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Supplementary Fig. S1: Characterization of the model used for whole genome silencing, individual screen lethality scores and list of candidate genes with differential lethality scores ; Supplementary Fig. S2: Validation of the RRM1 and RRM2 knockdown; Supplementary Fig. S3: Expression of RRM1 and RRM2 in MPM; Supplementary Fig. S4: Modulation of BAP1 expression, effect on gene expression, response to olaparib, and rescue of gemcitabine cytotoxicity by dNMPs; Supplementary Fig. S5: Schematic representation of the potential role of BAP1 in the regulation of RRM2 on transcriptional level under normal and replicative stress condition; Supplementary Fig. S6: Gemcitabine induced increase of RRM1 and RRM2 in NCI60 cell line panel and high RRM1 and RRM2 expression predicts poor overall survival in malignant pleural mesothelioma patients; Supplementary Table S1: Primer sequences; Supplementary Table S2: Gene ontology analysis on genes with differential lethality score.

List of primers for quantitative real time PCR

Reduced fibronectin protein levels in tumor treated with vismodegib

No correlation between Dhh and Gli1 expression levels in rat MPM cells

Supernatant from rat and human MPM cells could stimulate Gli1 Ptch1 and Fn1 expression of NIH3T3 cells

Supplementary figure legends

Vismodegib does not interfere with in vivo Bli measurement

An autocrine-driven upregulation of the Hedgehog (Hh) signaling pathway has been described in malignant pleural mesothelioma (MPM), in which the ligand, desert Hh (DHH), was produced from tumor cells. However, our investigation revealed that the Hh pathway is activated in both tumor and stroma of MPM tumor specimens and an orthotopic immunocompetent rat MPM model. This was demonstrated by positive immunohistochemical staining of Glioma-associated oncogene 1 (GLI1) and Patched1 (PTCH1) in both tumor and stromal fractions. DHH was predominantly expressed in the tumor fractions. To further investigate the role of the Hh pathway in MPM stroma, we antagonized Hh signaling in the rat model of MPM using a Hh antagonist, vismodegib, (100 mg/kg orally). Daily treatment with vismodegib efficiently downregulated Hh target genes Gli1, Hedgehog Interacting Protein (Hhip), and Ptch1, and caused a significant reduction of tumor volume and tumor growth delay. Immunohistochemical analyses revealed that vismodegib treatment primarily downregulated GLI1 and HHIP in the stromal compartment along with a reduced expression of previously described fibroblast Hh-responsive genes such as Fibronectin (Fn1) and Vegfa. Primary cells isolated from the rat model cultured in 3% O2 continued to express Dhh but did not respond to vismodegib in vitro. However, culture supernatant from these cells stimulated Gli1, Ptch1, and Fn1 expression in mouse embryonic fibroblasts, which was suppressed by vismodegib. Our study provides new evidence regarding the role of Hh signaling in MPM stroma in the maintenance of tumor growth, emphasizing Hh signaling as a treatment target for MPM. Mol Cancer Ther; 15(5); 1095–105. ©2016 AACR.

Primary cells isolated from rat tumor did not response to SAG and vismodegib treatment

Survival analysis for human-based TF scores on H&E stained sections by univariate and multivariate cox regressions. OS=overall survival; RFS=relapse-free survival; CI=95% confidence interval; TF (low/high): scores dichotomized at the median (categorical); TF (continuous): score as continuous variable.

Purpose:Combination of immune checkpoint inhibitors with chemotherapy is under investigation for cancer treatment.Experimental Design:We studied the rationale of such a combination for treating mesothelioma, a disease with limited treatment options.Results:The combination of gemcitabine and immune checkpoint inhibitors outperformed immunotherapy alone with regard to tumor control and survival in a preclinical mesothelioma model; however, the addition of dexamethasone to gemcitabine and immune checkpoint inhibitors nullified the synergistic clinical response. Furthermore, treatment with gemcitabine plus anti-PD-1 resulted in an objective clinical response in two patients with mesothelioma, who were resistant to gemcitabine or anti-PD-1 as monotherapy.Conclusions:Thus, treatment of mesothelioma with a combination of gemcitabine with immune checkpoint inhibitors is feasible and results in synergistic clinical response compared with single treatment in the absence of steroids.

