Virtual Library

Background:
Variant splicing isoforms of CD44 (CD44v) are a marker of cancer stem cells (CSCs) in solid tumors. CD44v stabilizes the glutamate-cystine transporter subunit xCT and thereby promotes synthesis of the intracellular antioxidant glutathione and protects CSCs from oxidative stress. Salazosulfapyridine (SASP) is an inhibitor of xCT activity, and, in combination with cisplatin (CDDP), it attenuates the increase in the proportion of CD44v-positive tumor cells during the growth of tumor xenografts in mice.

Methods:
Individuals with advanced (stage IIIB or IV) nonsquamous non–small cell lung cancer are eligible to enroll in a phase I dose-escalation study (standard 3+3 design) of SASP in combination with CDDP and pemetrexed (PEM) as first-line treatment. Patients receive SASP daily as well as CDDP (75 mg/m[2]) and PEM (500 mg/m[2]) on day 1 of a 21-day cycle. The primary end point is the percentage of patients who experience dose-limiting toxicity (DLT) between administration of the first dose of SASP (day 1) and day 21.

Results:
From April 2015 to January 2016, 15 patients were enrolled in the study (mean age, 64 years; age range, 42–74 years; male/female ratio, 10/5; ECOG performance status 0/1 ratio, 6/9). Immunohistochemical staining of tumor biopsy specimens revealed that the proportion of CD44v-positive cells was >10% in 9 patients before SASP treatment. No DLT was observed in the first three patients treated at SASP dose level 1 (500 mg TID) or those treated at dose level 2 (1000 mg TID). At dose level 3 (1500 mg TID), two of three patients experienced a DLT (anorexia of grade 3). We enrolled additional patients at dose level 2 and two of the total of five patients treated at this dose level experienced DLTs (hypotension or pneumonitis, each of grade 3). To confirm the safety of dose level 1, we enrolled additional patients at this dose level and one of the total of six patients treated at this dose level experienced DLTs (AST and ALT elevation, each of grade 3). Exposure of SASP following oral administration varied markedly among individuals according to ABCG2 and NAT2 genotypes as previously reported.

Conclusion:
SASP 500 mg TID was the recommended dose when administered with CDDP plus PEM.

Background:
Tumors originating in lung tissues or in the bronchi invade adjacent tissue and cause infiltration beyond the lung. Lung macrophages express mannose-specific endocytosis receptor that might binds or internalize mannose terminated dendrimer. Therefore, it is hypothesized that incorporation of anticancer drug into mannose anchored dendrimer will transport the drug effectively to the tumor cells via receptor mediated endocytosis. Dendrimer are easy to synthesis and better stability, Nanoscopic size range, High drug loading propensity, Dose reduction possible, Number of free surface groups available for further conjugation. The project aimed to investigate the targeting potential of mannose conjugated Poly Propyl Imine (PPI) dendrimer having potent anticancer drug, Gemcitabine in lung cancer cells. The dendrimers were conjugated so as to enhance the therapeutic potential and reduce adverse effect of anticancer drug.

Methods:
The 5.0 generation dendrimers were synthesized and were characterized by FTIR and Nuclear Magnetic Resonance (NMR). The PPI dendrimers prepared were then conjugated with mannose and drug was loaded. The shape and size were characterized by Transmission Electron Microscopy (TEM), drug loading efficiency, In-vitro drug release and stability studies. The ex-vivo studies constituted Hemolytic toxicity study and Cell cytotoxic study by MTT Cytotoxicity Assay on A-549 (Lung adenocarcinoma epithelial) cell line. The in-vivo studies were performed on albino rats and Pharmakokinetic parameters were studied, also Biodistribution Studies were done to access gemcitabine level attained in different organs.

Results:
Thus Mannosylated PPI dendrimers showed high gemcitabine loading, sustained release and excellent biocompatibility as evident by low hemolytic toxicity. MTT assay suggested high cytotoxicity of GmcH-MPPI against A549 cancer cell lines. The Presence of ligand on dendrimer molecule, elevated receptor mediated binding or internalization in AM. The developed ligand conjugated dendritic system targeted higher concentration of GmcH to lung than the free drug.

Conclusion:
Thus, we concluded that GmcH loaded mannosylated PPI dendritic system could have higher potential to target anticancer drug to lungs for effective chemotherapy of lung tumor.

Background:
The dual mTORC1/2 inhibitor AZD2014 has multiple effects on cell growth, apoptosis, angiogenesis and metabolism in cancer cells. AZD2014 increases the efficacy of paclitaxel in preclinical models, including patient derived xenografts. These data and clinical responses in the dose escalation arm of the TAX-TORC study led to an expansion cohort of 40 patients with squamous non-small cell lung cancer (sqNSCLC).

Methods:
Patients, of ECOG performance status 0-1, with sqNSCLC who had received at least one line of platinum-based chemotherapy were eligible for the study. Paclitaxel was dosed once weekly at 80mg/m[2], 6 weeks out of 7. AZD2014 was dosed BD, 3 days per week starting with the paclitaxel dosing. The cohort was started at 50mg AZD2014 BD.

Results:
Thirty-two patients have been treated, 24 male/8 female with median age 68 years. The median number of previous treatments was 1 with 6/32 (19%) having received a prior taxane (docetaxel or paclitaxel). Analysis of data from the first 17 patients, by the safety review committee, showed that fatigue, skin rash and diarrhoea were the most common toxicities in 59%, 47% and 41% patients respectively. The majority of toxicities were CTCAE grades 1 or 2 (112/123, 91%) and reversible with AZD2014 interruption or reduction. However, there were 9 grade 3 and 4 toxicities and 2 incidences of grade 5 respiratory infection. There were 2/17 (12%) responses though patients often stopped early due to toxicity. Following the safety review, the dose of AZD2014 was reduced to 25mg BD which is a pharmacodynamically active dose associated with fewer toxicities. Fifteen additional patients have subsequently been treated at this lower dose. Their most common toxicities were anaemia, alopecia and fatigue in 47%, 47% and 40% patients respectively. There have been no grade 5 events and only 8/78 (10%) grade 3 or 4 toxicities. The response rate in this cohort is 5/15 (33%) and recruitment is ongoing. Archival samples and circulating free DNA at baseline are being assessed with targeted next generation sequencing to explore putative predictive biomarkers for response and resistance.

Conclusion:
We have established a tolerable dose and schedule for the combination of weekly paclitaxel and AZD2014. The promising response rate of 33% in previously treated sqNCSLC patients warrants further investigation. The study is supported by AstraZeneca, Cancer Research UK, Experimental Cancer Medicine Centre and NIHR Biomedical Research Centre Initiatives.

Background:
Autophagy is a catabolic process that regulates the lysosomal turnover of damaged proteins and organelles in order to maintain cellular homeostasis. Dysregulation of autophagy is often observed in lung cancer. Kinase inhibitors have proved successful in the clinic. The fact that uncoordinated (Unc) 51-like kinase (Ulk1) is the only conserved serine/threonine kinase in the autophagy cascade makes it a very attractive target for therapeutic development. Up-regulation of Ulk1 has been shown to promote cell survival of several cancer cell lines. Moreover, overexpression of Ulk1 has been shown to be negatively correlated with the prognosis of several types of human cancer. However, the role of Ulk1 in NSCLC is largely unknown.

Methods:
We evaluated Ulk1 expression levels in human normal lung epithelial cell line BEAS-2B and five NSCLC cell lines, A549, H460, H1299, H292 and HCC827. We analyzed the correlation between Ulk1 expression levels and the prognosis of NSCLC patients. We evaluated the effect of SBI0206965 alone or in combination with cisplatin on cell proliferation, apoptosis and autophagy in two human NSCLC cell lines, A549 and H460. Cell proliferation of untreated or drug-treated cells was measured by CCK8 assay. Percentage of apoptotic cells was measured using PE-conjugated Annexin V with a flow cytometer. Autophagy was determined by conversion of LC3I to LC3II and p62 degradation using Western blot.

Results:
Ulk1 is overexpressed in NSCLC cell lines and negatively correlated with the prognosis of NSCLC patients. The inhibition of Ulk1 by a selective inhibitor, SBI0206965, blocks the proliferation of NSCLC cells and induces apoptosis. SBI0206965 treatment augments the efficacy of cisplatin to kill NSCLC cells by inhibiting cisplatin induced cell protective autophagy. SBI0206965 can also destabilize Bcl2/Bclxl to promote cisplatin induced apoptosis in NSCLC cell lines.

Conclusion:
Our results suggest that the inhibition of Ulk1 may be a promising strategy for the treatment of NSCLC. SBI0206965 may be a promising agent for the treatment of NSCLC by modulating autophagy and apoptosis pathways. Furthermore, the combination of SBI0206965 with classical chemotherapy agents represents a promising therapeutic strategy that warrants further clinical evaluation in NSCLC.

Background:
MET exon 14 (METex14) skipping has been reported as a potentially targetable driver mutation in lung adenocarcinoma. We aimed to evaluate the prevalence and clinicopathologic characteristics of lung adenocarcinoma harboring METex14 skipping in patients with lung adenocarcinoma in which targetable genomic alterations are not available.

Methods:
We screened 795 patients with lung adenocarcinoma and 45 patients with quintuple-negative (EGFR-/KRAS-/ALK-/ROS1-/RET-) lung adenocarcinomas were finally included to identify the patients harboring METex14 skipping by using RT-PCR with probes overlapping an exon 13–15 junction. In addition, we summarized recent articles reported about METex14 skipping in lung cancer.

Results:
Based on the present study, seventeen patients (37.8%) had tumors harboring METex14 skipping alterations. Diverse genomic sequence variants causing METex14 skipping were identified. The median age of the METex14 skipping-positive patients was 73 years (range, 55–81 years), 8 patients (47.1%) were female, and 7 (41.2%) had never smoked. The predominant subtype was acinar followed by the solid type. The MET immunohistochemistry test for METex14 skipping demonstrated 100% (95% CI, 79.6–100) sensitivity and 70.4% (95% CI, 51.5–84.2) specificity. In literature reviews, we included 619 patients with lung cancer harboring METex14 skipping. The median age of the patients was 68 years (range, 41-84), 60% (251/418) were female, and 50% (58/116) were never smoker. MET immunochemical stain was positive in 62.3% (48/77), MET amplification was identified in 14.6% (45/309) of the patients. The prevalences of METex14 skipping were 12.9% (20/155) in sarcomatoid carcinoma, 3.9% (11/282) in adenosquamous carcinoma, 2.6% (398/15050) in adenocarcinoma, 2.1% (26/1226) in squamous cell carcinoma, and 0.8% (2/243) in large cell carcinoma, respectively.

Conclusion:
The prevalence of METex14 skipping was relatively high in patients with quintuple-negative (EGFR-/KRAS-/ALK-/ROS1-/RET-) lung adenocarcinomas. Lung adenocarcinomas harboring METex14 skipping were associated with old age, acinar or solid histolgy, and MET protein overexpression. Identification of subpopulation harboring METex14 skipping can be an important step toward developing targeted therapies for patients with lung cancer.

