Gene fusions during the early evolution of mesothelioma correlate with impaired DNA repair and Hippo pathways.

Malignant pleural mesothelioma (MPM), a rare cancer a long latency period (up to 40 years) between asbestos exposure and disease presentation. The mechanisms coupling asbestos to recurrent somatic alterations are poorly defined. Gene fusions arising through genomic instability may create novel drivers during early MPM evolution. We explored the gene fusions that occurred early in the evolutionary history of the tumor. We conducted multiregional whole exome sequencing (WES) of 106 samples from 20 patients undergoing pleurectomy decortication and identified 24 clonal nonrecurrent gene fusions, three of which were novel (FMO9P-OR2W5, GBA3, and SP9). The number of early gene fusion events detected varied from zero to eight per tumor, and presence of gene fusions was associated with clonal losses involving the Hippo pathway genes and homologous recombination DNA repair genes. Fusions involved known tumor suppressors BAP1, MTAP, and LRP1B, and a clonal oncogenic fusion involving CACNA1D-ERC2, PARD3B-NT5DC2, and STAB2-NT5DC2 fusions were also identified as clonal fusions. Gene fusions events occur early during MPM evolution. Individual fusions are rare as no recurrent truncal fusions event were found. This suggests the importance of early disruption of these pathways in generating genomic rearrangements resulting in potentially oncogenic gene fusions.

Cytoreductive surgery with hyperthermic intrathoracic chemotherapy for malignant pleural mesothelioma: a systematic review.

INTRODUCTION: Cytoreductive surgery has been used a part of multimodality treatment in patients with malignant pleural mesothelioma (MPM). The residual microscopic disease that remains will lead to disease progression in the majority of patients. Delivery of hyperthermic intrathoracic chemotherapy at the time of surgery has been used to address this microscopic disease, however it's effect and place in the multimodality treatment sphere is unknown. The aim of this systematic review was to assess the effect of surgery and hyperthermic intrathoracic chemotherapy in patients with MPM on overall survival and disease-free interval. METHODS: Ovid MEDLINE, Embase, Web of Science and the Cochrane Database of Systematic Reviews were searched from database inception through to June 2021. Studies reporting overall survival and/or disease-free interval in patients with MPM undergoing cytoreductive surgery with hyperthermic intrathoracic chemotherapy were considered. Study quality was assessed using the Newcastle-Ottawa Scale. A narrative review was performed. RESULTS: Fifteen studies were eligible for inclusion comprising 598 patients. Surgery with hyperthermic intrathoracic chemotherapy was associated with a median overall survival and disease-free interval ranging from 11 to 75 months and 7.2 to 57 months, respectively. These appeared to be superior to patients not receiving hyperthermic intrathoracic chemotherapy (overall survival: 5-36 months and disease-free interval: 12.1-21 months). A higher dose of hyperthermic intrathoracic chemotherapy was associated with an improvement in overall survival compared with a lower dose: 18-31 months versus 6-18 months, respectively. The most common morbidity was atrial fibrillation followed by renal complications. CONCLUSION: Surgery with hyperthermic intrathoracic chemotherapy offers a safe and effective therapy with an improvement in disease-free interval and overall survival, particularly when hyperthermic intrathoracic chemotherapy is administered at a higher dose. PROSPERO REGISTRATION NUMBER: CRD42019129002.

BAP1 loss induces mitotic defects in mesothelioma cells through BRCA1-dependent and independent mechanisms.

The tumour suppressor BRCA1-associated protein 1 (BAP1) is the most frequently mutated cancer gene in mesothelioma. Here we report novel functions for BAP1 in mitotic progression highlighting the relationship between BAP1 and control of genome stability in mesothelioma cells with therapeutic implications. Depletion of BAP1 protein induced proteasome-mediated degradation of BRCA1 in mesothelioma cells while loss of BAP1 correlated with BRCA1 loss in mesothelioma patient tumour samples. BAP1 loss also led to mitotic defects that phenocopied the loss of BRCA1 including spindle assembly checkpoint failure, centrosome amplification and chromosome segregation errors. However, loss of BAP1 also led to additional mitotic changes that were not observed upon BRCA1 loss, including an increase in spindle length and enhanced growth of astral microtubules. Intriguingly, these consequences could be explained by loss of expression of the KIF18A and KIF18B kinesin motors that occurred upon depletion of BAP1 but not BRCA1, as spindle and astral microtubule defects were rescued by re-expression of KIF18A and KIF18B, respectively. We therefore propose that BAP1 inactivation causes mitotic defects through BRCA1-dependent and independent mechanisms revealing novel routes by which mesothelioma cells lacking BAP1 may acquire genome instability and exhibit altered responses to microtubule-targeted agents.

