Novel pharmacological strategies for antagonizing anti-apoptosis protein function in malignancy.

Historically, cytotoxic therapies have provided the greatest advances in the treatment of malignant disease. Although some cancers are curable, many are not. Chemotherapeutic drugs rely upon the induction of a phylogenically old, cell suicide programme termed apoptosis for their efficacy. Apoptotic sensitivity is associated with curability, whereas, intrinsic resistance plays a major role in limiting therapeutic effectiveness. Mitochondria, the centres for aerobic respiration in the cell also play a pivotal role in regulating apoptosis. The anti-death proteins Bcl-2 and Bc1-XL localize to the outer mitochondrial membrane, and are expressed at high levels in many resistant malignacies compared with normal tissues. These proteins contribute to resistance by blocking apoptosis, and therefore represent valid targets for the development of novel inhibitory strategies. This thesis presents two strategies with therapeutic potential for antagonizing the anti-death action of Bcl-2 and Bc1-XL in haematological and epithelial malignancies. The first, involves the suppression of Bcl-2 and Bc1-XL gene expression by antisense oligodeoxynucleotides in vitro and in vivo. Mathematical models of antisense pharmacology are presented. The second, targets the mitochondrial megachannel that is intimately involved in apoptosis, and is regulated by binding to Bcl-2 and Bc1-XL In order to quantitatively measure the putative apoptosis sensitizing efficacy of these approaches at single cell resolution, stochastic models are described, enabling robust estimation of the distribution of tolerances and latency preceding apoptosis. The peripheral benzodiazepine receptor interacts with the mitochondrial megachannel. Evidence is provided, that Bcl-2 resistant apoptosis sensitization is mediated in vitro, in vivo, and ex vivo, by the ligand PK11195, through a mechanism involving direct megachannel regulation. This occurs not via the peripheral benzodiazepine receptor as previously thought, but through de novo generation of reactive oxygen species. Investigations of PK11195 pharmacodynamics, and molecular structural studies using proton nuclear magnetic resonance spectroscopy support a novel mechanism of action

Gene Expression Meta-Analysis Identifies VDAC1 as a Predictor of Poor Outcome in Early Stage Non-Small Cell Lung Cancer

The bioenergetic status of non-small cell lung cancer correlates with tumour aggressiveness. The voltage dependent anion channel type 1 (VDAC1) is a component of the mitochondrial permeability transition pore, regulates mitochondrial ATP/ADP exchange suggesting that its over-expression could be associated with energy dependent processes including increased proliferation and invasiveness. To test this hypothesis, we conducted an in vivo gene-expression meta-analysis of surgically resected non-small cell lung cancer (NSCLC) using 602 individual expression profiles, to examine the impact of VDAC1 on survival.High VDAC1 expression was associated with shorter overall survival with hazard ratio (HR) = 0.6639 (95% confidence interval (CI) 0.4528 to 0.9721), p = 0.035352 corresponding to 52 versus 101 months. VDAC1 predicted shorter time to recurrence and was shown to be an independent prognostic factor compared with histology, gender, age, nodal stage and tumour stage in a Cox multivariate analysis. Supervised analysis of all the datasets identified a 6-gene signature comprising HNRNPC, HSPA4, HSPA9, UBE2D2, CSNK1A1 and G3BP1 with overlapping functions involving regulation of protein turnover, RAS-RAF-MEK pathway and transcription. VDAC1 predicted survival in breast cancer and myeloma and an unsupervised analysis revealed enrichment of the VDAC1 signature in specific subsets.In summary, gene expression analysis identifies VDAC1 gene expression as a predictor of poor outcome in NSCLC and other cancers and is associated with dysregulation of a conserved set of biological pathways, which may be causally associated with aggressive tumour behaviour

A TMA De-Arraying Method for High Throughput Biomarker Discovery in Tissue Research

BACKGROUND: Tissue MicroArrays (TMAs) represent a potential high-throughput platform for the analysis and discovery of tissue biomarkers. As TMA slides are produced manually and subject to processing and sectioning artefacts, the layout of TMA cores on the final slide and subsequent digital scan (TMA digital slide) is often disturbed making it difficult to associate cores with their original position in the planned TMA map. Additionally, the individual cores can be greatly altered and contain numerous irregularities such as missing cores, grid rotation and stretching. These factors demand the development of a robust method for de-arraying TMAs which identifies each TMA core, and assigns them to their appropriate coordinates on the constructed TMA slide. METHODOLOGY: This study presents a robust TMA de-arraying method consisting of three functional phases: TMA core segmentation, gridding and mapping. The segmentation of TMA cores uses a set of morphological operations to identify each TMA core. Gridding then utilises a Delaunay Triangulation based method to find the row and column indices of each TMA core. Finally, mapping correlates each TMA core from a high resolution TMA whole slide image with its name within a TMAMap. CONCLUSION: This study describes a genuine robust TMA de-arraying algorithm for the rapid identification of TMA cores from digital slides. The result of this de-arraying algorithm allows the easy partition of each TMA core for further processing. Based on a test group of 19 TMA slides (3129 cores), 99.84% of cores were segmented successfully, 99.81% of cores were gridded correctly and 99.96% of cores were mapped with their correct names via TMAMaps. The gridding of TMA cores were also extensively tested using a set of 113 pseudo slide (13,536 cores) with a variety of irregular grid layouts including missing cores, rotation and stretching. 100% of the cores were gridded correctly

