ROS1 fusions in resected stage I-III adenocarcinoma: Results from the European Thoracic Oncology Platform Lungscape project.

BACKGROUND: ROS1 fusion is a relatively low prevalence (0.6-2.0%) but targetable driver in lung adenocarcinoma (LUAD). Robust and low-cost tests, such as immunohistochemistry (IHC), are desirable to screen for patients potentially harboring this fusion. The aim was to investigate the prevalence of ROS1 fusions in a clinically annotated European stage I-III LUAD cohort using IHC screening with the in vitro diagnostics (IVD)-marked clone SP384, followed by confirmatory molecular analysis in pre-defined subsets. METHODS: Resected LUADs constructed in tissue microarrays, were immunostained for ROS1 expression using SP384 clone in a ready-to-use kit and Ventana immunostainers. After external quality control, analysis was performed by trained pathologists. Staining intensity of at least 2+ (any percentage of tumor cells) was considered IHC positive (ROS1 IHC + ). Subsequently, ROS1 IHC + cases were 1:1:1 matched with IHC0 and IHC1 + cases and subjected to orthogonal ROS1 FISH and RNA-based testing. RESULTS: The prevalence of positive ROS1 expression (ROS1 IHC + ), defined as IHC 2+/3+, was 4 % (35 of 866 LUADs). Twenty-eight ROS1 IHC + cases were analyzed by FISH/RNA-based testing, with only two harboring a confirmed ROS1 gene fusion, corresponding to a lower limit for the prevalence of ROS1 gene fusion of 0.23 %. They represent a 7 % probability of identifying a fusion among ROS1 IHC + cases. Both confirmed cases were among the only four with sufficient material and H-score ≥ 200, leading to a 50 % probability of identifying a ROS1 gene fusion in cases with an H-score considered strongly positive. All matched ROS1 IHC- (IHC0 and IHC1 + ) cases were also found negative by FISH/RNA-based testing, leading to a 100 % probability of lack of ROS1 fusion for ROS1 IHC- cases. CONCLUSIONS: The prevalence of ROS1 fusion in an LUAD stage I-III European cohort was relatively low. ROS1 IHC using SP384 clone is useful for exclusion of ROS1 gene fusion negative cases.

PD-1-expressing macrophages and CD8 T cells are independent predictors of clinical benefit from PD-1 inhibition in advanced mesothelioma.

BACKGROUND: Few tissue biomarkers exist to date that could enrich patient with cancer populations to benefit from immune checkpoint blockade by programmed cell death protein 1/ligand-1 (PD-/L-1) inhibitors. PD-L1 expression has value in this context in some tumor types but is an imperfect predictor of clinical benefit. In malignant pleural mesothelioma, PD-L1 expression is not predictive of the benefit from PD-1 blockade. We aimed to identify novel markers in malignant pleural mesothelioma to select patients better. METHODS: We performed a multiplex-immune histochemistry analysis of tumor samples from the phase III PROMISE-meso study, which randomized 144 pretreated patients to receive either pembrolizumab or standard second-line chemotherapy. Our panel focused on CD8+T cell, CD68+macrophages, and the expression of PD-1 and PD-L1 on these and cancer cells. We analyzed single and double positive cells within cancer tissues (infiltrating immune cells) and in the stroma. In addition, we performed cell neighborhood analysis. The cell counts were compared with clinical outcomes, including responses, progression-free and overall survivals. RESULTS: We confirmed the absence of predictive value for PD-L1 in this cohort of patients. Furthermore, total CD8 T cells, CD68+macrophages, or inflammatory subtypes (desert, excluded, inflamed) did not predict outcomes. In contrast, PD-1-expressing CD8+T cells (exhausted T cells) and PD-1-expressing CD68+macrophages were both independent predictors of progression-free survival benefit from pembrolizumab. Patients with tumors simultaneously harboring PD1+T cells and PD-1+macrophages benefited the most from immune therapy. CONCLUSION: We analyzed a large cohort of patients within a phase III study and found that not only PD-1+CD8 T cells but also PD-1+CD68+ macrophages are predictive. This data provides evidence for the first time for the existence of PD-1+macrophages in mesothelioma and their clinical relevance for immune checkpoint blockade.

European Epidemiology of Pleural Mesothelioma-Real-Life Data From a Joint Analysis of the Mesoscape Database of the European Thoracic Oncology Platform and the European Society of Thoracic Surgery Mesothelioma Database.