Because of the refractory nature of mutant KRAS lung adenocarcinoma (LUAD) to current therapies, identification of new molecular targets is essential. Genes with a prognostic role in mutant KRAS LUAD have proven to be potential molecular targets for therapeutic development. Here we determine the clinical, functional, and mechanistic role of inhibitor of differentiation-1 (Id1) in mutant KRAS LUAD. Analysis of LUAD cohorts from TCGA and SPORE showed that high expression of Id1 was a marker of poor survival in patients harboring mutant, but not wild-type KRAS. Abrogation of Id1 induced G2–M arrest and apoptosis in mutant KRAS LUAD cells. In vivo, loss of Id1 strongly impaired tumor growth and maintenance as well as liver metastasis, resulting in improved survival. Mechanistically, Id1 was regulated by the KRAS oncogene through JNK, and loss of Id1 resulted in downregulation of elements of the mitotic machinery via inhibition of the transcription factor FOSL1 and of several kinases within the KRAS signaling network. Our study provides clinical, functional, and mechanistic evidence underscoring Id1 as a critical gene in mutant KRAS LUAD and warrants further studies of Id1 as a therapeutic target in patients with LUAD.Significance:These findings highlight the prognostic significance of the transcriptional regulator Id1 in KRAS-mutant lung adenocarcinoma and provide mechanistic insight into how it controls tumor growth and metastasis.

Supplementary Materials and Methods, References

A) Percentage distribution of computed TF scores for TMA and correlation between matching cores. B) Percentage distribution of computed TF scores for WS and correlation between matching whole sections. C) Percentage distribution of human-based TF scores for TMA and correlation between matching cores. B) Percentage distribution of human-based TF scores for WS and correlation between matching whole sections. E) Percentage distribution of human-based TF scores for TCGA images from observer 1 (Obs1) and correlations between the three observers on a subset of pictures (n=67). Dashed line indicates the median.

Flow chart of transcriptome profiling after antigen-specific stimulation of redirected T cells with different co-stimulations

Purpose: The aim of this study was to assess the activity of hedgehog signaling pathway in malignant pleural mesothelioma (MPM).Experimental Design: The expression of hedgehog signaling components was assessed by quantitative PCR and in situ hybridization in 45 clinical samples. Primary MPM cultures were developed in serum-free condition in 3% oxygen and were used to investigate the effects of smoothened (SMO) inhibitors or GLI1 silencing on cell growth and hedgehog signaling. In vivo effects of SMO antagonists were determined in an MPM xenograft growing in nude mice.Results: A significant increase in GLI1, sonic hedgehog, and human hedgehog interacting protein gene expression was observed in MPM tumors compared with nontumoral pleural tissue. SMO antagonists inhibited GLI1 expression and cell growth in sensitive primary cultures. This effect was mimicked by GLI1 silencing. Reduced survivin and YAP protein levels were also observed. Survivin protein levels were rescued by overexpression of GLI1 or constitutively active YAP1. Treatment of tumor-bearing mice with the SMO inhibitor HhAntag led to a significant inhibition of tumor growth in vivo accompanied by decreased Ki-67 and nuclear YAP immunostaining and a significant difference in selected gene expression profile in tumors.Conclusions: An aberrant hedgehog signaling is present in MPM, and inhibition of hedgehog signaling decreases tumor growth indicating potential new therapeutic approach. Clin Cancer Res; 18(17); 4646–56. ©2012 AACR.

PDF file, 96K, 1A. Expression of HH pathway components in non-tumoral pleural tissue and mesothelioma tumors. Quantitative real-time PCR analysis of SMO, GLI2, IHH, DHH gene expression in non-tumoral pleural tissue (NT) and tumor (T). 1B. Overall survival is inversely correlated with high GLI1 expression (delta Ct below the median) (p=0.042, low GLI1: median survival 22.9 months, 95% confidence interval 0.4- 45.4 months; high GLI1: median survival 17.0 months, 95% confidence interval 9.0- 25.1 months ).

Immune modulatory ligand expression on the investigated tumor cell line

An Id1 gene signature is dependent of KEAP1-related genes and a FOSL1 network.

Clinical implication of Id1 expression in LUAD patients.

Series of cox univariate survival analyses are represented for the WS cohort. Data points represent single survival analyses repeated for computed TF scores using different tumor fragments size cut-offs. X-axis indicates the minimal size threshold for tumor fragments. Y-axis are the corresponding p-values. Black line indicates p=0.05.