Background:
Invasive mucinous adenocarcinoma of the lung (IMA) accounts for 2 to 10% of all lung adenocarcinomas and usually presents as a multifocal and unresectable disease for which no effective treatment exists. Recently, rearrangements of the HER3 ligand gene NRG1 have been identified in IMA such as NRG1-SLC3A2 and NRG1-CD74 leading to activation of HER3 and PI3K-AKT signaling pathways. Therefore, IMA harboring NRG1 fusion genes may serve as a biologically attractive target for HER3-targeted therapies.

Methods:
Study NCT01482377 is a phase Ib study analyzing the safety and preliminary efficacy of lumretuzumab, a monoclonal anti-HER3 antibody, in combination with erlotinib in patients with HER3 protein-positive tumors. Lumretuzumab IV was given every 2 weeks at 800 mg and erlotinib was given at standard dose of 150 mg/d po. A pretreatment tumor biopsy was mandated for the assessment of membranous HER3 protein by IHC. NRG1 fusion genes were identified by RT-PCR and sequencing. Tumor assessments were performed by CT scans every 8 weeks. Therapy was given until progressive disease or unacceptable toxicity. Here we describe the clinical course of two patients with IMA harboring a SLC3A2-NRG1 fusion gene treated within this study.

Results:
Patient 1 is a 55-year-old Asian female who was diagnosed in 2011. Previous lines of therapy included gemcitabine and cisplatin, erlotinib, pemetrexed, docetaxel and irinotecan and cisplatin. After enrolling into the study the first CT scan showed a decrease of 16% of the target lesion qualifying for stable disease per RECIST 1.1. At the following tumor assessment progressive disease was documented resulting in a disease stabilization of 16.4 weeks. Patient 2 is a 42-year-old Asian female who was diagnosed in 2013. Previous lines of therapy included pemetrexed and cisplatin, erlotinib, docetaxel, vinorelbine, and gemcitabine and cisplatin. After enrolling into the study, the patient showed stable disease as a best overall RECIST response that lasted 16.3 weeks. Both patients experienced mild to moderate rash and diarrhea (grade 1 & 2). No ≥ grade 3 adverse events were observed.

Conclusion:
This is the first report of a novel targeted therapy approach in IMA patients harboring NRG1 gene rearrangements - a histological entity that is generally considered to be extremely difficult to treat. The combination of lumretuzumab and erlotinib was well tolerated and showed signs of tumor shrinkage in a heavily pretreated IMA patient. Further studies are warranted to elucidate the clinical relevance of HER3-targeted therapy in IMA patients with NRG1 fusion genes.

Background:
Non-small cell lung cancer (NSCLC) is treated with surgery and chemotherapy with either tyrosine kinase inhibitors (TKIs) or platinum-based regimens, but drug-resistance is frequent and long-term prognosis poor. Dianhydrogalactitol (VAL-083) is a bi-functional alkylating agent with proven activity against NSCLC in clinical studies. VAL-083 has demonstrated superior activity to cisplatin in both in vitro and in vivo NSCLC models, including TKI-resistant NSCLC, and circumvents cisplatin-resistance in ovarian cancer cells. VAL-083 is approved for the treatment of lung cancer in China; however, clinical adoption is limited by lack of modern data related to mechanism-of-action and utility in the context of standard-of-care platinum-based chemotherapy. Here we aimed to investigate in vitro i) the distinct mechanism-of-action of VAL-083, ii) VAL-083 cytotoxicity in a panel of NSCLC cell-lines with varying p53 status, and iii) the combination of VAL-083 with cisplatin or oxaliplatin.

Methods:
VAL-083 cytotoxicity was investigated in a panel of 11 human NSCLC cell-lines: 3 wild-type (H460, A549, H226), 6 mutant (H1975, SkLU1, H2122, H157, H1792, H23) and 2 null (H838, H1299) for p53. Cell-cycle and DNA damage was investigated by propidium iodide and immunofluorescent staining in synchronized cultures of H2122, H1792, A549. Cytotoxicity was determined by MTT assay. Combination potential for VAL-083 with cisplatin or oxaliplatin was investigated in H460, A549, H1975 (TKI-resistant) by determining superadditivity and synergy using the combination index (CI)<1 criteria.

Results:
VAL-083 treatment caused persistent S/G2 cell-cycle arrest and cell-death. Furthermore, one-hour pulse treatment led to phosphorylation of DNA double-strand break sensors ATM, single-strand DNA-binding Replication Protein A (RPA32), and histone variant H2A.X, suggesting activation of the homologous repair pathway. S/G2 phase cell-cycle arrest and increased γH2A.X in cancer cells persisted >72 hours after treatment, indicating irreversible DNA lesions. Importantly, VAL-083 was active against all cell-lines tested, irrespective of their p53 status, suggesting a mechanism-of-action that differs from other alkylating agents, including cisplatin. When combined with either cisplatin or oxaliplatin in vitro, VAL-083 demonstrated significant superadditivity (p<0.05) and synergism (CI<1) for both combinations in all NSCLC cell-lines. This strongly suggests non-overlapping modes-of-action between the platinum drugs and VAL-083 and demonstrates synergism in TKI-resistant cell-lines.

Conclusion:
This preclinical data strongly suggests VAL-083 as a potential treatment for platinum and TKI-resistant NSCLC. An open-label post-market clinical trial in China will investigate the activity of VAL-083 in relapsed/refractory NSCLC. Results will provide guidance to physicians under the context of VAL-083’s current approval in China, as well as serve as proof-of-concept for expanded development worldwide.

Background:
Amplification of the mesenchymal-epithelial transition factor (MET) gene plays a vital role in non-small cell lung cancer (NSCLC). The anti-MET therapeutic strategies are still unclear in epidermal growth factor receptor (EGFR) mutant patients and EGFR-naive patients. Aims of our study are to discuss role of MET amplification in Chinese NSCLC patients, and evaluate the antitumor activity of crizotinib (MET inhibitor) in Chinese NSCLC patients with MET gene amplification.

Methods:
From Jun 2015 to Jan 2016, we detected 11 metastatic NSCLC patients with MET amplification by fluorescence in situ hybridization (FISH). MET amplification was defined as gene focal amplification or high polysomy (at least 15% cells with ≥5 copy numbers). Patients with MET de novo amplification received crizotinib, patients with concomitant MET acquired amplification and EGFR mutation received combined therapy of EGFR-tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, icotinib)and crizotinib. All enrolled subjects received tumor measurement according to RECIST1.1

Results:
The frequency of MET de novo amplification was 54.5%(6/11), and that of concomitant MET acquired amplification and EGFR mutation was 45.5%(5/11) respectively. 4 of 6 patients with MET de novo amplification received crizotinib, 2 patients had partial response (PR), 1 patient had stable disease (SD), 1 patient died due to heart disease. Response rate (RR) of crizotinib was 50%(2/4). Encouraging response was observed in one case, a CT scan performed 31 days after starting crizotinib revealed 42.2% decrease in tumor measurement, until now, a 7-month CT revealed 60.0% decrease. 3 of 5 patients with concomitant MET acquired amplification and EGFR mutation received the combined therapy of EGFR-TKIs and crizotinib. 1 patient achieved PR, 2 patients had SD. RR of combined therapy was 33.3%(1/3). Dramatic response was observed in one case with combined therapy, a 2-month CT revealed 31.0% decrease in tumor measurement.

Conclusion:
According to our study, patients with MET amplification benefited from crizotinib, and RR was inspiring. Patients with concomitant MET acquired amplification and EGFR mutation need combined targeted therapy.

Background:
Lung cancer is a main cause of death by cancer worldwide and adenocarcinoma the most common cell type. Most of patients present pleural effusion at an advanced stage of the disease with high morbidity and mortality, however its pathogenesis is still poorly understood and therapeutic options are limited. OBJECTIVE: Evaluate the effects of intrapleural anti-VEGF and anti-EGFR in malignant pleural effusion induced in an experimental model.

Methods:
One hundred and twenty C57BL/6 mice received intrapleural injection of 0.5x10[5] of LLC cells and were divided into four groups that received, after 3, 7, 10 and 14 days, anti-VEGF, anti-EGFR, anti-VEGF+anti-EGFR or PBS (control) intrapleurally. Ten animals for each group were followed until death to evaluate the survival curve. Eighty animals were euthanized after 7, 10, 14 or 21 days after LLC injection and had weight (g), mobility (score 0-3), pleural fluid volume evaluated. Presence of tumor in pleura and pericardium, inflammatory cells in lung parenchyma, histological changes in kidney, liver and spleen and tumor apoptosis (TUNEL) and proliferation (PCNA) were evaluated by score (0-4). Statistical analysis: One Way ANOVA, Kaplan–Meier curve, p<0.05.

Results:
In the survival analysis, pleural carcinomatosis was lethal showed maximum survival of 25 days without statistical differences among groups (p=0.739). Reduction of body weight mice was observed in all groups after 21 days (p<0.05). However, the animals mobility was better in the groups that received anti-EGFR (p=0.026). The fluid volume was higher in all control groups no matter the study time (p=0.010). Tumor implants in the pleura were more evident in control groups compared to treated groups after 14 days (p=0.001). Neoplastic infiltration of lung parenchyma was observed only in a few animals. However, lung parenchymal inflammation was minimal in all groups. Histological evaluation of pericardium and heart muscle showed tumor implants mainly in the 21-day in the control group. In liver and kidney steatosis were observed after 14 days in control group (p<0.001). Hyperplasia of the white pulp of the spleen was observed at all evaluation time points with greater evidence at 21-day in the control group (p<0.001). High scores of apoptosis and lower scores of tumor proliferation were observed in the groups that received treatment with anti-EGFR and anti-VEGF+antiEGFR.

Conclusion:
In this experimental model, the target therapies reduced significantly the pleural fluid volume, morbidity and histological parameters mainly in the therapies with EGFR, although its action did not increased the survival of the animals.

Background:
Non-small cell lung cancer (NSCLC) is a leading cause of cancer mortality globally, having a 5 year survival rate of less than 15%. PI3K-mTOR signalling has been implicated in various hallmarks of cancer and this pathway is often dysregulated in NSCLC. Efforts to therapeutically target the PI3K-mTOR pathway have been hindered by emerging drug resistance. In this study, mechanisms of drug resistance were investigated in a H1975 cell line model of acquired resistance, following chronic exposure to a phase II PI3K-mTOR inhibitor (GDC-0980), Additionally, short term exposure of BEZ235 (another phase II PI3K-mTOR inhibitor) and IBL-301 (a novel PIM/PI3K/mTOR inhibitor) were investigated for effects on cell viability/proliferation and downstream signalling pathways.

Methods:
Alterations to the mRNA expression profile of GDC-0980 acquired resistant H1975 cells versus matched parent cells were examined using an 84-gene IL-6/STAT3 signalling-specific profiler array. Subsequently, selected genes were validated by qPCR. The effectiveness of BEZ235 and IBL-301 on cell viability of two lung cancer cell lines (H1975 and H1838) following 72 hour treatment were investigated by Cell Titre Blue. pAkt and p4E-BP1 expression were examined by Western blot analyses following treatment with BEZ235 and IBL-301 at 3, 6 and 24 hours.