INTRODUCTION: Pleural mesothelioma (PM) is an aggressive malignancy with increasing prevalence and poor prognosis. Real-life data are a unique approach to reflect the reality of PM epidemiology, treatment, and prognosis in Europe. METHODS: A joint analysis of the European Thoracic Oncology Platform Mesoscape and the European Society of Thoracic Surgeons (ESTS) databases was performed to better understand the characteristics and epidemiology of PM, including histologic subtype, staging, and treatment. Overall survival (OS) was assessed, adjusting for parameters of clinical interest. RESULTS: The analysis included 2766 patients (Mesoscape: 497/10 centers/ESTS: 2269/77 centers). The primary histologic subtype was epithelioid (71%), with 57% patients on stages III to IV. Within Mesoscape, the patients received either multimodality (59%) or palliative intention treatment (41%). The median follow-up was 47.2 months, on the basis of 1103 patients (Mesoscape: 491/ESTS: 612), with 823 deaths, and median OS was 17.4 months. In multivariable analysis, female sex, epithelioid subtype, and lower stage were associated with longer OS, when stratifying by cohort, age, and Eastern Cooperative Oncology Group Performance Status. Within Mesoscape, multimodality treatment including surgery was predictive of longer OS (hazard ratio = 0.56, 95% confidence interval: 0.45-0.69), adjusting for sex, histologic subtype, and Eastern Cooperative Oncology Group Performance Status. Overall, surgical candidates with a macroscopic complete resection had a significantly longer median OS compared with patients with R2 (25.2 m versus 16.4 m; log-rank p < 0.001). CONCLUSIONS: This combined European Thoracic Oncology Platform/ESTS database analysis offers one of the largest databases with detailed clinical and pathologic outcome. Our finding reflects a benefit for selected patients that undergo multimodality treatment, including macroscopic complete resection, and represents a valuable resource to inform the epidemiology and treatment options for individual patients.

Assessment of RANK/RANK-L prevalence and clinical significance in NSCLC European Thoracic Oncology Platform Lungscape cohort and SPLENDOUR randomized clinical trial.

BACKGROUND: The primary objective of this study is to evaluate the clinical significance of RANK/L expression, in both a retrospective cohort of surgically resected stage I-III NSCLC (Lungscape) and a randomized clinical trial-cohort (SPLENDOUR) of advanced NSCLC treated with chemotherapy alone or in combination with denosumab. METHODS: RANK-L expression was assessed on tissue microarrays (TMAs) in Lungscape and whole sections in SPLENDOUR, using immunohistochemistry, with H-scores values > 0 indicating positivity. Prevalence of RANK positivity and its association with clinicopathological characteristics, and patient outcome was explored in a subset of the ETOP Lungscape cohort and in SPLENDOUR. Also investigated were the prevalence of RANK overexpression (proportion of positive cancer cells ≥ 50%) in the Lungscape cohort, and RANK-L in the SPLENDOUR trial. RESULTS: In the Lungscape cohort, RANK expression was assessed at a median follow-up of 46 months (N = 488 patients; 4 centers); 35% were female, 44/49/6% adenocarcinomas (AC)/squamous cell carcinomas (SCC)/other, 48/27/25% with stage I/II/III. Median RFS/TTR/OS were 58/Not reached/74 months. Prevalence of RANK expression was 31% (95%CI:27%-35%); significantly higher in AC: 50% (95%CI:43%-57%) vs SCC: 12% (95%CI:8%-16%) (p < 0.001); more frequent in females (42% vs 25%, p < 0.001) and tumors ≤ 4 cm (35.3% vs 23.3%, p = 0.0065). No association with outcome was found. In the SPLENDOUR trial (463 patients), the prevalence of membranous and cytoplasmic RANK positivity was 34% (95%CI:30%-38%) and 9% (95%CI:7%-12%), respectively, while prevalence for RANK-L was 5% (95%CI:3%-7%) and 36% (95%CI:31%-40%), respectively. Cytoplasmic RANK-L positivity was more common among females (47% vs 31%, p = 0.001) and in non-SCC histology (45% vs 10%, p < 0.0001). At the pre-specified 1% significance level, no prognostic or predictive effect was found. CONCLUSIONS: Both cohorts indicate that RANK expression is more common in adenocarcinoma/non-squamous NSCLC and in female patients. No prognostic effect is found, and in the clinical trial involving addition of denosumab to chemotherapy no predictive effect is detected.

Prognostic impact of tumour mutational burden in resected stage I and II lung adenocarcinomas from a European Thoracic Oncology Platform Lungscape cohort.

