancer heterogeneity seen in patients. We aimed to establish a patient-derived tumor xenograft (PDX) resource from surgically resected non-small cell lung cancer (NSCLC). Fresh tumor tissue from surgical resection was implanted and grown in the subcutaneous pocket of non-obese severe combined immune deficient (NOD SCID) gamma mice. Subsequent passages were in NOD SCID mice. A subset of matched patient and PDX tumors and non-neoplastic lung tissues were profiled by whole exome sequencing, single nucleotide polymorphism (SNP) and methylation arrays, and phosphotyrosine (pY)-proteome by mass spectrometry. The data were compared to published NSCLC datasets of NSCLC primary and cell lines. 127 stable PDXs were established from 441 lung carcinomas representing all major histological subtypes: 52 adenocarcinomas, 62 squamous cell carcinomas, one adeno-squamous carcinoma, five sarcomatoid carcinomas, five large cell neuroendocrine carcinomas, and two small cell lung cancers. Somatic mutations, gene copy number and expression profiles, and pY-proteome landscape of 36 PDXs showed greater similarity with patient tumors than with established cell lines. Novel somatic mutations on cancer associated genes were identified but only in PDXs, likely due to selective clonal growth in the PDXs that allows detection of these low allelic frequency mutations. The results provide the strongest evidence yet that PDXs established from lung cancers closely mimic the characteristics of patient primary tumors.

Spatial genomic heterogeneity within localized, multifocal prostate cancer.

Herein we provide a detailed molecular analysis of the spatial heterogeneity of clinically localized, multifocal prostate cancer to delineate new oncogenes or tumor suppressors. We initially determined the copy number aberration (CNA) profiles of 74 patients with index tumors of Gleason score 7. Of these, 5 patients were subjected to whole-genome sequencing using DNA quantities achievable in diagnostic biopsies, with detailed spatial sampling of 23 distinct tumor regions to assess intraprostatic heterogeneity in focal genomics. Multifocal tumors are highly heterogeneous for single-nucleotide variants (SNVs), CNAs and genomic rearrangements. We identified and validated a new recurrent amplification of MYCL, which is associated with TP53 deletion and unique profiles of DNA damage and transcriptional dysregulation. Moreover, we demonstrate divergent tumor evolution in multifocal cancer and, in some cases, tumors of independent clonal origin. These data represent the first systematic relation of intraprostatic genomic heterogeneity to predicted clinical outcome and inform the development of novel biomarkers that reflect individual prognosis.

Appropriateness of using patient-derived xenograft models for pharmacologic evaluation of novel therapies for esophageal/gastro-esophageal junction cancers.

The high morbidity and mortality of patients with esophageal (E) and gastro-esophageal junction (GEJ) cancers, warrants new pre-clinical models for drug testing. The utility of primary tumor xenografts (PTXGs) as pre-clinical models was assessed. Clinicopathological, immunohistochemical markers (p53, p16, Ki-67, Her-2/neu and EGFR), and global mRNA abundance profiles were evaluated to determine selection biases of samples implanted or engrafted, compared with the underlying population. Nine primary E/GEJ adenocarcinoma xenograft lines were further characterized for the spectrum and stability of gene/protein expression over passages. Seven primary esophageal adenocarcinoma xenograft lines were treated with individual or combination chemotherapy. Tumors that were implanted (n=55) in NOD/SCID mice had features suggestive of more aggressive biology than tumors that were never implanted (n=32). Of those implanted, 21/55 engrafted; engraftment was associated with poorly differentiated tumors (p=0.04) and older patients (p=0.01). Expression of immunohistochemical markers were similar between patient sample and corresponding xenograft. mRNA differences observed between patient tumors and first passage xenografts were largely due to loss of human stroma in xenografts. mRNA patterns of early vs late passage xenografts and of small vs large tumors of the same passage were similar. Complete resistance was present in 2/7 xenografts while the remaining tumors showed varying degrees of sensitivity, that remained constant across passages. Because of their ability to recapitulate primary tumor characteristics during engraftment and across serial passaging, PTXGs can be useful clinical systems for assessment of drug sensitivity of human E/GEJ cancers.

Integrating RAS status into prognostic signatures for adenocarcinomas of the lung.