Results:
Thirty candidate gene alterations were identified from the array profile and six genes were chosen for validation by qPCR (n=3). The pro-proliferative and pro-metabolic regulator mTOR and the anti-apoptotic protein BCL-2 were increased in GDC-0980 resistant cells (p<0.05 and p<0.001). Similarly, TNF-α and its receptor co-stimulatory molecule CD40 were increased (p<0.05 and p<0.01). Furthermore, the cell cycle inhibitor, CDKN1, and JAK-signalling blocker, SOCS1 were downregulated (both p<0.01) in GDC-0980 resistant cells. BEZ235 and IBL-301 had a dose-dependent effect on the viability of NSCLC cell lines with respective IC~50~ values of 9.42nM and 136.55nM in H1975 cells and 103.35nM and 159.27nM in H1838 cells. Treatments of 250nM BEZ235 or IBL-301 inhibited pAKt at all time points in the lung cancer cell lines. BEZ235 blocked translation repressor protein (p4E-BP1) across all 3 cell lines and time points while IBL-301 treatment resulted in a reduction in p4E-BP1 at 24 hours.

Conclusion:
This study highlights a number of genes involved in IL-6/STAT3 signalling that may contribute to PI3K-mTOR inhibitor resistance. BEZ235 and IBL-301 both decrease cell viability and inhibit PI3K pathway signalling and cap-dependent translation in NSCLC cell lines that warrant further investigation.

Background:
Hyaluronan (HA) is a megadalton polysaccharide found in the tumor microenvironment (TME). HA accumulation in the TME increases tumor interstitial pressure, which promotes vascular collapse and limits access of chemotherapy and immune cells to tumor sites. In animal models, HA-High tumors exhibit increased growth and metastasis, treatment resistance, and reduced survival. PEGPH20 is a pegylated recombinant human hyaluronidase that enzymatically degrades tumor HA. Pembrolizumab (PEM) is a humanized monoclonal antibody targeting PD-1 and demonstrating tolerability and activity in patients with non-small cell lung cancer (NSCLC). This study evaluates the safety and activity of PEGPH20 plus PEM in patients with HA-High tumors.

Methods:
This is a Phase 1b study comprising a dose escalation portion (up to 30 patients without regard to HA status) followed by a cohort expansion portion in up to 51 patients with HA-High tumors, determined using a companion diagnostic assay developed in collaboration with Ventana Medical Systems. Eligible patients are ≥18 years, ECOG PS 0-1, with either relapsed/refractory stage IIIB/IV NSCLC who failed ≥1 previous platinum-based chemotherapy regimen or relapsed/refractory locally advanced or metastatic gastric adenocarcinoma who failed ≥1 previous chemotherapy regimen. Patients with NSCLC known to be epidermal growth factor receptor (EGFR)- or anaplastic lymphoma kinase (ALK)-positive must have received an EGFR inhibitor or ALK inhibitor, respectively. PEGPH20 (1.6, 2.2, 2.6, 3.0, 4.0 μg/kg) is administered intravenously (IV) over 10 minutes on days 1, 8, and 15 of each 21-day cycle followed by PEM 2 mg/kg IV on day 1, 4 to 6 hours after PEGPH20 is completed. Piroxicam will be given prophylactically for possible musculoskeletal events. Prophylactic proton pump inhibitors will be given to all patients. The primary endpoint for the dose escalation portion is the recommended Phase 2 dose for PEGPH20 in combination with PEM. In the cohort expansion portion, the primary endpoint is objective response rate per RECIST v1.1. Secondary endpoints are duration of response, disease control rate, progression-free survival per RECIST and immune-related response criteria, pharmacokinetics, and adverse events. Exploratory endpoints in patients with HA-High NSCLC include HA levels in plasma and tumor tissue and imaging parameters of tumor blood flow (dynamic contrast-enhanced magnetic resonance imaging [DCE-MRI]) and tumor metabolic activity (positron emission tomography/computed tomography [PET/CT] scans). ClinicalTrials.gov Identifier: NCT02563548.

Results:
Section not applicable

Conclusion:
Section not applicable

Background:
Cancer stem cells (CSCs) represent a small fraction of stem-like cells in the cancer that are characterized by their ability for self-renewal, proliferation, resistance to chemotherapy and radiation therapy, multipotent capability, and expression of stem cell markers. CSCs play essential role in cancer progression, chemoresistance, recurrence, and metastasis. Apatinib is a new small molecular VEGFR2 inhibitor which has been approved for the treatment of advanced or metastatic, chemo-refractory gastric cancer patients in China, and a phase II study evaluating its efficacy in patients with advanced NSCLC after second-line chemotherapy is currently ongoing. To date, however, no information is available regarding the action of Apatinib on CSCs. Therefore, the present study aimed to investigate the inhibitory effects of Aptinib on lung CSCs.

Methods:
Turmorsphere formation assay via serum-free medium (SFM) culturing was utilized to isolate and enrich lung CSCs from human lung cancer cell lines A549 and H1299. Protein and mRNA expression of lung CSCs markers, including CD133, CD44, ALDH1, Nanog, Sox2 and Oct4, was determined by Western blotting and qRT-PCR in sphere-forming A549 and H1299 cells. The number of CD133[+] cells was measured by Flow cytometry assay. Following the treatment of sphere-forming A549 and H1299 cells with Apatinib at various concentrations (0-10 µM), the inhibitory effects of Apatinib on lung CSCs were determined by tumorsphere formation, CSCs markers’ expression, number of CD133[+] cells, cell proliferation and apoptosis, activation of CSCs regulating signal pathways including Wnt/β-catenin and Sonic Hedgehog pathways, as well as the expression of VEGFR2 and ABC drug resistance proteins.

Results:
We revealed that sphere-forming A549 and H1299 cells in SFM culturing exhibited lung CSCs properties. Apatinib suppressed the sphere formation capacity of these cells in a concentration-dependent manner. The expression levels of lung CSCs markers and the number of CD133[+] cells were significantly downregulated by Apatinib. Our results further showed that Apatinib significantly inhibited the activation of Wnt/β-catenin and Sonic Hedgehog pathways, inhibited cell proliferation, and induced apoptosis in sphere-forming cells. Moreover, we demonstrated that Apatinib markedly reduced the expression levels of VEGFR2, as well as drug resistance proteins p-gp and ABCG2.

Conclusion:
Taken together, these data suggest for the first time the inhibitory effects of Apatinib on lung CSCs. Findings from this study could provide an important molecular basis for the application of Apatinib in chemo-refractory lung cancer patients.

Background:
Despite the development of a number of new targeted therapies, lung cancer remains the leading cause of cancer-related death worldwide. The use of oncolytic herpes simplex virus type 1 (HSV-1) has been shown to be an effective therapeutic approach for a variety of cancer in preclinical models. A third generation oncolytic HSV-1, G47Δ, is currently used in multiple clinical trials in Japan.

Methods:
In this study, we evaluated the efficacy and safety of G47Δ when used in combination with erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor, in lung cancer xenograft models in mice. Human lung cancer cell line A549 subcutaneous tumor-bearing mice were treated with G47Δ and erlotinib, each alone or in combination, and effects on tumor volume were determined. The toxicity was evaluated by body weight changes.

Results:
In this subcutaneous tumor model, combination therapy resulted in the inhibition of tumor growth without toxicity to a greater extent than that using each agent alone.

Conclusion:
These findings suggest that combination therapy of G47Δ and erlotinib may be a new treatment strategy against human lung cancer.

Background:
The benefit of the continuation of bevacizumab (BEV) beyond over progressive disease (PD) in patients with non-small cell lung cancer (NSCLC) has not been clarified yet. We present our experience of chemotherapy with BEV continuation beyond over PD in patients with recurrent NSCLC after surgery.

Methods:
They were consisted of 19 patients. There were 10 males and 9 females, and their age at surgery was 69±10 (41-85) years old. Lobectomy was done in 18 patients, and segmentectomy in 1. Pathological stage was IA in 5, IB in 3, IIB in 3, IIIA in 5, IIIB in 1, and IV in 2. Recurrence was observed at 630±460 days after surgery. Sixteen patients among them had been received some chemotherapy protocols before usage of BEV for 507±448 days. Performance status before treatment was grade 0 in 11 patients, 1 in 7, and 3 in 1. Chemotherapy was performed intending to continue BEV beyond over PD in these patients.

Results:
The average number of protocols with BEV was 3±1 (1-5). BEV was used for 1734±413 days. Side effects (≥ grade 2) due to BEV were hypertension in 6 patients, proteinuria in 4, and hemoptysis in 1. Seven patients were died of cancer, and 1 of COPD worsening. Five-year survival rate after surgery, after recurrence, and after initiation of BEV was 81.2%, 45.0%, and 31.2%, and median survival time was 2384 days, 1661 days, and 1105 days, respectively.

Conclusion:
The majority of patients with operable NSCLC have good performance status. Moreover, we can detect their recurrence in the early periods at most before the symptoms appear, because of the regular examinations. Therefore, these patients are at an advantage that they can receive more chemotherapy protocols. In these selected patients, their prognosis may be prolonged by the continuation of BEV beyond over PD.

Background:
S-1 plus cisplatin is a standard chemotherapy regimen for advanced non-small cell lung cancer (NSCLC) (Ann. Oncol. 2015; 26:1401-8). The addition of bevacizumab significantly improved overall survival (OS) in patients with advanced non-squamous (non-SQ) NSCLC who received carboplatin plus paclitaxel but failed to show an OS advantage in patients who received cisplatin plus gemcitabine. Few studies of bevacizumab have evaluated quality of life (QOL) in patients with non-SQ NSCLC.

Methods:
Chemotherapy-naïve patients with stage IIIB, IV, or recurrent non-SQ NSCLC, Eastern Cooperative Oncology Group (ECOG) performance status (PS) 0–1, age 20–74 years, and measurable lesions were treated with a 3-week cycle of S-1 40 mg/m[2] twice a day on days 1–14, cisplatin 60 mg/m[2] on day 8, and bevacizumab 15 mg/kg on day 8, for 4–6 cycles. Patients without progressive disease received maintenance bevacizumab 15 mg/kg on day 1 with a 3-week cycle and S-1 40 mg/m[2] twice a day every other day. The primary endpoint was progression-free survival (PFS). Secondary endpoints were objective response rate (ORR), OS, toxicity profile, and QOL.

Results:
From June 2013 to January 2015, 39 evaluable patients were enrolled from 8 institutions: 10 women and 29 men; median age 65 (range, 38–74) years; epidermal growth factor receptor positive/anaplastic lymphoma kinase positive: two patients/two patients; performance status 0/1: 22/17; stage IIIB/IV/recurrence: 1/35/3; adeno/large cell/other: 35/1/3. Thirty one patients (80%) completed 4 cycles of induction chemotherapy, and 23 patients (59%) were started on maintenance chemotherapy. Median PFS, OS, and ORR were 7.3 months (95% confidence interval (CI): 5.9–8.7), 21.4 months (95% CI: 14.7–not reached), and 64%, respectively. The worst hematologic and non-hematologic adverse events were as follows (%): grade 3/4 leukopenia: 13/0; neutropenia: 18/5; thrombocytopenia: 0/0; anemia: 0/0; neutropenic fever: 3/0; and hypertension: 28/0; diarrhea: 3/0. QOL data will be presented at the meeting.