BACKGROUND: The primary objective of this study is to evaluate tumor mutational burden (TMB), its associations with selected clinicopathological and molecular characteristics as well as its clinical significance, in a retrospective cohort of surgically resected stage I-II lung adenocarcinomas, subset of the ETOP Lungscape cohort. METHODS: TMB was evaluated on tumor DNA extracted from resected primary lung adenocarcinomas, based on FoundationOne®CDx (F1CDx) genomic profiling, centrally performed at the University Hospital Zurich. The F1CDx test sequences the complete exons of 324 cancer-related genes and detects substitutions, insertions and deletions (indels), copy number alterations and gene rearrangements. In addition, the genomic biomarkers TMB and microsatellite instability (MSI) are analyzed. RESULTS: In the Lungscape cohort, TMB was assessed in 78 surgically resected lung adenocarcinomas from two Swiss centers (62 % males, 55 %/45 % stage I/II). Median TMB was 7.6 Muts/Mb, with TMB high (≥10 Muts/Mb) in 40 % of cases (95 %CI:29 %-52 %). The most frequently mutated genes were TP53/KRAS/EGFR/MLL2 detected in 58 %/38 %/33 %/30 % of samples, respectively. TMB was significantly higher among males (TMB high: 50 % vs 23 % in females, p = 0.032), as well as among current/former smokers (TMB high: 44 % vs 8 % in never smokers, p = 0.023). Furthermore, TMB was significantly higher in TP53 mutated than in non-mutated patients (TMB high: 60 % vs 12 %, p < 0.001), while it was higher in EGFR non-mutated patients compared to EGFR mutated (TMB high: 48 % vs 23 %, p = 0.049). At a median follow-up time of 56.1 months (IQR:38.8-72.0), none of the three outcome variables (OS, RFS, TTR) differed significantly by TMB status (all p-values > 5 %). This was also true when adjusting for clinicopathological characteristics. CONCLUSIONS: While presence of TP53 mutations and absence of EGFR mutations are associated with high TMB, increased TMB had no significant prognostic impact in patients with resected stage I/II lung adenocarcinoma beyond T and N classification, in both unadjusted and adjusted analyses.

Expression of phosphorylated ribosomal protein S6 in mesothelioma patients - correlation with clinico-pathological characteristics and outcome: results from the European Thoracic Oncology Platform (ETOP) Mesoscape project.

Pleural mesothelioma (PM) is an aggressive malignancy with poor prognosis. Although histology and pathologic stage are important prognostic factors, better prognostic biomarkers are needed. The ribosomal protein S6 is a downstream target of the phosphatidylinositol 3-kinase (PI3K) pathway involved in protein synthesis and cell proliferation. In previous studies, low phosphorylated S6 (pS6) immunoreactivity was significantly correlated with longer progression-free survival (PFS) and overall survival (OS) in PM patients. We aimed to correlate pS6 expression to clinical data in a large multi-centre PM cohort as part of the European Thoracic Oncology Platform (ETOP) Mesoscape project. Tissue Micro Arrays (TMAs) of PM were constructed and expression of pS6 was evaluated by a semi-quantitatively aggregate H-score. Expression results were correlated to patient characteristics as well as OS/PFS. pS6 IHC results of 364 patients from 9 centres, diagnosed between 1999 and 2017 were available. The primary histology of included tumours was epithelioid (70.3%), followed by biphasic (24.2%) and sarcomatoid (5.5%). TMAs included both treatment-naïve and tumour tissue taken after induction chemotherapy. High pS6 expression (181 patients with H-score>1.41) was significantly associated with less complete resection. In the overall cohort, OS/PFS were not significantly different between pS6-low and pS6-high patients. In a subgroup analysis non-epithelioid (biphasic and sarcomatoid) patients with high pS6 expression showed a significantly shorter OS (p < 0.001, 10.7 versus 16.9 months) and PFS (p < 0.001, 6.2 versus 10.8 months). In subgroup analysis, in non-epithelioid PM patients high pS6 expression was associated with significantly shorter OS and PFS. These exploratory findings suggest a clinically relevant PI3K pathway activation in non-epithelioid PM which might lay the foundation for future targeted treatment strategies.

Extended resection for potentially operable patients with stage III non-small cell lung cancer after induction treatment.

OBJECTIVE: Surgical treatment of locally advanced non-small cell lung cancer including single or multilevel N2 remains a matter of debate. Several trials demonstrate that selected patients benefit from surgery if R0 resection is achieved. We aimed to assess resectability and outcome of patients with locally advanced clinical T3/T4 (American Joint Committee on Cancer 8th edition) tumors after induction treatment followed by surgery in a pooled analysis of 3 prospective multicenter trials. METHODS: A total of 197 patients with T3/T4 non-small cell lung cancer of 368 patients with stage III non-small cell lung cancer enrolled in the Swiss Group for Clinical Cancer Research 16/96, 16/00, 16/01 trials were treated with induction chemotherapy or chemoradiation therapy followed by surgery, including extended resections. Univariable and multivariable analyses were applied for analysis of outcome parameters. RESULTS: Patients' median age was 60 years, and 67% were male. A total of 38 of 197 patients were not resected for technical (81%) or medical (19%) reasons. A total of 159 resections including 36 extended resections were performed with an 80% R0 and 13.2% pathological complete response rate. The 30- and 90-day mortality were 3% and 7%, respectively, without a difference for extended resections. Morbidity was 32% with the majority (70%) of minor grading complications. The 3-, 5-, and 10-year overall survivals for extended resections were 61% (95% confidence interval, 43-75), 44% (95% confidence interval, 27-59), and 29.5% (95% confidence interval, 13-48), respectively. R0 resection was associated with improved overall survival (hazard ratio, 0.41; P < .001), but pretreatment N2 extension (177/197) showed no impact on overall survival. CONCLUSIONS: Surgery after induction treatment for advanced T3/T4 stage including single and multiple pretreatment N2 disease resulted in 80% R0 resection rate and 7% 90-day mortality. Favorable overall survival for extended and not extended resection was demonstrated to be independent of pretreatment N status.