CONFIRM: a double-blind, placebo controlled phase III clinical trial investigating the effect of nivolumab in patients with relapsed mesothelioma: study protocol for a randomised controlled trial

Background: Mesothelioma is an incurable, apoptosis-resistant cancer caused in most cases by previous exposure to asbestos and is increasing in incidence. It represents a growing health burden but remains under-researched, with limited treatment options. Early promising signals of activity relating to both PD-L1- and PD-1-targeted treatment in mesothelioma implicate a dependency of mesothelioma on this immune checkpoint. There is a need to evaluate checkpoint inhibitors in patients with relapsed mesothelioma where treatment options are limited. Methods: The addition of 12 months of nivolumab (anti-PD1 antibody) to standard practice will be conducted in the UK using a randomised, placebo-controlled phase III trial (the Cancer Research UK CONFIRM trial). A total of 336 patients with pleural or peritoneal mesothelioma who have received at least two prior lines of therapy will be recruited from UK secondary care sites. Patients will be randomised 2:1 (nivolumab:placebo), stratified according to epithelioid/non-epithelioid, to receive either 240 mg nivolumab monotherapy or saline placebo as a 30-min intravenous infusion. Treatment will be for up to 12 months. We will determine whether the use of nivolumab increases overall survival (the primary efficacy endpoint). Secondary endpoints will include progression-free survival, objective response rate, toxicity, quality of life and cost-effectiveness. Analysis will be performed according to the intention-to-treat principle using a Cox regression analysis for the primary endpoint (and for other time-to-event endpoints). Discussion: The outcome of this trial will provide evidence of the potential benefit of the use of nivolumab in the treatment of relapsed mesothelioma. If found to be clinically effective, safe and cost-effective it is likely to become the new standard of care in the UK

CDKN2A Determines Mesothelioma Cell Fate to EZH2 Inhibition

Malignant pleural mesothelioma is an aggressive cancer, heterogeneous in its presentation and behaviour. Despite an increasing knowledge about molecular markers and their diagnostic and prognostic value, they are not used as much as they might be for treatment allocation. It has been recently reported that mesothelioma cells that lack BAP1 (BRCA1 Associated Protein) are sensitive to inhibition of the EZH2 (Enhancer of Zeste Homolog 2) histone methyltransferase. Since we observed strong H3K27me3 (histone H3 lysine 27 trimetylation) immunoreactivity in BAP1 wild-type mesothelioma biopsies, we decided to characterize in vitro the response/resistance of BAP1 wild-type mesothelioma cells to the EZH2 selective inhibitor, EPZ-6438. Here we demonstrate that BAP1 wild-type mesothelioma cells were rendered sensitive to EPZ-6438 upon SIRT1 (Sirtuin 1) silencing/inhibition or when cultured as multicellular spheroids, in which SIRT1 expression was lower compared to cells grown in monolayers. Notably, treatment of spheroids with EPZ-6438 abolished H3K27me3 and induced the expression of CDKN2A (Cyclin-Dependent Kinase Inhibitor 2A), causing cell growth arrest. EPZ-6438 treatment also resulted in a rapid and sustained induction of the genes encoding HIF2α (Hypoxia Inducible Factor 2α), TG2 (Transglutaminase 2) and IL-6 (Interleukin 6). Loss of CDKN2 is a common event in mesothelioma. CDKN2A silencing in combination with EPZ-6438 treatment induced apoptotic death in mesothelioma spheroids. In a CDKN2A wild-type setting apoptosis was induced by combining EPZ-6438 with 1-155, a TG2 selective and irreversible inhibitor. In conclusion, our data suggests that the expression of CDKN2A predicts cell fate in response to EZH2 inhibition and could potentially stratify tumors likely to undergo apoptosis

Inclusion of multiple high-risk histopathological criteria improves the prediction of adjuvant chemotherapy efficacy in lung adenocarcinoma.