Conclusion:
S-1 plus cisplatin in combination with bevacizumab met the primary endpoint in patients with advanced non-SQ NSCLC in the present trial. Additionally, the response rate is anticipated to be high, and the regimen was well tolerated. Clinical trial information: UMIN000009476

Background:
Malignant pleural effusion(MPE) is a common complication in advanced lung cancer, with multiple symptoms and poor prognosis. Vascular endothelial growth factor (VEGF) was considered important in the formation of MPE. We aimed to investigate the efficacy of bevacizumab plus chemotherapy in the treatment of MPE due to lung adenocarcinoma.

Methods:
Data of 18 lung adenocarcinoma patients with MPE were analyzed retrospectively. All the patients were treated with bevacizumab plus chemotherapy with 3 weeks in one cycle, up to 6 cycles. Those who exhibited response or stable disease received maintenance therapy of bevacizumab till disease progression. The primary outcomes were control rate of MPE, progression free survival(PFS), pleural progression-free survival (PPFS) and changes in the lung volumn and thoracic cage. Evaluation by CT scan was done every 6 weeks.

Results:

Observation Items	Outcomes
Response in Measurable Lesions	CR	PR	SD	PD	RR
n(%)	0(0)	8(44.4)	9(50.0)	1(5.6)	8(44.4)
Response in MPE	CR	PR	NC	PD	RR
n(%)	7(38.9)	7(38.9)	3(16.7)	1(5.6)	14(77.8)
Control Rate of MPE	6weeks	12weeks	24weeks	48weeks	96weeks
n(%)	17(94.4)	16(88.9)	15(88.2)	10(66.7)	6(40.0)
MPE Conditions at the Time of Measurable Lesions PD	CR	PR	NC	PD
n(%)	5(35.7)	6(42.9)	0(0)	3(21.4)
Changes in the Lung Volume and Thoracic Cage	Lung Reexpansion ≥ 70%		Thoracic Cage Diminishment
n(%)	16(88.9)		1(5.6)
Survival	PFS		PPFS		OS
Median(day)	254		734		773

All the 18 cases were evaluable for response. Median age was 59 years(29 years-74 years). There were 14 chemotherapy-naïve patients and 4 relapsing patients. Median numbers of cycles were 7(1-42) for bevacizumab and 5(2-6) for chemotherapy. Median duration of follow-up was 596 days(76-1752 days). Control rate of MPE at 6 weeks, 12 weeks, 24 weeks, 48 weeks, 96weeks were 94.4%, 88.9%, 88.2%, 66.7%, 40.0% respectively. The response rate of MPE to combine treatment reached 77.8%. Median PPFS was significantly longer than PFS(734days vs 254 days, P=0.039). The overall survival was 773 days. 16(88.9%) patients experienced lung reexpansion after treatment. Only one(5.6%) patient suffered from thoracic cage diminishment in treatment and follow-up period. Side effects of bevacizumab-based regimens included myelosuppression, digestive symptoms, bleeding, hypertension, proteinuria, etc. Most of them were grade 1-2.

Conclusion:
Bevacizumab combined with chemotherapy demonstrated efficacious, persistent and safety in controlling MPE caused by lung adenocarcinoma. It was suggested that lung adenocarcinoma patients complicated with MPE are more likely to get benefit from bevacizumab-based chemotherapy.

Background:
RET-rearrangement in unselected non-small cell lung cancer (NSCLC) is expected in 1-2%, most common in adenocarcinomas, young, never or former light smoker. Multikinase Inhibitors such as cabozantinib or vandetanib have shown acitivity against RET-rearranged lung cancer in vitro and in vivo. ORR is about 40% and median duration of response about 8 month with cabozantinib. Progression after RET-inhibition warrants further strategies. 2nd line RET-inhibition might overcome resistance.

Methods:
We report about a 77 year old female patient with primary diagnosis of adenocarcinoma of the lung with malignant pleural effusion, stage IV, in 08/13.

Results:
EFGR-mutation, ALK- and ROS1-translocation were negative at initial diagnosis. 1[st] line therapy with carboplatin and paclitaxel resulted in stable disease and clinical deterioration. A VATS was performed with partial resolution of pleural effusion. 2[nd] line therapy with pemetrexed for 16 months resulted in a clinical benefit (cough and dyspnoe, ECOG raised from 2 to 0). In 07/15 CT scan revealed a dissiminated progression with multiple intrapulmonary nodules, clinically corresponding with cough and dyspnoe again. Molecular diagnostic for cMET, BRAF, KRAS, HER2, PD-L1 revealed no new targets, but a positive RET-FISH testing from initial biopsy. From 10/15 to 03/16 the patient had a good clinical benefit from cabozantinib (multikinase inhibition, including RET). Clinical symptoms resolved in 10 days and ECOG raised from 1 to 0. In 03/16 new hepatic and lymphonodular lesions occurred and the patient suffered again from former clincical symptoms. One cycle gemcitabine was without any benefit. In 04/16 we started with docetaxel 35mg/m[2] on day 1 and 8, q3w, combined with nintedanib 200mg twice daily (except day 1 and 8). Again, the patient had an immediate clinical benefit (within 1 week; cough and dyspnoe) and the ECOG raised from 1 to 0. FISH analysis from initial biopsy revealed a non-KIF5B-RET-fusion. This type might show a substatial benefit in the preliminary literature.

Conclusion:
Conclusion: Nintedanib (plus docetaxel) in a patient with PD on cabozantinib resulted in good clinical response in a patient with a non-KIF5B-RET-mutation. This case illustrates that treatment with a 'second-line' TKI (such as nintedanib) can be effective in RET-rearranged NSCLC.

Background:
Lung cancer represents the leading cause of cancer-related deaths worldwide and novel therapeutic approaches targeting crucial pathways are urgently needed to improve its treatment. Inflammation plays a critical role in multistage tumor development and increased evidence has supported the involvement of cyclooxygenase-2 expression in carcinogenesis. We investigated the potential use of COX-2 inhibitors in cancer proliferation and apoptosis.

Methods:
Celecoxib, rofecoxib, etoricoxib, meloxicam, ibufrofen and indomethacin are the COX-2 inhibitors included in this study. Docetaxel and Cisplatin are the chemotherapeutic agents that we combined with COX-2 inhibitors. Lung cancer cell lines (NCI-H1048-Small cell lung cancer, A549- Non-small cell lung cancer) were purchased from ATCC LGC Standards. At indicated time-point, following 24h and 48h incubation, cell viability and apoptosis were measured with Annexin V staining by flow cytometry. Statistical analysis was performed by GraphPad Prism (version 6).

Results:
In Small cell lung cancer cells, following 24h incubation, combinations of docetaxel and meloxicam, docetaxel and ibuprofen, docetaxel and indomethacin, showed increased apoptosis when compared to docetaxel alone (p<0.0001). In Non-small cell lung cancer cells, the 24h incubation was not enough to induce satisfactory apoptosis, but following 48h incubation, docetaxel plus indomethacin showed more cytotoxicity when compared to docetaxel alone (p<0.0001). In addition, the combination of cisplatin plus indomethacin was the only combination to be found with higher cytotoxicity when compared to cisplatin alone after 48h treatment (p<0.0001).

Conclusion:
Depending on the drug, the synergistic effect of COX-2 inhibitors plus chemotherapeutic agents has been demonstrated in lung cancer. Our suggestion is that COX-2 inhibitors could be used as additive and maintenance treatment in combination to antineoplastic agents in lung cancer patients.

Background:
Lung cancer is often insidious disease. It usually produces only a few symptoms until the disease is advanced. At initial diagnosis 20% of patients have localized disease, 25% of patients have regional metastasis and 55% of patients have distant spread of disease. Metastasis is a process by which a small number of cancer cells undergo numerous alterations, which enables them to form secondary tumors at another and often multiple sites in the host. Recently, studies have suggested that cancer stem cells are the originators of metastasis. Cancer stem cells are small populations of slowly dividing, tretment – resistant , undifferentiated cancer cells that are being discovered in a different cancers. Metformin has proved to be effective in the treatment of glioblastomas and neuroblastomas, in vitro, by targeting their cancer stem cell population. Recently, studies have shown that metformin use is not associated with a decreased risk of lung cancer in patients with type 2 diabetes, but it has been suggested that metformin use is associated with improved survival among patients with stage IV NSCLC patients.

Methods:
The aim of our study was to compare incidence of metastasis in lung cancer patients (NSCLC and SCLC) that were treated with metformin and patients with lung cancer that were not treated with metformin. It is a retrospective analysis of lung cancer patients diagnosed at our department between January 1, 2012 and December 31 2013. and data were collected from our computerized base.

Results:
During the two-year period in our department there were 335 newly diagnosed lung cancer patients. Among them there were 25 (7%) patients with diabetes mellitus that were on therapy with metformin prior to lung cancer diagnosis for at least six months. We have proved significant difference between two groups in the incidence of patients with distant spread of disease (stage IV) at the time of diagnosis. Metformin group had a lower inicidence of stage IV at the time of diagnosis ( 44% vs 64%; x2 =4.14; p=0.041). The results did neither revealed a significant difference in total number of patients with distant spread nor in the type of metastasis.

Conclusion:
We have shown that patients that were treated with metformin had lower incidenece of distant metastases at the time of diagnosis. Further research should evaluate biologic mechanisms and test the effect of metformin on inhibiting the cancer spread in prospective clinical trials.

Background:
Bevacizumab has proven efficacy in extending OS and PFS as first-line treatment for advanced nonsquamous NSCLC.

Methods:
There were three advanced NSCLC patients received maintenance with bevacizumab for more than 3 years in our hospital, and here we share the data of these patients.

Results:
All three patients with advanced-stage lung adenocarcinoma received bevacizumab (15mg/kg) plus paclitaxel and carboplatin for 6 cycles as first-line treatment and bevacizumab maintenance. In the maintenance, proteinuria occurred in all three patients after 6 months of treatment or longer and caused cessation of bevacizumab in two patients. It’s noteworthy that two patients presented spleen changes after long-term maintenance. In patient 01, proteinuria occurred after 8 cycles of bevacizumab, caused cessation of 7 doses, and lasted till one year after discontinuation of bevacizumab. In patient 02, splenomegaly was found after 44 cycles of bevacizumab, caused treatment discontinuation, and reversed after 6 months of discontinuation. In patient 03, proteinuria occurred after 29 cycles of bevacizumab and caused cessation of 5 doses of bevacizumab. Besides, increased serum creatinine and blood urea nitrogen were found after 18 months of protienuria, and CT scan indicated wedge-shaped defects in spleen after 55 cycles. After disease progression or discontinuation of bevacizumab, two patients were confirmed harboring EGFR mutations and received EGFR TKI treatment. The other patient, EGFR mutation and ALK-arrangement negative, .received chemotherapy after disease progression. Figure 1



Conclusion:
These patients received bevacizumab maintenance for more than 3 years were all enrolled in clinical trials, and the long-term maintenance brought them adverse effects as well as clinical benefit. In the real world，the cycles of maintenance and the best total dosage of bevacizumab for NSCLC remain uncertain. Is it true that the longer the treatment lasts, the more benefit the patients get with the maintenance treatment of bevacizumab ?