A prognostic score for patients with malignant pleural mesothelioma (MPM) receiving second-line immunotherapy or chemotherapy in the ETOP 9-15 PROMISE-meso phase III trial.

INTRODUCTION: Clinical and laboratory parameters associated with response for patients with advanced pre-treated malignant pleural mesothelioma (MPM) are lacking. We aimed to identify prognostic and predictive markers among patients with relapsed MPM who were randomised into the ETOP 9-15 PROMISE-meso phase III trial, evaluating pembrolizumab and chemotherapy. METHODS: Baseline clinical and laboratory parameters were investigated for prognostic or predictive value on progression-free survival (PFS) and overall survival (OS) in a retrospective analysis, based on the full cohort of 144 MPM patients. These consisted of immune-inflammatory indexes (neutrophil-lymphocyte ratio [NLR], systemic immune-inflammatory index [SII], lactate dehydrogenase [LDH]) along with other already known prognostic baseline characteristics and laboratory values. Cut-offs were chosen independently of outcome. Based on Cox multivariable analysis for PFS in the whole cohort, a risk factor model was built to illustrate the prognostic stratification of patients by the combination of the derived independent prognostic factors, taking into account the EORTC score, a validated prognostic score in MPM. All models were stratified by histology and adjusted by treatment. RESULTS: In the stratified multivariable analysis in the whole cohort, high SII (hazard ratio (HR) 2.06; 95%CI 1.39-3.05) and low haemoglobin (HR 1.62; 95%CI 1.06-2.50) were associated with worse PFS. Based on these two prognostic factors, a mesothelioma risk score (MRS) was constructed with three PFS risk prognosis categories: favourable, intermediate and poor with 0, 1 and 2 risk factors, respectively (corresponding percent of cohort: 24%, 34% and 42% and median PFS: 5.8, 4.2 and 2.1 months). The derived MRS stratified the prognosis for PFS and OS, overall and within each of the EORTC groups. No significant predictors of treatment benefit were identified. CONCLUSIONS: The proposed MRS is prognostic of patient outcome and it fine-tunes the prognosis of patients with pre-treated MPM alone or when used with the already established EORTC score.

Combined, patient-level, analysis of two randomised trials evaluating the addition of denosumab to standard first-line chemotherapy in advanced NSCLC - The ETOP/EORTC SPLENDOUR and AMGEN-249 trials.

INTRODUCTION: The efficacy of adding denosumab to standard first-line chemotherapy for advanced NSCLC patients has been evaluated in two separate randomised trials (SPLENDOUR and AMGEN-249). In this pooled analysis, we will assess the combination-treatment effect in the largest available population, in order to conclude about the potential impact of denosumab in NSCLC. METHODS: Both trials included in this combined analysis, were randomised (SPLENDOUR 1:1, AMGEN-249 2:1) multi-centre trials stratified by histology, bone metastasis, geographical region and for SPLENDOUR only, ECOG PS. Cox proportional hazards models, were used to assess the treatment effect with respect to overall survival (OS; primary endpoint) and progression-free survival (PFS; secondary endpoint). Heterogeneity between trials was assessed, and subgroup analyses were performed. RESULTS: The pooled analysis was based on 740 randomised patients (SPLENDOUR:514; AMGEN-249:226), with 407 patients in the chemotherapy-denosumab arm and 333 in the chemotherapy-alone arm. In the chemotherapy-denosumab arm, at a median follow-up of 22.0 months, 277 (68.1%) deaths were reported with median OS 9.2 months (95%CI:[8.0-10.7]), while in the chemotherapy-alone arm, with similar median follow-up of 20.3 months, 230 (69.1%) deaths with median OS 9.9 months (95%CI:[8.2-11.2]). No significant denosumab effect was found (HR = 0.98; 95%CI:[0.82-1.18]; P = 0.85). Among subgroups, interaction was found between treatment and histology subtypes (P = 0.020), with a statistically significant benefit in the squamous group (HR = 0.70; 95%CI:[0.49-0.98]; P = 0.038), from 7.6 to 9.0 months median OS. With respect to PFS, 363 (89.2%) and 298 (89.5%) events were reported in the chemotherapy-denosumab and chemotherapy-alone arms, respectively, with corresponding medians 4.8 months (95%CI:[4.4-5.3]) and 4.9 months (95%CI:[4.3-5.4]). HR for PFS was 0.97(95%CI:[0.83-1.15]; P = 0.76), indicating that no significant denosumab benefit existed for PFS. CONCLUSION: In this pooled analysis, no statistically significant improvement was shown in PFS/OS with the combination of denosumab and chemotherapy for advanced NSCLC and no meaningful benefit in any of the subgroups.