PURPOSE: While the dysregulation of specific pathways in cancer influences both treatment response and outcome, few current prognostic markers explicitly consider differential pathway activation. Here we explore this concept, focusing on K-Ras mutations in lung adenocarcinoma (present in 25%-35% of patients). EXPERIMENTAL DESIGN: The effect of K-Ras mutation status on prognostic accuracy of existing signatures was evaluated in 404 patients. Genes associated with K-Ras mutation status were identified and used to create a RAS pathway activation classifier to provide a more accurate measure of RAS pathway status. Next, 8 million random signatures were evaluated to assess differences in prognosing patients with or without RAS activation. Finally, a prognostic signature was created to target patients with RAS pathway activation. RESULTS: We first show that K-Ras status influences the accuracy of existing prognostic signatures, which are effective in K-Ras-wild-type patients but fail in patients with K-Ras mutations. Next, we show that it is fundamentally more difficult to predict the outcome of patients with RAS activation (RAS(mt)) than that of those without (RAS(wt)). More importantly, we demonstrate that different signatures are prognostic in RAS(wt) and RAS(mt). Finally, to exploit this discovery, we create separate prognostic signatures for RAS(wt) and RAS(mt) patients and show that combining them significantly improves predictions of patient outcome. CONCLUSIONS: We present a nested model for integrated genomic and transcriptomic data. This model is general and is not limited to lung adenocarcinomas but can be expanded to other tumor types and oncogenes.

The transcriptomic profile of ovarian cancer grading.

Ovarian carcinoma is the leading cause of gynecological malignancy, with the serous subtype being the most commonly presented subtype. Recent studies have demonstrated that grade does not yield significant prognostic information, independent of TNM staging. As such, several different grading systems have been proposed to reveal morphological characteristics of these tumors, however each yield different results. To help address this issue, we performed a rigorous computational analysis to better understand the molecular differences that fundamentally explain the different grades and grading systems. mRNA abundance levels were analyzed across 334 total patients and their association with each grade and grading system were assessed. Few molecular differences were observed between grade 2 and 3 tumors when using the International Federation of Gynecology and Obstetrics (FIGO) grading system, suggesting their molecular similarity. In contrast, grading by the Silverberg system reveals that grades 1-3 are molecularly equidistant from one another across a spectrum. Additionally, we have identified a few candidate genes with good prognostic information that could potentially be used for classifying cases with similar morphological appearances.

Hypoxia promotes stem cell phenotypes and poor prognosis through epigenetic regulation of DICER.

MicroRNAs are small regulatory RNAs that post transcriptionally control gene expression. Reduced expression of DICER, the enzyme involved in microRNA processing, is frequently observed in cancer and is associated with poor clinical outcome in various malignancies. Yet, the underlying mechanisms are not well understood. Here we identify tumour hypoxia as a regulator of DICER expression in large cohorts of breast cancer patients. We show that DICER expression is suppressed by hypoxia through an epigenetic mechanism that involves inhibition of oxygen-dependent H3K27me3 demethylases KDM6A/B and results in silencing of the DICER promoter. Subsequently, reduced miRNA processing leads to derepression of the miR-200 target ZEB1, stimulates the epithelial to mesenchymal transition and ultimately results in the acquisition of stem cell phenotypes in human mammary epithelial cells. Our study uncovers a previously unknown relationship between oxygen-sensitive epigenetic regulators, miRNA biogenesis and tumour stem cell phenotypes that may underlie poor outcome in breast cancer.

Ensemble analyses improve signatures of tumour hypoxia and reveal inter-platform differences.

BACKGROUND: The reproducibility of transcriptomic biomarkers across datasets remains poor, limiting clinical application. We and others have suggested that this is in-part caused by differential error-structure between datasets, and their incomplete removal by pre-processing algorithms. METHODS: To test this hypothesis, we systematically assessed the effects of pre-processing on biomarker classification using 24 different pre-processing methods and 15 distinct signatures of tumour hypoxia in 10 datasets (2,143 patients). RESULTS: We confirm strong pre-processing effects for all datasets and signatures, and find that these differ between microarray versions. Importantly, exploiting different pre-processing techniques in an ensemble technique improved classification for a majority of signatures. CONCLUSIONS: Assessing biomarkers using an ensemble of pre-processing techniques shows clear value across multiple diseases, datasets and biomarkers. Importantly, ensemble classification improves biomarkers with initially good results but does not result in spuriously improved performance for poor biomarkers. While further research is required, this approach has the potential to become a standard for transcriptomic biomarkers.

