Publications
Steroid hormone receptors are ligand-activated transcription factors, acting as master regulators of gene expression. Steroid receptors mediate formation of large protein complexes by recruiting coregulatory proteins and transcriptional machinery to specific genomic regions. Unlike the structured ligand-binding or DNA-binding domains, the N-terminal domain (NTD), where many of these protein-protein interactions occur, contains extended regions of intrinsic disorder. Interactions in the NTD and allosteric binding elsewhere induce temporary and reversible changes in the NTD structure, substantially influencing the repertoire of potential binding partners. This structural plasticity is key for the steroid receptors to coordinate intra- and intercellular signals into a tissue specific response. Designing small molecule inhibitors against intrinsically disordered proteins (IDP) in general has proven difficult as structural information is limited. While some progress has been made in this area, only recently has any molecule targeting IDPs progressed beyond the preclinical stage. Here we summarize the discovery and development of sintokamides, niphatenones, and EPI compounds which target the intrinsically disordered NTD of the androgen receptor. These are the first drugs to target the NTD of any steroid receptor, and EPI-506 and EPI-7386 remain the only compounds that bind to an IDP to have been tested in clinical trials.
PURPOSE
Nearly all men with prostate cancer treated with androgen receptor (AR) signaling inhibitors (ARSIs) develop resistance via diverse mechanisms including constitutive activation of the AR pathway, driven by AR genomic structural alterations, expression of AR splice variants (AR-Vs), or loss of AR dependence and lineage plasticity termed neuroendocrine prostate cancer. Understanding these de novo acquired ARSI resistance mechanisms is critical for optimizing therapy.
MATERIALS AND METHODS
A novel liquid biopsy technology was used to collect mRNA from circulating tumor cells (CTCs) to measure expression of AR-Vs, AR targets, and neuroendocrine prostate cancer markers. An institutional review board–approved prospective cohort (N = 99) was used to identify patterns of gene expression. Two prospective multicenter phase II clinical trials of ARSIs for men with castration-resistant prostate cancer (ClinicalTrials.gov: NCT01942837 [enzalutamide, N = 21] and NCT02025010 [abiraterone, N = 27]) were used to further validate these findings.
RESULTS
Hierarchical clustering of CTC transcripts identified two distinct clusters. Cluster 2 (C2) exhibited increased expression of AR-regulated genes and was associated with worse overall survival (median 8.6 v 22.4 months; P < .01; hazard ratio [HR] = 3.45 [1.9 to 6.14]). In multivariable analysis, C2 was prognostic independent of other clinicopathologic variables. AR-V status was not significant when accounting for C2. Upon further validation in pooled multicenter phase II trials, C2 was associated with worse overall survival (15.2 months v not reached; P < .01; HR = 8.43 [2.74 to 25.92]), prostate-specific antigen progression-free survival (3.6 v 12 months; P < .01; HR = 4.64 [1.53 to 14.11]), and radiographic progression-free survival (2.7 v 40.6 months; P < .01; HR = 4.64 [1.82 to 17.41]).
CONCLUSION
We demonstrate that a transcriptional profile detectable in CTCs obtained from liquid biopsies can serve as an independent prognostic marker beyond AR-V7 in patients with metastatic prostate cancer and can be used to identify the emergence of multiple ARSI resistance mechanisms. This is currently being investigated in additional prospective trials.
Purpose: Nearly all men with prostate cancer treated with androgen receptor (AR) signaling inhibitors (ARSIs) develop resistance via diverse mechanisms including constitutive activation of the AR pathway, driven by AR genomic structural alterations, expression of AR splice variants (AR-Vs), or loss of AR dependence and lineage plasticity termed neuroendocrine prostate cancer. Understanding these de novo acquired ARSI resistance mechanisms is critical for optimizing therapy.
Materials and methods: A novel liquid biopsy technology was used to collect mRNA from circulating tumor cells (CTCs) to measure expression of AR-Vs, AR targets, and neuroendocrine prostate cancer markers. An institutional review board-approved prospective cohort (N = 99) was used to identify patterns of gene expression. Two prospective multicenter phase II clinical trials of ARSIs for men with castration-resistant prostate cancer (ClinicalTrials.gov: NCT01942837 [enzalutamide, N = 21] and NCT02025010 [abiraterone, N = 27]) were used to further validate these findings.