Background:
The acquired resistance of EGFR-TKIs in non-small cell lung cancer (NSCLC) is a big challenge in the targeted therapy. It is necessary to investigate whether CDK4/6 inhibitor PD 0332991 could contribute to reverse the drug resistance of EGFR TKI resistant human lung cancer cell in vitro and in vivo and to explore the underlying mechanisms.

Methods:
Cell viability, proliferation, cell cycle and apoptosis were measured by MTT assay, EDU assay, cell cycle and apoptosis assay, respectively. The underlying mechanisms were assessed by real-time PCR, western-blot and microarray analysis. Tumor xenograft animal study was performed to verify the effect of PD 0332991 in vivo. Lung adenocarcinoma patients with acquired resistance to EGFR-TKIs were given a tentative treatment of PD 0332991.

Results:
Our study showed that PD 0332991 could potentiate significantly the gefitinib induced growth inhibition of both EGFR-TKI sensitive PC-9 and EGFR-TKI resistant PC-9/AB2 cells, through down-regulating the proliferation, inducing cell apoptosis and G0/G1 cell cycle arrest. In the mice experiments in vivo, we found the mice treated by PD 0332991 and gefitinib showed a fastest tumor regression, and a most delayed relapse pattern. The tumor from the mice treated by the combination showed a significantly down-regulated proliferation, an up-regulated apoptosis and a less angiogenesis confirmed by Ki67, TUNEL and CD34 staining, respectively. In addition, we reported an adenocarcinoma patient with gefitinib resistance had a remarkable clinical remission after administrated with PD 0332991.

Conclusion:
Our research showed, for the first time, that PD 0332991 could contribute EGFR-TKI resistant NSCLC cells to overcome the acquired resistance in vivo and in vitro. This might provide a novel treatment strategy for the patients with EGFR-TKI resistance.

Background:
The highly selective multi-targeted agent sorafenib is an inhibitor of a number of intracellular signaling kinases with anti-proliferative, anti-angiogenic and pro-apoptotic effects in various types of tumors, including human non-small cell lung cancer (NSCLC). Betulin displays a broad spectrum of biological and pharmacological properties including the anticancer and chemopreventive activity. Combination of drugs with different targets is a logical approach to overcome multilevel cross-stimulation among key pathways in NSCLC progression.

Methods:
The NSCLC cell lines A549, H358, A427, HCC827, H1703, normal lung microvascular endothelial cells HLMEC and normal human PBMC were treated with sorafenib and betulinic acid alone and in combination. We examined the effect of different combined treatment on viability (MTS test), proliferation and apoptotic susceptibility analyzed by flow cytometry, alterations in signaling pathways by western blotting and as well colony-forming ability.

Results:
The combination of sorafenib with betulinic acid had strong effect on induction of apoptosis of different non-small cell lung cancer cell lines but not effect on normal cells. Combination treatment inhibited phosphorylation of ERK, AKT and mTOR in the A549 cells line and in low concentrations significantly reduced the colony-forming ability of A549, H358 and A427 cells as compared to either compound alone.

Conclusion:
In this study we show that sorafenib significantly suppressed the proliferation of lung cancer cells and treatment with the combination of low concentrations of sorafenib with betulinic acid exhibited ability to induce a high level of cell death in some non-small cell lung cancer cell lines.

Background:
PIK3CA mutation represents a clinical subset of diverse carcinomas. We explored the status of PIK3CA mutation and evaluated its genetic variability, treatment and prognosis in patients with lung adenocarcinoma.

Methods:
A total of 810 patients with completely resected lung adenocarcinoma were recruited between 2008 and 2013. The status of PIK3CA mutation and other three genes, i.e. EGFR mutation, KRAS mutation & ALK fusion, was examined by reverse transcription-polymerase chain reaction (RT-PCR). Survival curves were plotted with the Kaplan-Meier method and log-rank for comparison. Cox proportional hazard model was performed for multivariate analysis.

Results:
Among the 810 patients, 23 cases of PIK3CA mutation were identified with a frequency of 2.8%. There were 14 males and 9 females with a median age of 61 years. Seventeen tumors revealed concurrent gene abnormalities of EGFR mutation (n=12), KRAS mutation (n=3) and ALK fusion (n=2). Seven patients with EGFR & PIK3CA mutations recurred and the dosing of EGFR-TKIs yielded a median progression free-survival of 6.0 months. Among 4 eviromous-treated patients, stable disease was obtained in three patients with a median PFS of 3.5 months. Patients with and without PIK3CA mutation had different overall survivals (32.2 vs. 49.6 months, P=0.003). Multivariate analysis revealed that PIK3CA mutation was an independent predictor of poor overall survival (HR=2.37, P=0.017).

Conclusion:
The frequency of PIK3CA mutation was around 2.8% in Chinese patients of lung adenocarcinoma. PIK3CA mutation was associated with reduced PFS of EGFR-TKIs treatment and shorter overall survival.

Background:
Activation of the fibroblast growth factor receptor (FGFR) family through mutation, amplification , C-terminal truncation, and 3’ fusion has been described in multiple cancer types, and FGFR inhibitors are currently being evaluated in the clinic. Though FGFR1 amplification has been defined in several datasets, other FGFR alterations in NSCLC are not well defined.

Methods:
Hybrid-capture based comprehensive genomic profiling (CGP) was performed on 13,898 consecutive FFPE lung cancer specimens (adeno 71%; squamous 12%) to a mean coverage depth of >650X for 236 or 315 cancer-related genes plus 47 introns from 19 genes frequently rearranged in cancer.

Results:
CGP of 13,898 NSCLCs led to the identification of 53 cases (0.4%) with FGFR1-4 rearrangements, truncations or splice site mutations resulting in an intact kinase domain (KD). The median age was 63 years old (range 36-83 years). Patients with these alterations were 60% (26/53) male, and 72% (31/43) with available data were stage IV. 26 patients (49%) had adenocarcinomas and 18 patients (34%) had squamous histology. FGFR alterations identified included 19 FGFR3-TACC3 fusions, one FGFR2-KIAA1598 fusion, and 7 novel fusions involving FGFR2, FGFR3 or FGFR4. We also identified 16 cases with C-terminal truncations resulting in loss of exon 18, but retention of the KD, 9 cases with mutations predicted to result in alternative splicing in the FGFR extracellular domain (exons 3 or 4), and one case with deletion of exons 3-6. Genomic analysis revealed concurrent FGFR amplification in 13% (7/53) of cases. Co-occurring alterations were observed in known drivers including EGFR, ERBB2, MET, and BRAF in 15% of (8/53) cases, and KRAS mutation in an additional 15% (8/53) of cases. The average tumor mutation burden in cases with these FGFR alterations was relatively high (mean 16.9 mutations/Mb, median 10.1 mutations/Mb, range 0.9-86.5 mutations/Mb) as compared to a mean of 9.2 mutations/Mb in NSCLCs. One patient with a novel FGFR2-LZTFL1 fusion had a partial response to the pan-FGFR inhibitor JNJ-42756493 and remained progression free for 11 months.

Conclusion:
Diverse FGFR alterations were detected using CGP in 0.4% of NSCLCs. Of the 53 cases identified, 37 (70%) were negative for other known driver alterations. In cases with co-occurring drivers, including two with EGFR exon 19 deletion, the possibility of an FGFR fusion arising in the setting of acquired resistance will be evaluated. One patient with a novel FGFR2 fusion had clinical benefit from an investigational FGFR inhibitor, suggesting that these alterations may predict response to targeted therapies.

Background:
Apatinib is a tyrosine kinase inhibitor which selectively inhibits VEGFR-2 and also represents mild inhibition to PDGFR, c-Kit and c-src. It is an orally bioavailable, small molecule agent which is thought to inhibit angiogenesis and tumor cell proliferation. Previous clinical trials have demonstrated its obvious antitumor activity in various cancer type. Thus we designed this phase II, open-label, single-armed, prospective study (NCT02515435) to investigate the efficacy and safety of apatinib for heavily treated, advanced non-squamous NSCLC patients who are not applicable to current standardized therapy or other clinical trials.

Methods:
We prospectively enrolled 40 patients with previously heavily treated advanced non-squamous NSCLC. All patients received apatinib with a dose of 500mg, q.d., p.o. Efficiency was evaluated initially 4 weeks later and then every 8 weeks until disease progression, death, or unacceptable toxicity. The primary end point of this study was overall response rate (ORR). The secondary end points were progression-free survival (PFS), overall survival (OS).

Results:
Forty patients were enrolled in the study with a median age of 61. 15% of patients received apatinib as second-line therapy, 40% of patients received apatinib as third-line therapy, and 60% as forth-line to twelfth-line therapy. Nine patients were found with activated EGFR mutation. Among all the enrolled patients, 38 patients had clinical evaluation and the other 2 received treatment of apatinib less than one month. Within 33 patients who had available image efficiency, 6 were identified as PR,17 SD and 10 PD, no CR was observed. The ORR was 18.18 %, the DCR was 69.69 %. The ORR for patients with EGFR mutation positive and negative were 25% and 16% separately. The median PFS was 3.22 months (95% CI, 2.20-4.17 months). Among them, 6 patients received the treatment of apatinib more than five months. The 6-month OS rate was 76.98% (95% CI, 61.68%-92.27%), the 12-month OS rate was 57.48% (95% CI, 28.75%-86.20%). Common treatment-related adverse events were proteinuria (25%), hypertension (17.5%), and hand-foot-skin reaction (HFSR)(27.5%). Severe adverse events included grade 3 hypertension (5%), HFSR (5%), and thrombocytopenia (5%), no grade 4 or 5 adverse events were observed. No un-expected adverse events were found.

Conclusion:
Apatinib as a monotherapy had a promising overall response together with an acceptable side effect in patients with previously heavily treated advanced non-squamous NSCLC. A phase III study comparing apatinib monotherapy with placebo as 3rd/4th setting in advanced non-squamous NSCLC is ongoing to further evaluated the efficacy of apatinib (NCT02332512).

Background:
CheckMate 153 (NCT02066636) is an ongoing, predominantly community-based, phase 3B/4 safety study of nivolumab in patients with previously treated metastatic NSCLC in the US/Canada. Here we report safety, efficacy, and patient-reported outcome (PRO) data for subgroups of patients aged ≥70 years or with a poor baseline ECOG PS (PS2).

Methods:
Patients were enrolled in four subgroups based on histology and prior regimen number; one subgroup enrolled patients with squamous (SQ) or non-SQ NSCLC, PS2, and ≥1 prior therapies. Data on elderly patients were pooled across subgroups. The primary objective was assessment of high-grade (grade 3–4 and 5) select (those with a potential immunologic cause) treatment-related AE (TRAE) incidences. Exploratory endpoints included efficacy, biomarkers, pharmacokinetics, and PROs.