Epigenetics in radiotherapy: where are we heading?

Radiotherapy is an important component of anti-cancer treatment. However, not all cancer patients respond to radiotherapy, and with current knowledge clinicians are unable to predict which patients are at high risk of recurrence after radiotherapy. There is therefore an urgent need for biomarkers to guide clinical decision-making. Although the importance of epigenetic alterations is widely accepted, their application as biomarkers in radiotherapy has not been studied extensively. In addition, it has been suggested that radiotherapy itself introduces epigenetic alterations. As epigenetic alterations can potentially be reversed by drug treatment, they are interesting candidate targets for anticancer therapy or radiotherapy sensitizers. The application of demethylating drugs or histone deacetylase inhibitors to sensitize patients for radiotherapy has been studied in vitro, in vivo as well as in clinical trials with promising results. This review describes the current knowledge on epigenetics in radiotherapy.

Systematic analysis of 18F-FDG PET and metabolism, proliferation and hypoxia markers for classification of head and neck tumors.

BACKGROUND: Quantification of molecular cell processes is important for prognostication and treatment individualization of head and neck cancer (HNC). However, individual tumor comparison can show discord in upregulation similarities when analyzing multiple biological mechanisms. Elaborate tumor characterization, integrating multiple pathways reflecting intrinsic and microenvironmental properties, may be beneficial to group most uniform tumors for treatment modification schemes. The goal of this study was to systematically analyze if immunohistochemical (IHC) assessment of molecular markers, involved in treatment resistance, and 18F-FDG PET parameters could accurately distinguish separate HNC tumors. METHODS: Several imaging parameters and texture features for 18F-FDG small-animal PET and immunohistochemical markers related to metabolism, hypoxia, proliferation and tumor blood perfusion were assessed within groups of BALB/c nu/nu mice xenografted with 14 human HNC models. Classification methods were used to predict tumor line based on sets of parameters. RESULTS: We found that 18F-FDG PET could not differentiate between the tumor lines. On the contrary, combined IHC parameters could accurately allocate individual tumors to the correct model. From 9 analyzed IHC parameters, a cluster of 6 random parameters already classified 70.3% correctly. Combining all PET/IHC characteristics resulted in the highest tumor line classification accuracy (81.0%; cross validation 82.0%), which was just 2.2% higher (p = 5.2×10-32) than the performance of the IHC parameter/feature based model. CONCLUSIONS: With a select set of IHC markers representing cellular processes of metabolism, proliferation, hypoxia and perfusion, one can reliably distinguish between HNC tumor lines. Addition of 18F-FDG PET improves classification accuracy of IHC to a significant yet minor degree. These results may form a basis for development of tumor characterization models for treatment allocation purposes.

The role of Cancer-Testis antigens as predictive and prognostic markers in non-small cell lung cancer.

BACKGROUND: Cancer-Testis Antigens (CTAs) are immunogenic proteins that are poor prognostic markers in non-small cell lung cancer (NSCLC). We investigated expression of CTAs in NSCLC and their association with response to chemotherapy, genetic mutations and survival. METHODS: We studied 199 patients with pathological N2 NSCLC treated with neoadjuvant chemotherapy (NAC; n = 94), post-operative observation (n = 49), adjuvant chemotherapy (n = 47) or unknown (n = 9). Immunohistochemistry for NY-ESO-1, MAGE-A and MAGE-C1 was performed. Clinicopathological features, response to neoadjuvant treatment and overall survival were correlated. DNA mutations were characterized using the Sequenom Oncocarta panel v1.0. Affymetrix data from the JBR.10 adjuvant chemotherapy study were obtained from a public repository, normalised and mapped for CTAs. RESULTS: NY-ESO-1 was expressed in 50/199 (25%) samples. Expression of NY-ESO-1 in the NAC cohort was associated with significantly increased response rates (P = 0.03), but not overall survival. In the post-operative cohort, multivariate analyses identified NY-ESO-1 as an independent poor prognostic marker for those not treated with chemotherapy (HR 2.61, 95% CI 1.28-5.33; P = 0.008), whereas treatment with chemotherapy and expression of NY-ESO-1 was an independent predictor of improved survival (HR 0.267, 95% CI 0.07-0.980; P = 0.046). Similar findings for MAGE-A were seen, but did not meet statistical significance. Independent gene expression data from the JBR.10 dataset support these findings but were underpowered to demonstrate significant differences. There was no association between oncogenic mutations and CTA expression. CONCLUSIONS: NY-ESO-1 was predictive of increased response to neoadjuvant chemotherapy and benefit from adjuvant chemotherapy. Further studies investigating the relationship between these findings and immune mechanisms are warranted.