Results: Hierarchical clustering of CTC transcripts identified two distinct clusters. Cluster 2 (C2) exhibited increased expression of AR-regulated genes and was associated with worse overall survival (median 8.6 v 22.4 months; P < .01; hazard ratio [HR] = 3.45 [1.9 to 6.14]). In multivariable analysis, C2 was prognostic independent of other clinicopathologic variables. AR-V status was not significant when accounting for C2. Upon further validation in pooled multicenter phase II trials, C2 was associated with worse overall survival (15.2 months v not reached; P < .01; HR = 8.43 [2.74 to 25.92]), prostate-specific antigen progression-free survival (3.6 v 12 months; P < .01; HR = 4.64 [1.53 to 14.11]), and radiographic progression-free survival (2.7 v 40.6 months; P < .01; HR = 4.64 [1.82 to 17.41]).
Conclusion: We demonstrate that a transcriptional profile detectable in CTCs obtained from liquid biopsies can serve as an independent prognostic marker beyond AR-V7 in patients with metastatic prostate cancer and can be used to identify the emergence of multiple ARSI resistance mechanisms. This is currently being investigated in additional prospective trials.
Motivation: B-cells display remarkable diversity in producing B-cell receptors through recombination of immunoglobulin V-D-J genes. Somatic hypermutation of immunoglobulin heavy chain variable (IGHV) genes are used as a prognostic marker in B-cell malignancies. Clinically, IGHV mutation status is determined by targeted Sanger sequencing which is a resource intensive and low-throughput procedure. Here we describe a bioinformatic pipeline, CRIS (Complete Reconstruction of Immunoglobulin IGHV-D-J Sequences) that uses RNA sequencing (RNA-seq) datasets to reconstruct IGHV-D-J sequences and determine IGHV somatic hypermutation status.
Results: CRIS extracts RNA-seq reads aligned to immunoglobulin gene (Ig) loci, performs assembly of Ig-transcripts and aligns the resulting contigs to reference Ig sequences to enumerate and classify somatic hypermutations in the IGHV gene sequence. CRIS improves on existing tools that infer the B-cell receptor (BCR) repertoire from RNA-seq data using a portion IGHV gene segment by de novo assembly. We show that the somatic hypermutation status identified by CRIS using the entire IGHV gene segment is highly concordant with clinical classification in three independent chronic lymphocytic leukemia patient cohorts.
Short-read DNA sequencing instruments can yield over 10^12 bases per run, typically composed of reads 150 bases long. Despite this high throughput, de novo assembly algorithms have difficulty reconstructing contiguous genome sequences using short reads due to both repetitive and difficult-to-sequence regions in these genomes. Some of the short read assembly challenges are mitigated by scaffolding assembled sequences using paired-end reads. However, unresolved sequences in these scaffolds appear as "gaps". Here, we introduce GapPredict an implementation of a proof of concept that uses a character-level language model to predict unresolved nucleotides in scaffold gaps. We benchmarked GapPredict against the state-of-the-art gap-filling tool Sealer, and observed that the former can fill 65.6% of the sampled gaps that were left unfilled by the latter with high similarity to the reference genome, demonstrating the practical utility of deep learning approaches to the gap-filling problem in genome assembly.
The term diffuse large B-cell lymphoma (DLBCL) includes a heterogeneous collection of biologically distinct tumours. This heterogeneity currently presents a barrier to the successful deployment of novel, biologically targeted therapies. Molecular profiling studies have recently proposed new molecular classification systems. These have the potential to resolve the biological heterogeneity of DLBCL into manageable subgroups of tumours that rely on shared oncogenic programmes. In many cases these biological programmes straddle the boundaries of our existing systems for classifying B-cell lymphomas. Here we review the findings from these major molecular profiling studies with a specific focus on those that propose new genetic subgroups of DLBCL. We highlight the areas of consensus and discordance between these studies and discuss the implications for current clinical practice and for clinical trials. Finally, we address the outstanding challenges and solutions to the introduction of genomic subtyping and precision medicine in DLBCL.
Contexte: Il existe d’importantes iniquités en matière de santé chez les populations autochtones au Canada. La faible densité de la population canadienne et les populations en région éloignée posent un problème particulier à l’accès et à l’utilisation des soins chirurgicaux. Aucune synthèse des données sur les issues chirurgicales chez les Autochtones au Canada n’avait été publiée jusqu’à maintenant.
Méthodes: Nous avons interrogé 4 bases de données pour recenser les études comparant les issues chirurgicales et les taux d’utilisation chez les adultes des Premières Nations, inuits et métis et chez les adultes non autochtones au Canada. Des évaluateurs indépendants ont réalisé toutes les étapes en parallèle. L’issue primaire était la mortalité; les issues secondaires comprenaient le taux d’utilisation des chirurgies, les complications et la durée du séjour à l’hôpital. Nous avons effectué une méta-analyse pour l’issue primaire à l’aide d’un modèle à effets aléatoires. Nous avons évalué les risques de biais à l’aide de l’outil ROBINS-I.