Results:
Of 1,308 patients, 520 (40%) were aged ≥70 years and 108 (8%) had PS2. TRAE incidences for the age and PS subgroups were comparable with those for the overall population (table), as were select TRAE incidences for the subgroups. Estimated 6-month OS was lower with PS2 than PS0‒1, but similar between age subgroups and the overall population (table). Early PRO data revealed significant improvements overall in both age subgroups using LCSS and EQ-5D VAS, with younger patients showing greater improvement on some scales. Patients with SQ disease and PS2 generally reported stable quality-of-life/symptom control, whereas patients with non-SQ disease had statistically significant improvements on most scales. Updated data, including 1-year OS, will be presented.

Conclusion:
In this large study of advanced, previously treated, predominantly community-based patients with NSCLC, the nivolumab safety profile for age and PS subgroups was comparable with those for the overall population and from prior nivolumab NSCLC studies. OS was similar in younger and older patients, but lower in PS2 patients at early time points. Nivolumab appears to have similar risks/benefits in older and poorer PS patients as in the general population. Figure 1

Background:
EC1456 is composed of folic acid conjugated through a releasable linker system to a potent microtubule inhibitor, tubulysin B hydrazide. EC1456 targets folate receptor (FR)-expressing cancer cells, which occur in approximately 60% of NSCLC cases and 14% of SCLC.

Methods:
The objectives of the ongoing Phase 1 are to determine the safety, PK, and optimal dosing schedule of EC1456 in advanced cancer pts. FR expression (not required for enrollment) is characterized in all pts using [99m]Tc-etarfolatide.

Results:
63 pts were dosed weekly (QW) or twice-weekly (BIW), for 2 consecutive weeks of a 3-week cycle. 8 NSCLC and 3 SCLC pts received doses ranging from 1.0-12.5 mg/m[2]. Toxicities are primarily Grade (Gr) 1 and 2. Common adverse events (AE) are GI, fatigue, and metabolic changes. Gr 3 infusion reaction (4.5 mg/m[2]) and Gr 3 headache (10.0 mg/m[2]) were seen in the QW cohort. The safety profile in lung cancer pts was similar to the overall population.

	BIW (N=32)	QW (N=31)	BIW Lung (N=4)	QW Lung (N=7)
	All	Drug Related	All	Drug Related	All	Drug Related	All	Drug Related
≥ 1 AE	32 (100%)	25 (78%)	29 (94%)	23 (74%)	4 (100%)	4 (100%)	7 (100%)	4 (57%)
≥ 1 grade 3 or 4 AE	19 (59%)	6 (19%)	14 (45%)	4 (13%)	2 (50%)	0 (0%)	1 (14%)	0 (0%)
≥ 1 serious AE	12 (38%)	2 (6%)	14 (45%)	5 (16%)	1 (25%)	0 (0%)	2 (29%)	0 (0%)
Serious drug related AEs	Constipation (2/63 pts); Abd pain, Anemia, Headache, Infusion reactions, and SVT(1/63 pts each)

Response and durability of response is demonstrated in the figure: Figure 1



Conclusion:
EC1456 is well tolerated, with early indications of efficacy suggested by durable stable disease, and responses in this refractory population. Updated safety, PK, and efficacy data will be presented at the meeting.

Background:
In randomized trials of nivolumab in NSCLC, less than 10% of pts were ≥75 years old, and all had Eastern Cooperative Oncology Group Performance Status (ECOG PS) of 0-1. The effectiveness of nivolumab in elderly pts with NSCLC treated in routine practice has not been previously described.

Methods:
We conducted a retrospective cohort study of pts with advanced NSCLC treated with nivolumab outside of clinical trials at the University of Pennsylvania between March 2015 and March 2016. Logistic regression and Cox proportional hazards models were used to evaluate the association of age (≥75 vs. <75 years) with overall response rate (ORR), progression-free survival (PFS), and overall survival (OS), adjusting for ECOG PS (0-1 vs. ≥2), sex, smoking history [heavy (≥10 pack-years) vs. light/never (<10 pack-years)], and number of prior systemic therapies (1 vs. ≥2).

Results:
Of 175 pts treated with nivolumab, 43 (25%) were ≥75 years old and 42 (24%) had ECOG PS ≥2. Ninety-five pts (54%) were female, 147 (84%) had heavy smoking history, and 81 (46%) had received ≥2 prior systemic therapies. ORR was 19.4%, with median PFS and OS of 2.1 and 6.5 months, respectively. Age ≥75 years was not associated with ORR (OR 1.0, 95% CI 0.4-2.5; p=0.97), PFS (HR 0.71, 95% CI 0.5-1.1; p=0.12), or OS (HR 0.8, 95% CI 0.5-1.4; p=0.4; Figure 1). ECOG PS ≥ 2 was associated with lower ORR (7.1% vs. 23.3%; OR 0.25, 95% CI 0.07 – 0.88; p=0.03), inferior PFS (median 1.8 vs. 2.3 months; HR 1.9, 95% CI 1.3 – 2.8; p=0.001), and inferior OS (median 3.6 vs. 7.8 months; HR 2.6, 95% CI 1.6 – 4.1; p<0.001). Figure 1



Conclusion:
In a large NSCLC cohort treated outside of clinical trials, elderly pts gained similar benefit from nivolumab compared to younger pts. Pts with poor performance status had inferior outcomes regardless of age.

Background:
Immune checkpoint inhibitors are associated with immune-related adverse events (irAEs). While the incidence of irAEs in routine practice and their effect on outcomes have been well characterized in melanoma, a similar analysis has not been previously reported in NSCLC pts treated with anti-programmed death 1 (PD-1) therapy.

Methods:
We conducted a retrospective cohort study of pts with advanced NSCLC who received nivolumab outside of clinical trials between March 2015 and March 2016 at the University of Pennsylvania. irAEs were graded using the Common Terminology Criteria for Adverse Events version 4.0. Data collected included demographics, timing and treatment of irAEs, and dates of disease progression and death or last follow-up. To analyze the effect of irAEs on progression-free survival (PFS) and overall survival (OS), landmark analyses were used beginning from 3 months after start of treatment. Pts who reached the PFS or OS endpoints prior to 3 months were excluded. Cox proportional hazards models were used to assess for differences in PFS and OS according to the occurrence of an irAE, adjusting for age, sex, and Eastern Cooperative Oncology Group Performance Status (ECOG PS).

Results:
175 pts received a median of 5 cycles of nivolumab (range, 1-24, IQR, 3-9). Median age was 68 years (range, 33-88, IQR, 60-74). Forty-six percent of pts were male; 5% had an ECOG PS ≥ 2. Twenty-eight pts (16%) experienced an irAE of any grade and 6 (3%) had a grade 3/4 irAE. Median time to onset of the irAE was 3 cycles (range, 1-18, IQR, 2-6). Of the pts who experienced an irAE, 14 (50%) were treated with systemic corticosteroids. The most common irAEs were hypothyroidism/hyperthyroidism (n=8), pneumonitis (n=6; three grade 4), colitis (n=4; one grade 3), dermatitis (n=4), and arthritis (n=2). Less common irAEs (n=1 each) included hepatitis (grade 4), aseptic meningitis (grade 3), immune thrombocytopenia, and severe hypoalbuminemia that improved with steroids. Overall response rate was 19.4% (34 of 175), and median PFS and OS were 2.1 and 6.5 months, respectively. After adjusting for age, sex, and ECOG PS, landmark analyses revealed no difference in PFS (HR 1.3, 95% CI 0.4-3.8, p=0.7) or OS (HR 0.9, 95% CI 0.3-2.7, p=0.9) stratified by the presence or absence of an irAE.

Conclusion:
In NSCLC pts treated with nivolumab in typical practice, irAEs of any grade were uncommon, and grade 3/4 irAEs were rare. The occurrence of irAEs was not associated with PFS or OS.

Background:
Previous analyses showed no clinically significant exposure-efficacy relationship for pembrolizumab doses of 2-10 mg/kg. Population pharmacokinetics (popPK) modeling suggested weight-based or fixed pembrolizumab doses could maintain exposures within the established safety/efficacy bounds. Fixed dose advantages include increased convenience, reduced dosing error risk, and less discarded product. Pembrolizumab 200 mg Q3W was evaluated in the KEYNOTE-024 study of pembrolizumab versus platinum-doublet chemotherapy for treatment-naive advanced NSCLC with PD-L1 TPS ≥50% (NCT02142738).

Methods:
Pembrolizumab serum concentration was quantified with an electrochemiluminescence-based immunoassay (lower limit of quantitation, 10 ng/mL). The existing 2-compartment popPK model derived from studies of weight-based pembrolizumab dosing was extended with KEYNOTE-024 concentration-time data. Correlation between pembrolizumab exposure (ie, area under the serum-concentration curve over 6 weeks [AUC~ss-6weeks~]) and efficacy was assessed.

Results:
Median (range) weight was 69.7 kg (38-110) in KEYNOTE-024 and 75 kg (35.7-210) in the existing popPK model studies. In treatment-naive advanced NSCLC, there was a flat relationship between pembrolizumab exposure and efficacy for the 200-mg fixed dose and weight-based doses (linear regression P>0.05). Observed pembrolizumab concentrations for 200 mg (median 1976 μg·d/mL, 90% CI 1124-3322) were consistent with predictions (median 1751 μg·d/mL, 90% prediction interval 955-3136) and fell within the previously observed therapeutic window for 2 and 10 mg/kg (Figure). There was considerable overlap in exposures for 2 mg/kg and 200 mg, regardless of whether weight was >90 or <90 kg for 200 mg (Figure). Figure 1



Conclusion:
Pembrolizumab exposure at 200 mg Q3W is similar to that of 2 mg/kg Q3W. Including data from patients with advanced NSCLC treated with 200 mg did not change the flat exposure-efficacy relationship. Along with the superior PFS and OS provided by pembrolizumab over platinum-doublet chemotherapy as first-line therapy for advanced NSCLC with TPS ≥50%, these data support 200 mg Q3W as an alternative to the approved 2-mg/kg Q3W dose.

Background:
In non-squamous NSCLC PD-L1 negative patients (pts), IC might increase the risk of early death compared to docetaxel in the phase III study Checkmate 057. Tumor Growth Rate (TGR) is calculated using 2 CTscans and the time interval between the 2 exams. It integrates tumor dynamics and kinetics. We hypothesized that TGR could identify a subset of pts named PPD, in which IC could accelerate tumor progression, leading to early death.

Methods:
We performed a retrospective case study of all NSCLC pts treated by IC in a single institution between Dec. 12 and Feb. 16. CT scan were centrally reviewed by a senior radiologist and assessed according to RECIST 1.1 criteria. We calculated TGR at baseline of IC (baseline CTscan (n) vs n-1 CTscan) and TGR during IC (n+2 CTscan vs n+1 CTscan). We further estimated the difference (deltaTGR) between TGR during IC and TGR at baseline. DeltaTGR>0 means IC speeds up tumor growth. PPD was defined as deltaTGR>50%, corresponding to an absolute increase in TGR greater than 50% per month. PDL1 expression was assessed with the SP142 clone.