The autophagy associated gene, ULK1, promotes tolerance to chronic and acute hypoxia.

BACKGROUND AND PURPOSE: Tumor hypoxia is associated with therapy resistance and malignancy. Previously we demonstrated that activation of autophagy and the unfolded protein response (UPR) promote hypoxia tolerance. Here we explored the importance of ULK1 in hypoxia tolerance, autophagy induction and its prognostic value for recurrence after treatment. MATERIAL AND METHODS: Hypoxic regulation of ULK1 mRNA and protein was assessed in vitro and in primary human head and neck squamous cell carcinoma (HNSCC) xenografts. Its importance in autophagy induction, mitochondrial homeostasis and tolerance to chronic and acute hypoxia was evaluated in ULK1 knockdown cells. The prognostic value of ULK1 mRNA expression was assessed in 82 HNSCC patients. RESULTS: ULK1 enrichment was observed in hypoxic tumor regions. High enrichment was associated with a high hypoxic fraction. In line with these findings, high ULK1 expression in HNSCC patients appeared associated with poor local control. Exposure of cells to hypoxia induced ULK1 mRNA in a UPR and HIF1α dependent manner. ULK1 knockdown decreased autophagy activation, increased mitochondrial mass and ROS exposure and sensitized cells to acute and chronic hypoxia. CONCLUSIONS: We demonstrate that ULK1 is a hypoxia regulated gene and is associated with hypoxia tolerance and a worse clinical outcome.

A case report and genetic characterization of a massive acinic cell carcinoma of the parotid with delayed distant metastases.

We describe the presentation, management, and clinical outcome of a massive acinic cell carcinoma of the parotid gland. The primary tumor and blood underwent exome sequencing which revealed deletions in CDKN2A as well as PPP1R13B, which induces p53. A damaging nonsynonymous mutation was noted in EP300, a histone acetylase which plays a role in cellular proliferation. This study provides the first insights into the genetic underpinnings of this cancer. Future large-scale efforts will be necessary to define the mutational landscape of salivary gland malignancies to identify therapeutic targets and biomarkers of treatment failure.

Predicting outcomes in radiation oncology--multifactorial decision support systems.

With the emergence of individualized medicine and the increasing amount and complexity of available medical data, a growing need exists for the development of clinical decision-support systems based on prediction models of treatment outcome. In radiation oncology, these models combine both predictive and prognostic data factors from clinical, imaging, molecular and other sources to achieve the highest accuracy to predict tumour response and follow-up event rates. In this Review, we provide an overview of the factors that are correlated with outcome-including survival, recurrence patterns and toxicity-in radiation oncology and discuss the methodology behind the development of prediction models, which is a multistage process. Even after initial development and clinical introduction, a truly useful predictive model will be continuously re-evaluated on different patient datasets from different regions to ensure its population-specific strength. In the future, validated decision-support systems will be fully integrated in the clinic, with data and knowledge being shared in a standardized, instant and global manner.

Comparison of toxicity and outcomes of concurrent radiotherapy with carboplatin/paclitaxel or cisplatin/etoposide in stage III non-small cell lung cancer.