Baseline level of cytokines

Supplementary tables and figure's headings

Supplementary data

PDF file, 50K, GLI1 transfection (GLI1) leads to increased expression of GLI1 target HHIP compared to control vector (V) transfected ZL55SPT cells.

PDF file, 66K, Western blot analysis of NF2 protein in SDM103T, ZL55SPT cells. SDM71 cells were used as positive control (1). 1. Thurneysen C, Opitz I, Kurtz S, Weder W, Stahel RA, Felley-Bosco E. Functional inactivation of NF2/merlin in human mesothelioma. Lung Cancer. 2009;64:140-7.

PDF file, 60K, MPM primary cultures maintained without serum in 3% oxygen show a three-fold increase in expression of ABCG2, GLI1 and PATCH1 compared to cultures without serum in 20% oxygen.

List of differentially regulated cell cycle genes with Log2FC values.

PDF file, 36K, Table S1: Primers used for RT-PCR.

supplementary figure legends

Generation of humanized mice using leukapheresis or human fetal liver (HFL) derived CD34+ cells.

The table shows proportions of patients among low and high TF groups (dichotomized at the median TF scores) stratified by clinical parameters. Chi-square tests and Spearman's rank correlations were used for categorical (low/high TF) and continuous TF scores respectively.

A) gene ontology network visualization of RNAs overexpressed in high (red) and low TF (blue) groups. B) bar plot showing molecular pathways associated with low (blue) and high (red) TF, at the RNA level. C) bar plot showing biological processes (BP) associated with increasing (red) and decreasing (blue) TF, at the protein level. Numbers indicate protein counts per category; asterisks* indicate BPs also enriched at the RNA Level.

PDF file, 159K, HHIP protein is expressed in all tumor cells and its levels are increased by HhAntag. Immunohistochemistry analysis of HHIP expression was performed on xenograft tumors of control and HhAntag treated mice. H score was determined based as frequency* intensity ( frequency score 1=1-10% cells, 2=11-50% cells, 3=51-100% cells and intensity score1=low; 2= high). Bar represents 50 μm.

The list of proteins identified by mass spectrometry (n=2614) for 48 samples of the WS cohort.

The PRIDE (PRoteomics IDEntifications database) additional raw and search files of our experiment

A shRNA-resistant Id1 cDNA rescues Id1 loss.

Purpose:The clinical standard treatment for patients with malignant pleural mesothelioma (MPM) includes a cisplatin-based chemotherapy, leading to reduction of tumor size in only a minority of patients. Predicting response to chemotherapy in patients with MPM by using a genetic marker would, therefore, enable patient stratification.Experimental Design:In this retrospective biomarker study, eligible patients had resectable MPM, measurable disease, and available primary MPM tissue. All patients underwent first-line treatment with cisplatin and pemetrexed, followed by surgery. Thorough molecular analysis was performed (whole-exome and targeted deep sequencing, and copy-number analyses), and also mechanistic in vitro data (viability assays, Western blots, and immunoprecipitation) using mesothelioma cell lines with and without siRNA-mediated BRCA1-associated protein 1 (BAP1) knockdown were provided.Results:In a training cohort of patients with MPM (n = 28), mutations or deletions of BAP1 each predicted resistance to chemotherapy in patients with primary MPM. The negative predictive value of BAP1 loss in patients with MPM was confirmed by amplicon sequencing and copy-number array technology in an independent test cohort (n = 39). Preliminary mechanistic studies using siRNA-based knockdown of BAP1 in MPM cell culture models along with immunoprecipitation assays confirmed chemoresistance in vitro, possibly through inhibition of apoptosis and transcriptional regulation of the BAP1/HCF1/E2F1 axis.Conclusions:Alterations in BAP1 in MPM were a negative predictor for response to chemotherapy and could possibly be used as a companion biomarker for treatment decision.