Results:
89 pts were eligible. 58% were male, median age 60 (41-78); 15% never smokers. 62 pts had adenocarcinoma, 21 squamous and 6 other histologies. Mutational status was unknown for 14 pts; 36% wild type, 9 pts EGFRmut, 25 pts KRASmut. PDL1 expression was positive in 25 pts, unknown in 57 pts. 52 pts (58%) received nivolumab, 25 pembrolizumab and 12 atezolizumab. Treatement was received as 1-3[rd] line in 52 pts, and as ≥ 4[th] line in 37 pts. Overall, 25 pts (28%) had a response according to RECIST 1.1 criteria, 31 (35%) a stable disease. Median OS was 14.7 months. During IC, deltaTGR was <0 in 79 pts and >0 in 20 pts. Among the 20 pts with deltaTGR>0, 9 had a PPD. Characteristics (age, sex, smoking status, pathology, number of previous line, PDL1 status) of the 9 pts were not different from other pts. None of the PPD were pseudoprogression. Median OS of PPD vs others was 3.2 and 23 months, respectively. PPD was not more frequent in tumors with high baseline TGR.

Conclusion:
Our results suggest that PPD is a new subset of response criteria in which IC may increase tumor progression, leading to a poorer survival. Rapidly growing disease at study entry nor RECIST criteria could predict the occurrence of PPD.

Background:
Immunotherapy is now a standard of care in melanoma, lung cancer and is spreading across other tumours. Immune checkpoint inhibitors (ICI) are generally well tolerated but can also generate immune-related adverse effects. Since the first trials, pneumonitis has been identified as a rare but potentially life-threatening event.

Methods:
We conducted a retrospective study over a period of 5 months in centers experienced in ICI use in clinical trials, access programs or following national approval. We report the main features of possibly related pneumonitis occurring in patients treated with ICI with a particular focus on clinical presentation, radiologic patterns (with a double reviewing by radiologists and pulmonologists), pathology and therapeutic strategies.

Results:
We identified 71 patients with possibly related pneumonitis including 54 NSCLC and 13 melanoma. They mainly received PD1 inhibitors. Pneumonitis usually occurred in male, former or current smokers with a median age of 59 years. We observed grade 2/3 (n= 45, 65.2%) and grade 5 (n= 6, 8.7%) pneumonitis. The median duration time between the introduction of immunotherapy and the pneumonitis was 2.2 months [0.1-27.4]. Ground glass opacitiy on lung CT-scan were the most predominant lesion 80.9% (n=55), followed by consolidations 44.1% (n=30), reticulations 36.7% (n=25) and bronchiectasis in 20.6% (n=14). When performed, bronchoalveolar lavage (BAL) showed a T-lymphocytic alveolitis and transbronchial biopsy an inflammatory and lymphocytic infiltration. Pneumonitis treatment was steroids (86.6%) and/or antibiotics (67.6%). Immunotherapy was stopped after the pneumonitis for 65 cases (92.9%) and reintroduced for 12 (9.4%) cases. Twenty-four patients (34.3%) were dead at the last follow-up and 46 patients (65.7%) were still alive. Among the living patients, the pneumonitis outcome was a total recovery in 12 patients, improvement in 22 patients, stability in 10 patients, worsening evolution in 1 patient (1 unknown). Causality of immunotherapy was evaluated by investigators as “possible” for 34 patients (49.3%), “probable” for 17 (24.6%), “certain” for 15 (21.7%) other causes for 3 (4.3%) and 2 unknowns. Median overall survival from the onset of pneumonitis was 6 months.

Conclusion:
This serie, the largest to date, of immune-related pneumonitis demonstrates that it occurs usually during the first months and displays specific radiologic features. As there is no clearly identified risk factor, oncologists should be able to detect, diagnose (with CT-scan and bronchoscopy) and treat this adverse event. An early management is usually associated with a favourable outcome and requires a close collaboration between pulmonologists, radiologists and oncologists.

Background:
Anti-PD-1 antibodies (pembrolizumab and nivolumab) have shown improved overall survival in second-line treatment for metastatic non-small cell lung cancer (NSCLC) with durable responses. We aimed to assess the pattern of disease progression amongst patients who initially responded to anti-PD-1 agents and their subsequent management.

Methods:
We retrospectively assessed all patients who commenced single-agent anti-PD-1 antibodies between June 2012 and February 2016 at a single centre. Radiological responses were assessed by the investigator using RECIST 1.1 and irRC. Progressive disease (PD) patterns were defined as solitary, oligometastatic (2-3 lesions), generalised (>3 lesions), enlargement of existing or new lesions, visceral or non-visceral. Management and survival after progression were examined.

Results:
A total of 81 patients received single-agent pembrolizumab (N=43) or nivolumab (N=38). Of the seventeen (21.3%) patients achieving partial response, three were treatment-naïve, fifteen (88.2%) were former or current smokers, none had EGFR mutation or ALK translocation. The median number of disease sites at baseline was three, and two patients had stable brain metastases after radiotherapy at the commencement of anti-PD1 treatment. Ten (58.8%) responders developed acquired resistance, with a median time to progression of 20.2 months. Nine (90%) had solitary (N=4) or oligometastatic (N=5) progression. Five (50%) progressed only at existing sites, three (30%) developed new lesions only, and two (20%) progressed at both existing and new sites. Four (40%) progressed at non-visceral sites only, and one progressed in the brain at a previously treated site. Five (50%) patients underwent local treatment to solitary (N=2) or oligoprogressive disease (N=3) with all five achieving local control with radiotherapy. Seven(70%) continued anti-PD-1 agents beyond progression, while the three (30%) remaining patients did not receive any further therapy. With a median follow-up of 24.8 months, five (50%) of the patients had died, one from an infective exacerbation of COPD, one from type 1 respiratory failure, and three from disease progression. The median duration of treatment was 4.35 months (1.96 to 11.46) and the median overall survival after progression was 11.44 months.

Conclusion:
This study suggested that acquired resistance to anti-PD1 agents could often result in solitary or oligometastatic progression, and that CNS progression was uncommon. In a subset of patients, treatment beyond progression with or without local therapy to oligometastatic disease may provide ongoing and durable clinical benefit.

Background:
Lung cancer is the leading cause of cancer-related mortality globally. Recent trials results of checkpoint inhibitors have shown that immunotherapy represents a new standard option of care in pretreated patients compared with chemotherapy. Eficacy and toxicity data were assessed in 69 pretreated patients with metastatic NSCLC in our institution.

Methods:
A retrospective, non-interventional study was conducted. 69 patients with advanced NSCLC receiving immunotherapy after one or more prior treatment were enrolled between 2013 and 2015. Patients received PD1 and PDL1 checkpoints inhibitors as compassionate use treatment or as clinical trial therapy.

Results:
69 patients were analysed with a median age of 64y, including 82.6% males, 54.3% squamous histology and 8.7% never-smoker patients. Mutation profile was defined as negative EGFR/ALK in 95% and positive PDL1 in 30.4%. 68.3% of patients received anti-PD1 therapy vs anti-PDL1 inhibitors (31.7%) as clinical trial therapy (63.8%) vs compassionate use (36.2%). 40 patients (58%) received immnotherapy after two o more previous chemotherapy lines. With a median follow-up of 24.9 months, overall objective response rate was 5.8% with a disease control rate of 58%, with no responses seen at never smokers. Estimated 1year-PFS was 27.5%, with a median of 4.5months. There were no statistically significant differences according to histology (41.4% squamous vs 36.1% nonsquamous, P=0.14) or immunotherapy strategie (47.6% with PDL1 vs 38.6% with PDL1 inhibitors, p=0.55). Positive PDL1 was a prognostic factor for 6-month PFS in nonsquamous histology (64.3 % PDL1+ vs 18.8% PDL1-,p= 0.02, HR:0.24). OS was not reached as 37.7% of patients remain on treatment nowadays. The most common grade 1-2 adverse events were fatigue (55%) and anorexia (26%). 13 patients (17.3%) experienced grade 3 toxicity being pneumonitis the most common cause (5.8%).

Conclusion:
Our data are consistent with recent immunotherapy results, showing a clinically meaningful survival benefit vs chemotherapy with similar efficacy and toxicity between PD1 and PDL1 checkpoints inhibitors. PDL1 expression appears to be a prognostic and predictive factor, only for nonsquamous histology.

Background:
Immune check point inhibitor has become essential therapeutic option for advanced non-small cell lung cancer (NSCLC). Immune-related response in NSCLC has not been well evaluated, thus we assessed the tumor response using Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST v1.1) and immune-related response criteria (irRC) to identify atypical response in patients with advanced NSCLC treated with immunotherapeutic agents.

Methods:
Patients received immune check point inhibitors (pembrolizumab, atezolizumab, nivolumab, pembrolizumab plus tremelimumab) at Samsung Medical Center between July 2014 and October 2015. The tumor response was assessed according to both RECIST v1.1 and irRC. Pseudoprogression was defined as progressive disease at any time of assessment and not at next assessment per RECIST v1.1 or irRC.

Results:
Figure 1 Total 41 patients were analyzed, most of patients (80.5%) received anti-PD-1 agents (pembrolizumab or nivolumab) and 6 patients were treated with anti-PD-L1, atezolizumab, 2 patients received combination treatment with pembrolizumab and tremelimumab. Two patients showed pseudoprogression followed by regression per RECIST v1.1 not per irRC. 4 patients with progressive disease per RECIST v1.1 were partial response per irRC, objective response was 29.2% per RECIST v1.1 and 34.1% per irRC, respectively. There was no significant difference in response rate between two methods (p=0.923). The median duration of follow-up was 19.8 months, and the median progression-free survival of all patients was 4.5 months (95% CI 2.7-6.3)



Conclusion:
These results suggest that pseuoprogression is not frequently observed in NSCLC, and conventional RECIST v1.1 might underestimate the benefit of immune check point inhibitors. Given the small number of patients studied and short-term follow-up, further study will be warranted whether treatment with immune checkpoint inhibitor beyond RECIST progression can be benefit to patient with advanced NSCLC.

Background:
Immune check-point inhibitors have recently become a cornerstone of the management of advanced non-small cell lung cancer (NSCLC). The peculiar mechanism of action of this drug class implies the possibility to treat patients beyond progressive disease (PD) on the basis of parameters such as the observation of clinical benefit or mild progression at computed tomography scan (CT-scan); however, a guideline for managing early PD during cancer immunotherapy has not been clearly defined yet. The aim of this study is to evaluate the approaches to patients experiencing early PD during treatment with nivolumab for advanced NSCLC.