Résultats: Vingt-huit études ont été analysées, pour un total de 1 976 258 participants (10,2 % d’Autochtones). Aucune étude ne portait précisément sur les populations inuites et métisses. Quatre études portant sur 7 cohortes ont fourni des données corrigées sur la mortalité pour 7135 participants (5,2 % d’Autochtones); les Autochtones présentaient un risque de décès après une intervention chirurgicale 30 % plus élevé que les patients non autochtones (rapport de risque combiné 1,30; IC à 95 % 1,09–1,54; I2 = 81 %). Les complications étaient aussi plus fréquentes chez le premier groupe, notamment les infections (RC corrigé 1,63; IC à 95 % 1,13–2,34) et les pneumonies (RC 2,24; IC à 95 % 1,58–3,19). Les taux de différentes interventions chirurgicales étaient plus faibles, notamment pour les transplantations rénales, les arthroplasties, les chirurgies cardiaques et les accouchements par césarienne.
Interprétation: Les données disponibles sur les issues postopératoires et le taux d’utilisation de la chirurgie chez les Autochtones au Canada sont limitées et de faible qualité. Elles suggèrent que les Autochtones ont de plus hauts taux de décès et d’issues négatives postchirurgicales et qu’ils font face à des obstacles dans l’accès aux interventions chirurgicales. Ces conclusions indiquent qu’il y a un besoin de réévaluer en profondeur les soins chirurgicaux prodigués aux Autochtones au Canada pour leur assurer un accès équitable et améliorer les issues.
Numéro d’enregistrement du protocole: PROSPERO-CRD42018098757.
Tandem repeat (TR) expansion is the underlying cause of over 40 neurological disorders. Long-read sequencing offers an exciting avenue over conventional technologies for detecting TR expansions. Here, we present Straglr, a robust software tool for both targeted genotyping and novel expansion detection from long-read alignments. We benchmark Straglr using various simulations, targeted genotyping data of cell lines carrying expansions of known diseases, and whole genome sequencing data with chromosome-scale assembly. Our results suggest that Straglr may be useful for investigating disease-associated TR expansions using long-read sequencing.
Patients with non-small cell lung cancer (NSCLC) harboring ROS proto-oncogene 1 (ROS1) gene rearrangements show dramatic response to the tyrosine kinase inhibitor (TKI) crizotinib. Current best practice guidelines recommend that all advanced stage non-squamous NSCLC patients be also tested for ROS1 gene rearrangements. Several studies have suggested that ROS1 immunohistochemistry (IHC) using the D4D6 antibody may be used to screen for ROS1 fusion positive lung cancers, with assays showing high sensitivity but moderate to high specificity. A break apart fluorescence in situ hybridization (FISH) test is then used to confirm the presence of ROS1 gene rearrangement. The goal of Canadian ROS1 (CROS) study was to harmonize ROS1 laboratory developed testing (LDT) by using IHC and FISH assays to detect ROS1 rearranged lung cancers across Canadian pathology laboratories. Cell lines expressing different levels of ROS1 (high, low, none) were used to calibrate IHC protocols after which participating laboratories ran the calibrated protocols on a reference set of 24 NSCLC cases (9 ROS1 rearranged tumors and 15 ROS1 non-rearranged tumors as determined by FISH). Results were compared using a centralized readout. The stained slides were evaluated for the cellular localization of staining, intensity of staining, the presence of staining in non-tumor cells, the presence of non-specific staining (e.g. necrosis, extracellular mater, other) and the percent positive cells. H-score was also determined for each tumor. Analytical sensitivity and specificity harmonization was achieved by using low limit of detection (LOD) as either any positivity in the U118 cell line or H-score of 200 with the HCC78 cell line. An overall diagnostic sensitivity and specificity of up to 100% and 99% respectively was achieved for ROS1 IHC testing (relative to FISH) using an adjusted H-score readout on the reference cases. This study confirms that LDT ROS1 IHC assays can be highly sensitive and specific for detection of ROS1 rearrangements in NSCLC. As NSCLC can demonstrate ROS1 IHC positivity in FISH-negative cases, the degree of the specificity of the IHC assay, especially in highly sensitive protocols, is mostly dependent on the readout cut-off threshold. As ROS1 IHC is a screening assay for a rare rearrangements in NSCLC, we recommend adjustment of the readout threshold in order to balance specificity, rather than decreasing the overall analytical and diagnostic sensitivity of the protocols.