Prognostic Impact of KRAS G12C Mutation in Patients With NSCLC: Results From the European Thoracic Oncology Platform Lungscape Project.

INTRODUCTION: KRAS mutations, the most frequent gain-of-function alterations in NSCLC, are currently emerging as potential predictive therapeutic targets. The role of KRAS-G12C (Kr_G12C) is of special interest after the recent discovery and preclinical analyses of two different Kr_G12C covalent inhibitors (AMG-510, MRTX849). METHODS: KRAS mutations were evaluated in formalin-fixed, paraffin-embedded tissue sections by a microfluidic-based multiplex polymerase chain reaction platform as a component of the previously published European Thoracic Oncology Platform Lungscape 003 Multiplex Mutation study, of clinically annotated, resected, stage I to III NSCLC. In this study, -Kr_G12C mutation prevalence and its association with clinicopathologic characteristics, molecular profiles, and postoperative patient outcome (overall survival, relapse-free survival, time-to-relapse) were explored. RESULTS: KRAS gene was tested in 2055 Lungscape cases (adenocarcinomas: 1014 [49%]) with I or II or III stage respective distribution of 53% or 24% or 22% and median follow-up of 57 months. KRAS mutation prevalence in the adenocarcinoma cohort was 38.0% (95% confidence interval (CI): 35.0% to 41.0%), with Kr_G12C mutation representing 17.0% (95% CI: 14.7% to 19.4%). In the "histologic-subtype" cohort, Kr_G12C prevalence was 10.5% (95% CI: 9.2% to 11.9%). When adjusting for clinicopathologic characteristics, a significant negative prognostic effect of Kr_G12C presence versus other KRAS mutations or nonexistence of KRAS mutation was identified in the adenocarcinoma cohort alone and in the "histologic-subtype" cohort. For overall survival in adenocarcinomas, hazard ratio (HR)G12C versus other KRAS is equal to 1.39 (95% CI: 1.03 to 1.89, p = 0.031) and HRG12C versus no KRAS is equal to 1.32 (95% CI: 1.03 to 1.69, p = 0.028) (both also significant in the "histologic-subtype" cohort). For time-to-relapse, HRG12C versus other KRAS is equal to 1.41 (95% CI: 1.03 to 1.92, p = 0.030). In addition, among all patients, for relapse-free survival, HRG12C versus no KRAS is equal to 1.27 (95% CI: 1.04 to 1.54, p = 0.017). CONCLUSIONS: In this large, clinically annotated stage I to III NSCLC cohort, the specific Kr_G12C mutation is significantly associated with poorer prognosis (adjusting for clinicopathologic characteristics) among adenocarcinomas and in unselected NSCLCs.

Progression-Free and Overall Survival for Concurrent Nivolumab With Standard Concurrent Chemoradiotherapy in Locally Advanced Stage IIIA-B NSCLC: Results From the European Thoracic Oncology Platform NICOLAS Phase II Trial (European Thoracic Oncology Platform 6-14).

INTRODUCTION: The NICOLAS study is the first completed single-arm phase II trial in stage III NSCLC evaluating hierarchically first the safety and then the efficacy of adding nivolumab concurrently to standard definitive concurrent chemoradiotherapy. The safety end point was reported earlier; here, we present the efficacy results. METHODS: Stage IIIA-B unresectable treatment-naive patients with NSCLC received three cycles of platinum-based chemotherapy and concurrent radiotherapy (66 Gy, 33 fractions), along with nivolumab (360 mg, 3-weekly). Nivolumab was continued as monotherapy consolidation for a maximum of 1 year (480 mg, 4-weekly). The primary end point was 1-year progression-free survival (PFS), with a target improvement compared with historical data of at least 15%, from 45% to 60%. To test this efficacy hypothesis, a sample size of 74 assessable patients provided a power of 83% with a one-sided alpha of 5%. RESULTS: A total of 79 patients were enrolled with a median follow-up of 21.0 months (interquartile range: 15.8-25.8 mo) for the primary PFS analysis. A total of 35.4% of the patients had stage IIIA, and 63.3% had stage IIIB disease. The 1-year PFS was 53.7% (95% confidence interval [CI]: 42.0%-64.0%) and the median PFS was 12.7 months (95% CI: 10.1-22.8 mo). Because 37 PFS events occurred in the first year posttreatment among the first 74 assessable patients, a 1-year PFS rate of at least 45% could not be rejected (p = 0.23). At an extended follow-up (median 32.6 mo), 37 deaths have been recorded, with a median overall survival (OS) of 38.8 months (95% CI: 26.8 mo-not estimable) and a 2-year OS rate of 63.7% (95% CI: 51.9%-73.4%). The OS of patients with stage IIIA disease was found to be significantly higher than patients with stage IIIB disease, with a 2-year OS of 81% and 56%, respectively (p = 0.037). CONCLUSIONS: PFS and OS are arithmetically higher in studies involving the same population. However, on the basis of the formal hierarchical efficacy analysis, we could not reject that the 1-year PFS rate is at least 45%.