AIMS: The decision to consider adjuvant chemotherapy (AC) for non-small cell lung cancer is currently governed by clinical stage. This study aims to assess other routinely collected pathological variables related to metastasis and survival for their ability to predict the efficacy of AC in lung adenocarcinoma. METHODS AND RESULTS: A retrospective single-centre series of 620 resected lung non-mucinous adenocarcinoma cases from 2005 to 2015 was used. Digital images of all slides were subjected to central review, and data on tumour histopathology, AC treatment and patient survival were compiled. A statistical case matching approach was used to counter selection bias. Several high-risk pathological criteria predict both pathological nodal involvement and early death: positive vascular invasion status (VI+) (HR = 2.10, P < 0.001), positive visceral pleural invasion status (VPI+) (HR = 2.16, P < 0.001), and solid/micropapillary-predominant WHO tumour type (SPA/MPPA) (HR = 3.29, P < 0.001). Crucially, these criteria also identify patient groups benefiting from AC (VI + HR = 0.69, P = 0.167, VPI + HR = 0.44, P = 0.004, SPA/MPPA HR = 0.36, P = 0.006). Cases showing VI+/VPI+/SPA/MPPA histology in the absence of AC stage criteria were common (170 of 620 total), and 8 had actually received AC. This group showed much better outcomes than equivalent untreated cases in matched analysis (3-year OS 100.0% versus 31.3%). Inclusion of patients with VI+/VPI+/SPA/MPPA histology would increase AC-eligible patients from 51.0% to 84.0% of non-mucinous tumours in our cohort. CONCLUSIONS: Our data provide preliminary evidence that the consideration of AC in patients with additional high-risk pathological indicators may significantly improve outcomes in operable lung adenocarcinoma, and that AC may be currently underused

Inclusion of multiple high‐risk histopathological criteria improves the prediction of adjuvant chemotherapy efficacy in lung adenocarcinoma

Aims: The decision to consider adjuvant chemotherapy (AC) for non‐small cell lung cancer is currently governed by clinical stage. This study aims to assess other routinely collected pathological variables related to metastasis and survival for their ability to predict the efficacy of AC in lung adenocarcinoma. Methods and results: A retrospective single‐centre series of 620 resected lung non‐mucinous adenocarcinoma cases from 2005 to 2015 was used. Digital images of all slides were subjected to central review, and data on tumour histopathology, AC treatment and patient survival were compiled. A statistical case matching approach was used to counter selection bias. Several high‐risk pathological criteria predict both pathological nodal involvement and early death: positive vascular invasion status (VI+) (HR = 2.10, P &lt; 0.001), positive visceral pleural invasion status (VPI+) (HR = 2.16, P &lt; 0.001), and solid/micropapillary‐predominant WHO tumour type (SPA/MPPA) (HR = 3.29, P &lt; 0.001). Crucially, these criteria also identify patient groups benefiting from AC (VI + HR = 0.69, P = 0.167, VPI + HR = 0.44, P = 0.004, SPA/MPPA HR = 0.36, P = 0.006). Cases showing VI+/VPI+/SPA/MPPA histology in the absence of AC stage criteria were common (170 of 620 total), and 8 had actually received AC. This group showed much better outcomes than equivalent untreated cases in matched analysis (3‐year OS 100.0% versus 31.3%). Inclusion of patients with VI+/VPI+/SPA/MPPA histology would increase AC‐eligible patients from 51.0% to 84.0% of non‐mucinous tumours in our cohort. Conclusions: Our data provide preliminary evidence that the consideration of AC in patients with additional high‐risk pathological indicators may significantly improve outcomes in operable lung adenocarcinoma, and that AC may be currently underused

Ganetespib in combination with pemetrexed-platinum chemotherapy in patients with pleural Mesothelioma (MESO-02) : a phase Ib trial

Purpose: Ganetespib, a highly potent, small molecule Heatshock protein 90 inhibitor, has potential efficacy in malignant pleural mesothelioma (MPM) via activity on critical survival pathways and known synergies with antifolates and platinum chemotherapy. We conducted a dose-escalation study to identify the Maximum Tolerated Dose (MTD) of ganetespib in chemotherapy-naïve MPM patients. Experimental Design: MESO-02 (ClinicalTrials.gov: NCT01590160) was a non-randomized, multicentre, phase Ib trial of 3-weekly ganetespib (100 mg/m2, 150 mg/m2, 200 mg/m2; days 1 and 15) with pemetrexed (500 mg/m2; day 1) and cisplatin (75 mg/m2; day 1) or carboplatin (area under concentration-time curve 5; day 1) in MPM patients. Dose-escalation was performed using the 3+3 design (cisplatin) and accelerated titration design (carboplatin). Secondary endpoints included best response, progression-free survival (PFS) and pharmacogenomic analyses. Results: Of 27 patients enroled (cisplatin, n=16; carboplatin, n=11), 3 experienced dose-limiting toxicities: grade 3 nausea (cisplatin, n=1; carboplatin, n=1); grade 2 infusion-related reaction (carboplatin, n=1). Ganetespib's MTD was 200 mg/m2. Partial response was observed in 14/27 patients (52%; 61% in 23 response-evaluable patients) and 13/21 (62%) with epithelioid histology. At the MTD, 10/18 patients (56%) had partial response, 15/18 (83%) had disease control, and median PFS was 6.3 months (95% CI 5.0-10.0). One responder exhibited disease control beyond 50 months. Global Loss of Heterozygosity was associated with shorter time to progression (Hazard Ratio 1.12, 95% CI 1.02-1.24; p=0.018). Conclusions: Ganetespib can be combined safely with pemetrexed and platinum chemotherapy to treat patients with MPM. This class of agent should be investigated in larger randomized studies