Extracellular Vesicles Isolated from Malignant Mesothelioma Cancer-Associated Fibroblasts Induce Pro-Oncogenic Changes in Healthy Mesothelial Cells.

Malignant mesothelioma is an aggressive tumour of the pleura (MPM) or peritoneum with a clinical presentation at an advanced stage of the disease. Current therapies only marginally improve survival and there is an urgent need to identify new treatments. Carcinoma-associated fibroblasts (CAFs) represent the main component of a vast stroma within MPM and play an important role in the tumour microenvironment. The influence of CAFs on cancer progression, aggressiveness and metastasis is well understood; however, the role of CAF-derived extracellular vesicles (CAF-EVs) in the promotion of tumour development and invasiveness is underexplored. We purified CAF-EVs from MPM-associated cells and healthy dermal human fibroblasts and examined their effect on cell proliferation and motility. The data show that exposure of healthy mesothelial cells to EVs derived from CAFs, but not from normal dermal human fibroblasts (NDHF) resulted in activating pro-oncogenic signalling pathways and increased proliferation and motility. Consistent with its role in suppressing Yes-Associated Protein (YAP) activation (which in MPM is a result of Hippo pathway inactivation), treatment with Simvastatin ameliorated the pro-oncogenic effects instigated by CAF-EVs by mechanisms involving both a reduction in EV number and changes in EV cargo. Collectively, these data determine the significance of CAF-derived EVs in mesothelioma development and progression and suggest new targets in cancer therapy.

Comprehensive genomic and tumour immune profiling reveals potential therapeutic targets in malignant pleural mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) has a poor overall survival with few treatment options. Whole genome sequencing (WGS) combined with the immune features of MPM offers the prospect of identifying changes that could inform future clinical trials. METHODS: We analysed somatic mutations from 229 MPM samples, including previously published data and 58 samples that had undergone WGS within this study. This was combined with RNA-seq analysis to characterize the tumour immune environment. RESULTS: The comprehensive genome analysis identified 12 driver genes, including new candidate genes. Whole genome doubling was a frequent event that correlated with shorter survival. Mutational signature analysis revealed SBS5/40 were dominant in 93% of samples, and defects in homologous recombination repair were infrequent in our cohort. The tumour immune environment contained high M2 macrophage infiltrate linked with MMP2, MMP14, TGFB1 and CCL2 expression, representing an immune suppressive environment. The expression of TGFB1 was associated with overall survival. A small subset of samples (less than 10%) had a higher proportion of CD8 T cells and a high cytolytic score, suggesting a 'hot' immune environment independent of the somatic mutations. CONCLUSIONS: We propose accounting for genomic and immune microenvironment status may influence therapeutic planning in the future.

Transcriptional perturbation of protein arginine methyltransferase-5 exhibits MTAP-selective oncosuppression.

We hypothesized that small molecule transcriptional perturbation could be harnessed to target a cellular dependency involving protein arginine methyltransferase 5 (PRMT5) in the context of methylthioadenosine phosphorylase (MTAP) deletion, seen frequently in malignant pleural mesothelioma (MPM). Here we show, that MTAP deletion is negatively prognostic in MPM. In vitro, the off-patent antibiotic Quinacrine efficiently suppressed PRMT5 transcription, causing chromatin remodelling with reduced global histone H4 symmetrical demethylation. Quinacrine phenocopied PRMT5 RNA interference and small molecule PRMT5 inhibition, reducing clonogenicity in an MTAP-dependent manner. This activity required a functional PRMT5 methyltransferase as MTAP negative cells were rescued by exogenous wild type PRMT5, but not a PRMT5E444Q methyltransferase-dead mutant. We identified c-jun as an essential PRMT5 transcription factor and a probable target for Quinacrine. Our results therefore suggest that small molecule-based transcriptional perturbation of PRMT5 can leverage a mutation-selective vulnerability, that is therapeutically tractable, and has relevance to 9p21 deleted cancers including MPM.

Clonal architecture in mesothelioma is prognostic and shapes the tumour microenvironment.