Verification of a Blood-Based Targeted Proteomics Signature for Malignant Pleural Mesothelioma.

BACKGROUND: We have verified a mass spectrometry (MS)-based targeted proteomics signature for the detection of malignant pleural mesothelioma (MPM) from the blood. METHODS: A seven-peptide biomarker MPM signature by targeted proteomics in serum was identified in a previous independent study. Here, we have verified the predictive accuracy of a reduced version of that signature, now composed of six-peptide biomarkers. We have applied liquid chromatography-selected reaction monitoring (LC-SRM), also known as multiple-reaction monitoring (MRM), for the investigation of 402 serum samples from 213 patients with MPM and 189 cancer-free asbestos-exposed donors from the United States, Australia, and Europe. RESULTS: Each of the biomarkers composing the signature was independently informative, with no apparent functional or physical relation to each other. The multiplexing possibility offered by MS proteomics allowed their integration into a single signature with a higher discriminating capacity than that of the single biomarkers alone. The strategy allowed in this way to increase their potential utility for clinical decisions. The signature discriminated patients with MPM and asbestos-exposed donors with AUC of 0.738. For early-stage MPM, AUC was 0.765. This signature was also prognostic, and Kaplan-Meier analysis showed a significant difference between high- and low-risk groups with an HR of 1.659 (95% CI, 1.075-2.562; P = 0.021). CONCLUSIONS: Targeted proteomics allowed the development of a multianalyte signature with diagnostic and prognostic potential for MPM from the blood. IMPACT: The proteomic signature represents an additional diagnostic approach for informing clinical decisions for patients at risk for MPM.

A Randomized Open-Label Phase III Trial Evaluating the Addition of Denosumab to Standard First-Line Treatment in Advanced NSCLC: The European Thoracic Oncology Platform (ETOP) and European Organisation for Research and Treatment of Cancer (EORTC) SPLENDOUR Trial.

INTRODUCTION: Receptor activator of NF-kB ligand stimulates NF-kB-dependent cell signaling and acts as the primary signal for bone resorption. Retrospective analysis of a large trial comparing denosumab versus zoledronic acid in bone metastatic solid tumors suggested significant overall survival (OS) advantage for patients with lung cancer with denosumab (p = 0.01). The randomized open-label phase III SPLENDOUR trial was designed to evaluate whether the addition of denosumab to standard first-line platinum-based doublet chemotherapy improved OS in advanced NSCLC. METHODS: Patients with stage IV NSCLC were randomized in a 1:1 ratio to either chemotherapy with or without denosumab (120 mg every 3-4 wks), stratified by the presence of bone metastases (at diagnosis), Eastern Cooperative Oncology Group performance status, histology, and region. To detect an OS increase from 9 to 11.25 months (hazard ratio [HR] = 0.80), 847 OS events were required. The trial closed prematurely owing to decreasing accrual rate. RESULTS: A total of 514 patients were randomized, with 509 receiving one or more doses of the assigned treatment (chemotherapy: 252, chemotherapy-denosumab: 257). The median age was 66.1 years, 71% were men, and 59% were former smokers. Bone metastases were identified in 275 patients (53%). Median OS (95% confidence interval [CI]) was 8.7 (7.6-11.0) months in the control arm versus 8.2 (7.5-10.4) months in the chemotherapy-denosumab arm (HR = 0.96; 95% CI: 0.78-1.19; one-sided p = 0.36). For patients with bone metastasis, HR was 1.02 (95% CI: 0.77-1.35), whereas for those without, HR was 0.90 (95% CI: 0.66-1.23). Adverse events grade 3 or greater were observed in 40.9%, 5.2%, 8.7% versus 45.5%, 10.9%, 10.5% of patients. Conditional power for OS benefit was less than or equal to 10%. CONCLUSIONS: Denosumab was well-tolerated without unexpected safety concerns. There was no OS improvement for denosumab when added to chemotherapy in the intention-to-treat population and the subgroups with and without bone metastases. Our data do not provide evidence of a clinical benefit for denosumab in patients with NSCLC without bone metastases.

Current models, challenges and best practices for work conducted between European academic cooperative groups and industry.