Phenotypic and functional comparison of redirected T cells

Redirected T cells did not show persistence in the blood of tumor bearing humanized mice

Purpose: Combination therapy of adoptively transferred redirected T cells and checkpoint inhibitors aims for higher response rates in tumors poorly responsive to immunotherapy like malignant pleural mesothelioma (MPM). Only most recently the issue of an optimally active chimeric antigen receptor (CAR) and the combination with checkpoint inhibitors is starting to be addressed.Experimental Design: Fibroblast activation protein (FAP)–specific CARs with different costimulatory domains, including CD28, Δ-CD28 (lacking lck binding moiety), or 4-1BB were established. CAR-T cells were characterized in vitro and antitumor efficacy was tested in vivo in a humanized mouse model in combination with PD-1 blockade. Finally, the Δ-CD28 CAR was tested clinically in a patient with MPM.Results: All the three CARs demonstrated FAP-specific functionality in vitro. Gene expression data indicated a distinct activity profile for the Δ-CD28 CAR, including higher expression of genes involved in cell division, glycolysis, fatty acid oxidation, and oxidative phosphorylation. In vivo, only T cells expressing the Δ-CD28 CAR in combination with PD-1 blockade controlled tumor growth. When injected into the pleural effusion of a patient with MPM, the Δ-CD28 CAR could be detected for up to 21 days and showed functionality.Conclusions: Overall, anti-FAP-Δ-CD28/CD3ζ CAR T cells revealed superior in vitro functionality, better tumor control in combination with PD-1 blockade in humanized mice, and persistence up to 21 days in a patient with MPM. Therefore, further clinical investigation of this optimized CAR is warranted. Clin Cancer Res; 24(16); 3981–93. ©2018 AACR.

Regulation of kinases by Id1 expression

The table shows the list of proteins correlating with TF scores as a continuous response with a FDR

Osimertinib is a third-generation, irreversible oral EGFR-tyrosine kinase inhibitor), that potently inhibits EGFR-tyrosine kinase inhibitor-sensitizing mutations and T790M resistance mutations together with efficacy in CNS metastases in patients with non-small-cell lung cancer (NSCLC). Here we describe the rationale and design for the Phase III NeoADAURA study (NCT04351555), which will evaluate neoadjuvant osimertinib with or without chemotherapy versus chemotherapy alone prior to surgery, in patients with resectable stage II–IIIB N2 EGFR mutation-positive NSCLC. The primary end point is centrally assessed major pathological response at the time of resection. Secondary end points include event-free survival, pathological complete response, nodal downstaging at the time of surgery, disease-free survival, overall survival and health-related quality of life. Safety and tolerability will also be assessed. Trial Registration number: NCT04351555 (ClinicalTrials.gov), Lay abstract A plain language version of this article is available and is published alongside the paper online: www.futuremedicine.com/doi/suppl/10.2217/fon-2021-0549

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

OBJECTIVES Recent trials have begun to explore immune checkpoint inhibitors for non-small cell lung cancer in the neoadjuvant setting, but data on tumour response and surgical outcome remain limited. METHODS Retrospective evaluation of clinical data from patients with non-small cell lung cancer treated with immune checkpoint inhibitors followed by lung resection was performed at 2 large volume institutions (1 North American, 1 European). Data were analysed using Chi-squared, Fisher’s and Wilcoxon rank-sum tests where appropriate. RESULTS Thirty-seven patients were identified from 2017 to 2019. Forty-nine per cent were Stage IIIB and IV. Forty-six per cent received immunotherapy alone and 54% in combination with chemo- and/or radiotherapy. Sixteen per cent of cases were successfully performed minimally invasively. Twenty patients were operated with lobectomy (6 of these with wedges or segments of a neighbouring lobe, 2 with sleeve resections and 1 with a chest wall resection), 4 with bilobectomies, 11 with pneumonectomy (including 5 extrapleural pneumonectomies and 1 atrial resection) and 1 with a wedge resection. Overall, 10 patients (27%) developed postoperative complications and the 90-day mortality was zero. One-year recurrence-free survival was 73% for stage II/IIIA and 55% for stage IIIB/stage IV. The major pathologic response rate was 34%. CONCLUSION In this retrospective study, lung resection after immunotherapy (alone or in combination) is safe, although often requires complex surgery. Due to increasing number of clinical trials adopting immunotherapy in the neoadjuvant setting, it is likely that this therapy will become part of standard of care. Immunotherapy may also allow surgery to have a role for selected patients with advanced disease.