Malignant Pleural Mesothelioma (MPM) is typically diagnosed 20-50 years after exposure to asbestos and evolves along an unknown evolutionary trajectory. To elucidate this path, we conducted multi-regional exome sequencing of 90 tumour samples from 22 MPMs acquired at surgery. Here we show that exomic intratumour heterogeneity varies widely across the cohort. Phylogenetic tree topology ranges from linear to highly branched, reflecting a steep gradient of genomic instability. Using transfer learning, we detect repeated evolution, resolving 5 clusters that are prognostic, with temporally ordered clonal drivers. BAP1/-3p21 and FBXW7/-chr4 events are always early clonal. In contrast, NF2/-22q events, leading to Hippo pathway inactivation are predominantly late clonal, positively selected, and when subclonal, exhibit parallel evolution indicating an evolutionary constraint. Very late somatic alteration of NF2/22q occurred in one patient 12 years after surgery. Clonal architecture and evolutionary clusters dictate MPM inflammation and immune evasion. These results reveal potentially drugable evolutionary bottlenecking in MPM, and an impact of clonal architecture on shaping the immune landscape, with potential to dictate the clinical response to immune checkpoint inhibition.

Metastatic renal cell carcinoma extending to the left atrium through the inferior pulmonary vein.

Metastatic renal cell carcinoma with involvement through the pulmonary veins to the left atrium is very rare. We report the case of a 70-year-old male with metastatic renal cell carcinoma to the right lower lobe of the lung abutting the inferior pulmonary vein with extension to the left atrium without pre-operative evidence. Surgical resection was achieved through a posterolateral thoracotomy. Lung masses that abut the pulmonary veins should prompt further investigation with a pre-operative transoesophageal echocardiogram to minimize unexpected intraoperative findings.

BRCA1/MAD2L1 Deficiency Disrupts the Spindle Assembly Checkpoint to Confer Vinorelbine Resistance in Mesothelioma.

Mesothelioma is a universally lethal cancer lacking effective therapy. The spindle poison vinorelbine exhibits clinical activity in the relapsed setting, and in preclinical models requires BRCA1 to initiate apoptosis. However, the mechanisms underlying this regulation and the clinical implications have not been explored. Here, we show that BRCA1 silencing abrogated vinorelbine-induced cell-cycle arrest, recruitment of BUBR1 to kinetochores, and apoptosis. BRCA1 silencing led to codepletion of MAD2L1 at the mRNA and protein levels consistent with its status as a transcriptional target of BRCA1 Silencing of MAD2L1 phenocopied BRCA1 and was sufficient to confer resistance to vinorelbine. This was recapitulated in cell lines selected for resistance to vinorelbine, which acquired loss of both BRCA1 and MAD2L1 expression. Following ex vivo vinorelbine in 20 primary tumor explants, apoptotic response rate was 59% in BRCA1/MAD2L1-positive explants compared with 0% in BRCA1/MAD2L1-negative explants. In 48 patients, BRCA1 and/or MAD2L1 loss of expression was not prognostic; however, in a subset of patients treated with vinorelbine, survival was shorter for patients lacking BRCA1/MAD2L1 expression compared with double-positive patients (5.9 vs. 36.7 months, P = 0.03). Our data implicate BRCA1/MAD2L1 loss as a putative predictive marker of resistance to vinorelbine in mesothelioma and warrant prospective clinical evaluation.

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.

BTS guideline for the investigation and management of malignant pleural mesothelioma.

The full guideline for the investigation and management of malignant pleural mesothelioma is published in Thorax. The following is a summary of the recommendations and good practice points. The sections referred to in the summary refer to the full guideline.

Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells.

Malignant mesothelioma (MM) is poorly responsive to systemic cytotoxic chemotherapy and invariably fatal. Here we describe a screen of 94 drugs in 15 exome-sequenced MM lines and the discovery of a subset defined by loss of function of the nuclear deubiquitinase BRCA associated protein-1 (BAP1) that demonstrate heightened sensitivity to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand). This association is observed across human early passage MM cultures, mouse xenografts and human tumour explants. We demonstrate that BAP1 deubiquitinase activity and its association with ASXL1 to form the Polycomb repressive deubiquitinase complex (PR-DUB) impacts TRAIL sensitivity implicating transcriptional modulation as an underlying mechanism. Death receptor agonists are well-tolerated anti-cancer agents demonstrating limited therapeutic benefit in trials without a targeting biomarker. We identify BAP1 loss-of-function mutations, which are frequent in MM, as a potential genomic stratification tool for TRAIL sensitivity with immediate and actionable therapeutic implications.

Long-Fiber Carbon Nanotubes Replicate Asbestos-Induced Mesothelioma with Disruption of the Tumor Suppressor Gene Cdkn2a (Ink4a/Arf).