BACKGROUND: The academia-industry interface is important, and, despite challenges that inevitably occur, bears the potential for positive synergies to emerge. Perceived barriers to wider collaboration in academia-industry oncology research in Europe need to be addressed, current academic cooperative group and industry models for collaboration need to be discussed, and a common terminology to facilitate understanding of both sectors' concerns needs to be established with an eye towards improving academia-industry partnerships on clinical trials for the benefit of patients with cancer. METHODOLOGY: CAREFOR (Clinical Academic Cancer Research Forum), a multi-stakeholder platform formed to improve the direction for academic clinical trials in the field of oncology in Europe, formed the CAREFOR-Industry Working Group comprised of experienced professionals from European academic cooperative groups joined by industry representatives selected based on their activities in the area of medical oncology. They jointly discussed academic cooperative groups, clinical trials conducted between academic cooperative groups and industry, examples of successful collaborative models, common legal negotiation points in clinical trial contracts, data access, and principles of interaction. RESULTS: Four principles of interaction between the academia and industry are proposed: (1) clarify the roles and responsibilities of all partners involved in the study, (2) involve legal teams from an early stage; (3) acknowledge that data is an important output of the study, (4) agree on the intent of the trial prior to its start. CONCLUSIONS: The CAREFOR-Industry Working Group describes current models, challenges, and effective strategies for academia-industry research in Europe with an eye towards improving academia-industry partnerships on clinical trials for patients with cancer. Current perceived challenges are explained, and future opportunities/recommendations for improvement are described for the areas of most significant impact. Challenges are addressed from both the academic and industry perspectives, and principles of interaction for the optimal alignment between academia and industry in selected areas are proposed.

Evolution and Clinical Impact of EGFR Mutations in Circulating Free DNA in the BELIEF Trial.

INTRODUCTION: Longitudinal evaluation of mutations in blood samples was a prespecified secondary objective in the BELIEF trial of erlotinib and bevacizumab in advanced EGFR-positive NSCLC. Here, we report the testing results and explore the correlation of EGFR status in blood with clinical outcomes. METHODS: Blood samples were prospectively collected from patients at baseline, at response evaluation, and at progression and sent to a central laboratory. Circulating free DNA was purified and EGFR mutations were analyzed with a validated real-time quantitative polymerase chain reaction assay. RESULTS: EGFR exon 19/21 mutations were detected in 55 of 91 baseline blood samples (60.4%) and correlated with a significantly worse progression-free survival: 11.4 months (95% confidence interval [CI]: 9.0-14.8 mo) for the patients who were positive versus 22.9 months (95% CI: 9.5-33.9 mo) for those who were negative (log-rank p = 0.0020). Among the 74 samples at response, exon 19/21 mutations were detected only in three samples (4.1%). In contrast, 29 of 58 patients (50.0%) were exon 19/21 positive at progression and showed a significantly worse median overall survival of 21.7 months (95% CI: 17.0-30.9 mo) compared with 37.4 months (95% CI: 22.6-53.1 mo) for those who were negative (log-rank p = 0.011). Blood samples at the three time points were available for 48 patients. Of those, among 14 exon 19/21 EGFR-negative at presentation, 13 (93%) were persistently negative for the sensitizing mutations after progression and the p.T790M could only be detected in the blood of two patients. CONCLUSIONS: Longitudinal testing of EGFR mutations in blood can offer valuable clinical information. In patients of the BELIEF study, detection of EGFR mutations in circulating free DNA at presentation was associated with shorter progression-free survival, whereas positivity at progression correlated with shorter overall survival. Finally, patients negative in blood at presentation were almost invariably negative at relapse.

Functional Genomic Screen in Mesothelioma Reveals that Loss of Function of BRCA1-Associated Protein 1 Induces Chemoresistance to Ribonucleotide Reductase Inhibition.

Loss of function of BRCA1-associated protein 1 (BAP1) is observed in about 50% of malignant pleural mesothelioma (MPM) cases. The aim of this study was to investigate whether this aspect could be exploited for targeted therapy. A genetically engineered model was established expressing either functional or nonfunctional BAP1, and whole-genome siRNA synthetic lethality screens were performed assessing differentially impaired survival between the two cell lines. The whole-genome siRNA screen unexpectedly revealed 11 hits (FDR < 0.05) that were more cytotoxic to BAP1-proficient cells. Two actionable targets, ribonucleotide reductase (RNR) catalytic subunit M1 (RRM1) and RNR regulatory subunit M2 (RRM2), were validated. In line with the screen results, primary mesothelioma (BAP1 +/-) overexpressing BAP1 C91A (catalytically dead mutant) was more resistant to RNR inhibition, while BAP1 knockdown in the BAP1-proficient cell lines rescued the cells from their vulnerability to RNR depletion. Gemcitabine and hydroxyurea were more cytotoxic in BAP1-proficient cell line-derived spheroids compared with BAP1 deficient. Upregulation of RRM2 upon gemcitabine and hydroxyurea treatment was more profound in BAP1 mut/del cell lines. Increased lethality mediated by RNR inhibition was observed in NCI-H2452 cells reconstituted with BAP1-WT but not with BAP1 C91A. Upregulation of RRM2 in NCI-H2452-BAP1 WT spheroids was modest compared with control or C91A mutant. Together, we found that BAP1 is involved in the regulation of RNR levels during replication stress. Our observations reveal a potential clinical application where BAP1 status could serve as predictive or stratification biomarker for RNR inhibition-based therapy in MPM.