The treatment goal for patients with early-stage lung cancer is cure. Multidisciplinary discussions of surgical resectability and medical operability determine the modality of definitive local treatment (surgery or radiotherapy) and the associated systemic therapies to further improve the likelihood of cure. Trial evidence supports cisplatin-based adjuvant therapy either after surgical resection or concurrently with radiotherapy. Consensus guidelines support neoadjuvant chemotherapy in lieu of adjuvant chemotherapy and carboplatin-based regimens for patients who are ineligible for cisplatin. The incorporation of newer agents, now standard for patients with stage IV lung cancer, into the curative therapy paradigm has lagged owing to inefficient trial designs, the lengthy follow-up needed to assess survival end points and a developmental focus on the advanced-stage disease setting. Surrogate end points, such as pathological response, are being studied and might shorten trial durations. In 2018, the anti-PD-L1 antibody durvalumab was approved for patients with stage III lung cancer after concurrent chemoradiotherapy. Since then, the study of targeted therapies and immunotherapies in patients with early-stage lung cancer has rapidly expanded. In this Review, we present the current considerations in the treatment of patients with early-stage lung cancer and explore the current and future state of clinical research to develop systemic therapies for non-metastatic lung cancer. The authors of this Review present the current considerations in the treatment of patients with early-stage lung cancer, discussing the critical determination of resectability by thoracic surgical oncologists and the management of both resectable and unresectable disease with a focus on systemic therapy selection. They also address innovations in drug development, trial design and efforts to identify early-stage cancers.

Purpose: The clinical standard treatment for patients with malignant pleural mesothelioma (MPM) includes a cisplatin-based chemotherapy, leading to reduction of tumor size in only a minority of patients. Predicting response to chemotherapy in patients with MPM by using a genetic marker would, therefore, enable patient stratification. Experimental Design: In this retrospective biomarker study, eligible patients had resectable MPM, measurable disease, and available primary MPM tissue. All patients underwent first-line treatment with cisplatin and pemetrexed, followed by surgery. Thorough molecular analysis was performed (whole-exome and targeted deep sequencing, and copy-number analyses), and also mechanistic in vitro data (viability assays, Western blots, and immunoprecipitation) using mesothelioma cell lines with and without siRNA-mediated BRCA1-associated protein 1 (BAP1) knockdown were provided. Results: In a training cohort of patients with MPM (n = 28), mutations or deletions of BAP1 each predicted resistance to chemotherapy in patients with primary MPM. The negative predictive value of BAP1 loss in patients with MPM was confirmed by amplicon sequencing and copy-number array technology in an independent test cohort (n = 39). Preliminary mechanistic studies using siRNA-based knockdown of BAP1 in MPM cell culture models along with immunoprecipitation assays confirmed chemoresistance in vitro, possibly through inhibition of apoptosis and transcriptional regulation of the BAP1/HCF1/E2F1 axis. Conclusions: Alterations in BAP1 in MPM were a negative predictor for response to chemotherapy and could possibly be used as a companion biomarker for treatment decision.

BACKGROUND Cadaveric lobar lung transplantation (L-LTx) is developed to overcome donor-recipient size mismatch. Controversial short- and long-term outcomes following L-LTx have been reported compared to full-sized lung transplantation (F-LTx). This study reports long-term outcomes after L-LTx. METHODS We reviewed patients undergoing lung transplantation (LTx) between 2000 and 2016. The decision to perform L-LTx was made based mainly on donor-recipient height discrepancy and visual assessment of donor lungs. Predicted donor-recipient total lung capacity (TLC) ratio was calculated more recently. Primary outcome was overall survival. RESULTS In all, 370 bilateral LTx were performed during the study period, among those 250 (67%) underwent F-LTx and 120 (32%) underwent L-LTx, respectively. One- and 5-year survival rates were 85% vs. 90% and 53% vs. 63% for L-LTx and F-LTx, respectively (p = 0.16). Chronic lung allograft dysfunction (CLAD)-free survival at 5 years was 48% in L-LTx vs. 51% in F-LTx recipients (p = 0.89), respectively. Age, intraoperative extracorporeal membrane oxygenation (ECMO) use, intensive care unit (ICU) stay, and postoperative renal replacement therapy (RRT) were significant prognostic factors for survival using multivariate analysis. CONCLUSIONS Overall survival and CLAD-free survival following L-LTx were comparable to F-LTx. Given the ongoing donor organ shortage, cadaveric L-LTx remains as an important resource in LTx.