Mesothelioma is a fatal tumor of the pleura and is strongly associated with asbestos exposure. The molecular mechanisms underlying the long latency period of mesothelioma and driving carcinogenesis are unknown. Moreover, late diagnosis means that mesothelioma research is commonly focused on end-stage disease. Although disruption of the CDKN2A (INK4A/ARF) locus has been reported in end-stage disease, information is lacking on the status of this key tumor suppressor gene in pleural lesions preceding mesothelioma. Manufactured carbon nanotubes (CNTs) are similar to asbestos in terms of their fibrous shape and biopersistent properties and thus may pose an asbestos-like inhalation hazard. Here we show that instillation of either long CNTs or long asbestos fibers into the pleural cavity of mice induces mesothelioma that exhibits common key pro-oncogenic molecular events throughout the latency period of disease progression. Sustained activation of pro-oncogenic signaling pathways, increased proliferation, and oxidative DNA damage form a common molecular signature of long-CNT- and long-asbestos-fiber-induced pathology. We show that hypermethylation of p16/Ink4a and p19/Arf in CNT- and asbestos-induced inflammatory lesions precedes mesothelioma; this results in silencing of Cdkn2a (Ink4a/Arf) and loss of p16 and p19 protein, consistent with epigenetic alterations playing a gatekeeper role in cancer. In end-stage mesothelioma, silencing of p16/Ink4a is sustained and deletion of p19/Arf is detected, recapitulating human disease. This study addresses the long-standing question of which early molecular changes drive carcinogenesis during the long latency period of mesothelioma development and shows that CNT and asbestos pose a similar health hazard.

Extended pleurectomy decortication for malignant pleural mesothelioma in the elderly: the need for an inclusive yet selective approach.

OBJECTIVES: The median age at diagnosis of patients with pleural mesothelioma in the UK is 73 years. Recent series have shown the feasibility of extended pleurectomy decortication in the elderly, but with continuing debate about the efficacy of this treatment, we reviewed our experience to identify more detailed selection criteria. METHODS: We reviewed prospectively collected data on all patients from 1999 to 2016 undergoing extended pleurectomy decortication. We compared clinical and pathological outcomes and survival data from patients 70 years and older (≥70 years) with those younger than 70 years (<70 years). RESULTS: Eighty-two of the 300 (27.3%) patients were ≥70 years of age at the time of surgery. More patients in the elderly group required intensive care postoperatively (6.2 vs 16.7%, P = 0.01) and developed atrial fibrillation (14.4 vs 24.4%, P = 0.05). There was no intergroup difference in length of hospital stay or in in-hospital, 30-day or 90-day mortality. Elderly patients were less likely to receive neoadjuvant (<70 years 21.2%, ≥70 years 11.0%; P = 0.045) or adjuvant chemotherapy (<70 years 45.4%, ≥70 years 29.3%; P = 0.04). Median overall survival was similar: <70 years 14.0 months, ≥70 years 10.3 months; P = 0.29. However, in node-positive patients, survival was poorer in the elderly (13.0 vs 9.1 months, P = 0.05), particularly in those with non-epithelioid tumours (3.8 vs 6.7 months, P = 0.04). On multivariable analysis, age was not a significant prognostic factor, although lack of adjuvant therapy (P = 0.001) and admission to the intensive care unit (P < 0.001) remained poor prognostic factors. CONCLUSIONS: Although age in isolation should not be an exclusion criterion for extended pleurectomy decortication for mesothelioma, in the elderly, a more rigorous preoperative evaluation of nodal disease and an additional assessment of fitness for adjuvant chemotherapy are recommended.

How does the timing of chemotherapy affect outcome following radical surgery for malignant pleural mesothelioma?

OBJECTIVES: There is little evidence regarding the use of chemotherapy as part of multimodality treatment of malignant pleural mesothelioma (MPM). We aimed to determine whether, in those patients fit for chemotherapy, a delay in this treatment affected survival. MATERIALS AND METHODS: We analysed postoperative variables of 229 patients undergoing either extrapleural pneumonectomy (EPP) (81 patients) or extended pleurectomy-decortication (EPD) (197 patients) for MPM at a single centre. There was no standard protocol for additional chemotherapy and varied with referral centre. Outcome was compared between 4 chemotherapy strategies: true adjuvant therapy, neo-adjuvant therapy, therapy reserved until evidence of disease progression in those otherwise fit in the post-operative setting, and those unfit for chemotherapy. RESULTS: There was no effect of the timing of chemotherapy on overall or progression free survival in patients fit enough for treatment (p=0.39 and p=0.33 respectively). However delaying chemotherapy until evidence of disease progression in patients with non-epithelioid disease had a detrimental effect on overall survival (OS), and on progression free survival (PFS) in lymph node positive patients (15.6 vs. 8.2 months p=0.001, and 14.9 vs. 6.0 months p=0.016). Further analysis of 169 patients receiving platinum/pemetrexed as first line treatment, showed similar results; there was no effect of the timing of chemotherapy on OS or PFS (p=0.80 and p=0.53 respectively) and an improved OS in patients with non-epithelioid disease, and improved PFS in those with lymph node metastases, if chemotherapy was given in the immediate adjuvant setting (p=0.001 and 0.038) when therapy was not delayed until disease progression. CONCLUSION: Our results suggest that the timing of additional chemotherapy may be important in those with a poorer prognosis on the basis of cell type and nodal stage. In these patients additional postoperative chemotherapy should not be delayed.