Intracavitary cisplatin-fibrin chemotherapy after surgery for malignant pleural mesothelioma: A phase I trial.

OBJECTIVES: Intracavitary chemotherapy is a promising concept to improve local tumor control for malignant pleural mesothelioma with reported high morbidity rates. We have demonstrated that administration of cisplatin loaded to fibrin increased local drug concentration and reduced systemic toxicity in preclinical models. We present a phase I trial of intracavitary cisplatin-fibrin after surgical tumor resection. METHODS: A total of 12 patients (75% International Mesothelioma Interest Group stage III-IV) were treated with 4 dose levels of intracavitary cisplatin-fibrin (11-44 mg/m2 body surface area) in a dose-escalating design. Cisplatin-fibrin was sprayed on the resected surfaces after pleurectomy/decortication. Blood and tissue samples were taken to assess toxicity and pharmacokinetics. Patients were regularly followed up. RESULTS: No dose-limiting toxicity was observed. Major morbidity occurred in 4 patients (33%). The 30-day and 90-day mortality were both 0%. Of 80 adverse events, 9 were classified serious, but none of these were related to study treatment. Local cisplatin concentration in the chest wall tissue was high at all dose levels (median, 46.3 µg/g [12-133 µg/g]). In serum, median cisplatin area under the concentration time curve values were always below renal toxicity levels. The median overall survival with 95% confidence interval was 21 months (10-31 months). In 1 patient with epithelioid malignant pleural mesothelioma (International Mesothelioma Interest Group stage I), there was no sign of relapse 48 months after treatment (44 mg/m2 body surface area). CONCLUSIONS: The administration of intracavitary cisplatin-fibrin is safe with favorable pharmacokinetics. Although most patients had advanced disease, long-term outcomes are comparable to other multimodal concepts. A confirmation phase II trial is ongoing.

Programmed death-ligand 1 expression influenced by tissue sample size. Scoring based on tissue microarrays' and cross-validation with resections, in patients with, stage I-III, non-small cell lung carcinoma of the European Thoracic Oncology Platform Lungscape cohort.

PD-L1, as assessed by immunohistochemistry, is a predictive biomarker for immuno-oncology treatment in lung cancer. Different scoring methods have been used to assess its status, resulting in a wide range of positivity rates. We use the European Thoracic Oncology Platform Lungscape non-small cell lung carcinoma cohort to explore this issue. PD-L1 expression was assessed via immunohistochemistry on tissue microarrays (up to four cores per case), using the DAKO 28-8 immunohistochemistry assay, following a two-round external quality assessment procedure. All samples were analyzed under the same protocol. Cross-validation of scoring between tissue microarray and whole sections was performed in 10% randomly selected samples. Cutoff points considered: ≥1, 50 (primarily), and 25%. At the two external quality assessment rounds, tissue microarray scoring agreement rates between pathologists were: 73% and 81%. There were 2008 cases with valid immunohistochemistry tissue microarray results (50% all cores evaluable). Concordant cases at 1, 25, and 50% were: 85, 91, and 93%. Tissue microarray core results were identical for 70% of cases. Sensitivity of the tissue microarray method for 1, 25, and 50% was: 80, 78, and 79% (specificity: 90, 95, 98%). Complete agreement between tissue microarrays and whole sections was achieved for 60% of the cases. Highest sensitivity rates for 1% and 50% cutoffs were detected for higher number of cores. Underestimation of PD-L1 expression on small samples is more common than overestimation. We demonstrated that classification of PD-L1 on small biopsy samples does not represent the overall expression of PD-L1 in all non-small cell cancer carcinoma cases, although the majority of cases are 'correctly' classified. In future studies, sampling more and larger biopsies, recording the biopsy size and tumor load may permit further refinement, increasing predictive accuracy.

Pattern of failure after adjuvant radiotherapy following extrapleural pneumonectomy of pleural mesothelioma in the SAKK 17/04 trial.

Postoperative radiotherapy after extrapleural pneumonectomy of malignant pleural mesothelioma was investigated in the randomized phase II trial SAKK17/04. The relapse rate within the high and/or low-dose PTV without previous distant failure was 24%, the isolated in-field-relapse rate within the PTVs was 5% and the distant relapse rate outside of the PTVs was 81%. Clinical outcome was mainly determined by distant disease progression outside of the radiation field.