Concurrent chemoradiotherapy (CCRT) has become the standard of care for patients with unresectable stage III non-small cell lung cancer (NSCLC). The comparative merits of two widely used regimens: carboplatin/paclitaxel (PC) and cisplatin/etoposide (PE), each with concurrent radiotherapy, remain largely undefined. Records for consecutive patients with stage III NSCLC treated with PC or PE and ≥60 Gy chest radiotherapy between 2000 and 2011 were reviewed for outcomes and toxicity. Survival was estimated using the Kaplan-Meier method and Cox modeling with the Wald test. Comparison across groups was done using the student's t and chi-squared tests. Seventy-five (PC: 44, PE: 31) patients were analyzed. PC patients were older (median 71 vs. 63 years; P = 0.0006). Other characteristics were comparable between groups. With PE, there was significantly increased grade ≥3 neutropenia (39% vs. 14%, P = 0.024) and thrombocytopenia (10% vs. 0%, P = 0.039). Radiation pneumonitis was more common with PC (66% vs. 38%, P = 0.033). Five treatment-related deaths occurred (PC: 3 vs. PE: 2, P = 1.000). With a median follow-up of 51.6 months, there were no significant differences in relapse-free survival (median PC 12.0 vs. PE 11.5 months, P = 0.700) or overall survival (median PC 20.7 vs. PE 13.7 months; P = 0.989). In multivariate analyses, no factors predicted for improved survival for either regimen. PC was more likely to be used in elderly patients. Despite this, PC resulted in significantly less hematological toxicity but achieved similar survival outcomes as PE. PC is an acceptable CCRT regimen, especially in older patients with multiple comorbidities.

The prognostic value of temporal in vitro and in vivo derived hypoxia gene-expression signatures in breast cancer.

BACKGROUND AND PURPOSE: Recent data suggest that in vitro and in vivo derived hypoxia gene-expression signatures have prognostic power in breast and possibly other cancers. However, both tumour hypoxia and the biological adaptation to this stress are highly dynamic. Assessment of time-dependent gene-expression changes in response to hypoxia may thus provide additional biological insights and assist in predicting the impact of hypoxia on patient prognosis. MATERIALS AND METHODS: Transcriptome profiling was performed for three cell lines derived from diverse tumour-types after hypoxic exposure at eight time-points, which include a normoxic time-point. Time-dependent sets of co-regulated genes were identified from these data. Subsequently, gene ontology (GO) and pathway analyses were performed. The prognostic power of these novel signatures was assessed in parallel with previous in vitro and in vivo derived hypoxia signatures in a large breast cancer microarray meta-dataset (n=2312). RESULTS: We identified seven recurrent temporal and two general hypoxia signatures. GO and pathway analyses revealed regulation of both common and unique underlying biological processes within these signatures. None of the new or previously published in vitro signatures consisting of hypoxia-induced genes were prognostic in the large breast cancer dataset. In contrast, signatures of repressed genes, as well as the in vivo derived signatures of hypoxia-induced genes showed clear prognostic power. CONCLUSIONS: Only a subset of hypoxia-induced genes in vitro demonstrates prognostic value when evaluated in a large clinical dataset. Despite clear evidence of temporal patterns of gene-expression in vitro, the subset of prognostic hypoxia regulated genes cannot be identified based on temporal pattern alone. In vivo derived signatures appear to identify the prognostic hypoxia induced genes. The prognostic value of hypoxia-repressed genes is likely a surrogate for the known importance of proliferation in breast cancer outcome.

A Pilot Study Comparing HPV-Positive and HPV-Negative Head and Neck Squamous Cell Carcinomas by Whole Exome Sequencing.

Background. Next-generation sequencing of cancers has identified important therapeutic targets and biomarkers. The goal of this pilot study was to compare the genetic changes in a human papillomavirus- (HPV-)positive and an HPV-negative head and neck tumor. Methods. DNA was extracted from the blood and primary tumor of a patient with an HPV-positive tonsillar cancer and those of a patient with an HPV-negative oral tongue tumor. Exome enrichment was performed using the Agilent SureSelect All Exon Kit, followed by sequencing on the ABI SOLiD platform. Results. Exome sequencing revealed slightly more mutations in the HPV-negative tumor (73) in contrast to the HPV-positive tumor (58). Multiple mutations were noted in zinc finger genes (ZNF3, 10, 229, 470, 543, 616, 664, 638, 716, and 799) and mucin genes (MUC4, 6, 12, and 16). Mutations were noted in MUC12 in both tumors. Conclusions. HPV-positive HNSCC is distinct from HPV-negative disease in terms of evidence of viral infection, p16 status, and frequency of mutations. Next-generation sequencing has the potential to identify novel therapeutic targets and biomarkers in HNSCC.