The management of the diaphragm during radical surgery for malignant pleural mesothelioma.

OBJECTIVES: Macroscopic complete resection with lung preservation is the objective of radical management of pleural mesothelioma (MPM). Total removal of visceral and parietal pleura (pleurectomy/decortication) almost invariably proceeds to an extended pleurectomy/decortication (EPD) to ensure macroscopic complete resection. We suspected this may not always be necessary. METHODS: We reviewed 314 patients, 86.0% male, median age 62 years (range 14-81 years) undergoing radical surgery for MPM from 1999 to 2014, by either EPD or extrapleural pneumonectomy. The extent of diaphragmatic muscle involvement was recorded from postoperative pathology. Patients were divided into three groups: no involvement, non-transmural, transmural diaphragmatic invasion. RESULTS: A total of 213 (68%) patients underwent EPD, 237 (75.5%) had epithelioid disease and 57.6% were node positive. There was no difference between the three groups in terms of age, cell type, laterality, neoadjuvant chemotherapy and operation. There was a higher degree of diaphragm involvement in females (P = 0.01) and in patients with positive lymph nodes (P = 0.01). No evidence of diaphragmatic involvement was found following pathological assessment of the resection specimen in 119 patients (37.9%). The incidence of abdominal disease progression was 23.9%. There was no correlation with degree of diaphragmatic invasion (ρ = 0.01 P = 0.88). Overall survival of those with abdominal progression was similar to those with progression elsewhere: 14.5 vs 13.0 months (P = 0.79), and with those with no progression (16.7 months, P = 0.189). There was no difference in survival when stratified by diaphragmatic involvement (P = 0.44). CONCLUSIONS: In our cohort, there was no evidence of diaphragmatic invasion in over 30% of patients, and we have also failed to find evidence that peritoneal disease progression affects overall survival following radical management. It may therefore theoretically be unnecessary to resect the diaphragm in all cases, and a pleurectomy-decortication could suffice. However, there is an unknown risk of R2 resection which would prejudice survival, and as such we would advocate resecting the diaphragm in all cases to avoid an R2 resection.

The effects of an intentional transition from extrapleural pneumonectomy to extended pleurectomy/decortication.

OBJECTIVES: For many years, extrapleural pneumonectomy (EPP) was the operation of choice for the radical management of pleural mesothelioma in the UK. However, doubts surrounding the efficacy of EPP, and the change in demographics of the affected population, have prompted a transition in our practice towards extended pleurectomy/decortication (EPD). The aim of this study was to determine the effects an intentional transition from EPP to EPD has had on patient outcome. METHODS: Data from 362 patients undergoing radical surgery (229 EPD, 133 EPP) during 1999-2014 were included. Demographics and outcome were compared between the two groups; EPP versus EPD. RESULTS: The median age of patients undergoing EPD was significantly higher than those undergoing EPP [57 years (range 14-70 years) vs 65 years (range 42-81 years), P < 0.001]. There was a significantly higher proportion of patients with performance status ≥1 in the EPD group (46.3 vs 35.4%, P = 0.047). There was no difference in the median length of hospital stay between the two groups [14 days (range 1-133 days) vs 13 days (range 0-93 days), P = 0.409]. There was also no difference between the groups in terms of in-hospital mortality (EPP 5.3% and EPD 6.6%, P = 0.389), 30-day mortality [EPP 8 (6.0%) and EPD 8 (3.5%), P = 0.294] or 90-day mortality [EPP 18 (13.5%) and EPD 21 (9.2%), P = 0.220]. There was a significantly higher early reoperation rate in the EPP group (15.0 vs 6.2%, P = 0.008) but a significantly higher late reoperation rate in the EPD group (0.8 vs 5.3%, P = 0.037). There was no significant difference in overall survival or disease-free interval between the two groups (P = 0.899 and P = 0.399, respectively). However, overall survival was significantly greater in patients over the age of 65 undergoing EPD (12.5 vs 4.7 months, P = 0.001). CONCLUSION: The transition from EPP to EPD in our standard practice has enabled us to operate on more elderly, frail patients with no significant increase in use of hospital resources, and without detriment to overall survival.