Methods:
Patients treated with nivolumab (3 mg/Kg every 14 days) for advanced NSCLC between April 2015 and May 2016 within a single-institutional translational research study conducted in the San Martino Hospital – National Institute for Cancer Research, Genoa, Italy (approved by the local ethical committee) were considered eligible if their first response assessment (after 4 cycles) was PD. The response evaluation criteria in solid tumors (RECIST) and the immune-related response criteria (irRC) were employed. Since IRC imply the confirmation of PD after 2 further cycles, a cut-off of 6 cycles was set to define the patients who continued nivolumab beyond progression.

Results:
Globally, 31 patients were eligible: median age= 69 years (50-81); males/females= 74%/26%; current or former smokers= 90%; non-squamous/squamous histology= 67%/33%; 25 patients had PD as first evaluation with both criteria, while 4 had PD only with RECIST and 2 had PD only with IRC. With RECIST, 35% of the patients received nivolumab beyond progression (median= 10 cycles) and 80% of such patients were alive at the time of the analysis; on the contrary, only 53% of the patients who discontinued nivolumab at PD were still alive at the time of the analysis. With irRC, 30% of the patients received nivolumab beyond progression (median= 10 cycles) and 75% of such patients were alive at the time of the analysis, compared to only 47% of the patients who discontinued nivolumab at PD. The decision of continuing nivolumab beyond PD was based on the reported clinical benefit (67%), on the observation of a very limited progression at the CT-scan (22%) or on discordance between response criteria (11%).

Conclusion:
Administering nivolumab beyond progression might influence the outcomes of selected patients. Additional parameters for discriminating which patients are going to benefit from nivolumab continuation need to be investigated.

Background:
Newly approved anti-PD-1/PDL-1 and anti-CTLA-4 immune checkpoint monoclonal antibodies (ICM) significantly improve overall survival in advanced or metastasized melanoma and lung cancer. Viscum album L. (VA) may improve survival and supports health related quality of life in cancer patients. The primary objective of the present study was to determine the safety profile of combinatory ICM/VA therapy in advanced or metastasized cancer.

Methods:
Safety of ICM/VA therapy was examined in an observational open phase IV study in a certified Oncology Centre. ICM or combinational ICM/VA therapies were applied in patients with progressive or metastasized cancer (non-small cell lung cancer or melanoma) in an integrative oncology setting. Toxicity rates of both therapy groups were compared. Evaluation of disease response was performed.

Results:
A total of 16 cancer patients were treated with nivolumab (75%), ipilimumab (19%) or pembrolizumab (6%). The median age of the study population was 64 years; 44% were male. 11 patients were diagnosed with lung cancer (69%), 4 patients with malignant melanoma (25%) and one patient with a pleural mesothelioma (6%). 9 patients received VA (ICM/VA: 56%) and the remaining 7 received no VA treatment during ICM treatment (ICM: 44%). The adverse event rate for patients treated with combinational ICM/VA was not statistically different from the rate in patients treated with ICM therapy alone (67% vs. 71%, p = 0.060). 73% of adverse events in the ICM/VA group were suspected ICM reactions according to SmPCs. 19% of the total patient cohort showed partial disease response to ICM therapy in line with reported rates in literature. No statistical significant differences were seen between both groups with respect to partial disease response (ICM/VA: 22% vs. ICM: 14%, p > 0.999) and stable disease rates (ICM/VA: 33% vs. ICM: 86%; p = 0.060).

Conclusion:
This is the first study evaluating the clinical safety profile of immune checkpoint inhibitors in combination with VA in patients with advanced or metastasized cancer. These results indicate that concomitant VA application may not alter adverse event rates in patients treated with nivolumab, ipilimumab or pembrolizumab. Positive ICM-induced disease response has been maintained during additive VA therapy. Adverse event rates and partial disease response rates of the present study were within the range of reported rates. Further prospective studies in larger study cohorts should focus on the assessment of clinical efficacy, pharmacology and quality of life in patients with combinational ICM/VA therapy.

Background:
First-line treatments for patients with advanced NSCLC include targeted therapies and platinum-based doublet chemotherapy±bevacizumab and/or pemetrexed. Although immunotherapies targeting the PD-L1/PD-1 pathway are available for advanced NSCLC beyond the first line, chemotherapy is a key first-line option for patients, despite poor survival outcomes, highlighting the need for additional treatment options. Atezolizumab, a monoclonal anti–PD-L1 antibody, inhibits the binding of PD-L1 to its receptors PD-1 and B7.1, restoring tumor-specific T-cell immunity. Clinical efficacy has been reported with atezolizumab monotherapy in patients with squamous and nonsquamous NSCLC, with a survival benefit observed across all PD-L1 expression levels. Additionally, Phase Ib data showed the potential for chemotherapy to further enhance responses to atezolizumab, with tolerable safety, in patients with NSCLC. Bevacizumab in combination with atezolizumab may enhance efficacy in non-squamous NSCLC by inhibiting VEGF-mediated immunosuppression. Four global, Phase III, randomized, open-label trials are evaluating atezolizumab+platinum-based chemotherapy±bevacizumab in chemotherapy-naive patients with stage IV NSCLC.

Methods:
Eligible patients must have stage IV NSCLC, measurable disease (RECIST v1.1) and ECOG PS 0-1 and be chemotherapy naive. Exclusion criteria include untreated CNS metastases, autoimmune disease and prior exposure to immunotherapy. Patients will be enrolled regardless of PD-L1 expression status. Patients randomized to the experimental arm will receive atezolizumab 1200 mg with standard platinum-based chemotherapy in IMpower130 and 131 and also ±bevacizumab in IMpower150 for four or six 21-day cycles, then maintenance with atezolizumab in IMpower130 and 131 and atezolizumab+bevacizumab in IMpower150. In IMpower132, experimental-arm patients will receive atezolizumab+platinum-based chemotherapy+pemetrexed, then maintenance with atezolizumab+pemetrexed. Patients receiving atezolizumab may continue until loss of clinical benefit. Co-primary endpoints are progression-free survival and overall survival. Secondary endpoints include objective response rate and safety. Evaluation of predictive biomarkers associated with efficacy will be performed.

Trial	IMpower130	IMpower131	IMpower132	IMpower150
Histology	Nonsquamous	Squamous	Nonsquamous	Nonsquamous
Planned enrollment(N)	650	1025	568	1200
Experimental	Atezolizuma +carboplatin +nab-paclitaxel	Atezolizuma +carboplatin +paclitaxel or Atezolizumab +carboplatin +nab-paclitaxel	Atezolizuma +carboplatin or cisplatin +pemetrexed	Atezolizumab +carboplatin +paclitaxel or Atezolizumab +carboplatin +paclitaxel +bevacizumab
Comparator	Carboplatin +nab-paclitaxel	Carboplatin +nab-paclitaxel	Carboplatin or cisplatin +pemetrexed	Carboplatin +paclitaxel +bevacizumab
Stratification factors	Sex Liver metastases Centrally assessed PD-L1 expression by IHC	Sex Liver metastases Centrally assessed PD-L1 expression by IHC	Sex ECOG PS Chemotherapy type (carboplatin vs cisplatin) Smoking status	Sex Liver metastases Centrally assessed PD-L1 expression by IHC
Identifier	NCT02367781	NCT02367794	NCT02657434	NCT02366143

ECOG PS, Eastern Cooperative Oncology Group performance status; IHC, immunohistochemistry.

Results:
Section not applicable

Conclusion:
Section not applicable

Background:
Immune check-point inhibitors (ICPIs) are considered well-tolerated drugs. Previous experience with ipilimumab in advanced melanoma have shown possible endocrine toxicities, while less data have been collected about Nivolumab. ICPIs act by blocking inhibitory signaling and, therefore, enhancing T-cell activity against tumor cells. This mechanism might result in impaired self-tolerance with subsequent development of immune-related adverse events (irAEs), and endocrine toxicities are especially relevant.

Methods:
From May 2015 to April 2016, 74 patients with advanced pretreated NSCLC (52 Male, 22 Female, mean age: 64 years) received at least one dose of nivolumab (3 mg/Kg every 14 days) within a single-istitutional translational research trial . Blood samples were collected at baseline and at each cycle in order to monitor hormone (TSH, ACTH, cortisol, Prolactin[PRL] and testosterone) serum levels and autoantibodies (ATG, ATPO and anti-TSH) formation. Thyroid morphology was evaluated by ultrasonography at baseline, eventually repeated if TSH anomaly was observed.

Results:
Thyroid function was assessed in all 74 patients. At baseline, 6 patients had impaired thyroid function: 5 with reduced TSH, including two undergone previous thyroidectomy that required reduction in levothyroxine replacement therapy, and 1 with increased TSH. During treatment, 4 patients developed transient thyrotoxicosis evolving to hypothyroidism in 75% of cases. All patients with transient thyrotoxicosis reported increased thyroid autoantibodies; 8 patients developed hypothyroidism, with negative thyroid autoimmunity. Adrenocortical axis was evaluable in 55 subjects, of which 14 under steroid (equivalent of 10 mg of prednisone) therapy (one had partial hypopituitarism). Among the remaining 31 patients, 7 showed significant cortisol alterations (2 elevated, 5 reduced). Gonadal axis was evaluated in 38 male patients; among these, one was taking testosterone replacement therapy for partial hypopituitarism and one was receiving GnRH antagonists. No significant change on gonadal status was observed. PRL was assessed in 56 patients; among these, 10 were treated with drugs known to increase PRL levels; 20 patients had at least one elevated prolactin value, 7 from baseline and 13 developed during treatment. However, only 8 showed significantly increased values (>50 mcg/L in n=6, developed during therapy in 50% of cases; >100 mcg/L in n=2 from baseline).

Conclusion:
Thyroid function abnormalities, in particular non-autoimmune hypothyroidism and transient thyrotoxicosis on autoimmune basis, seem the major endocrine adverse event related to nivolumab. With respect to other hormonal axes, further conclusions might be drawn after a longer follow-up, due to the heterogeneity of available results and the presence of interfering factors.

Background:
Nivolumab, an anti-programmed death-1 antibody, is approved for previously treated metastatic NSCLC, advanced melanoma, advanced renal cell carcinoma (RCC), and relapsed/progressive classical Hodgkin lymphoma. In two phase 3 trials (CheckMate 017 and 057), nivolumab 3 mg/kg every 2 weeks (Q2W) demonstrated superior survival and favorable safety versus docetaxel in previously treated patients with metastatic NSCLC. Clinically meaningful efficacy and a manageable safety profile have been observed in studies in melanoma (CheckMate 037, 066, and 067), RCC (CheckMate 025), and Hodgkin lymphoma (CheckMate 205 and 039). On this basis, the currently approved nivolumab dose is 3 mg/kg Q2W. Decreasing the frequency of nivolumab administration may enhance convenience and compliance while maintaining efficacy and safety in patients who receive long-term nivolumab therapy. CheckMate 384 is a phase 3B/4 trial that will evaluate the efficacy and safety of nivolumab administered at two dosing frequencies in patients with advanced/metastatic NSCLC following ~4 months’ administration of nivolumab 3 mg/kg or 240 mg Q2W.

Methods:
Adult patients with advanced/metastatic squamous or nonsquamous NSCLC and ECOG performance status 0–2 are eligible; disease can be newly diagnosed or recurrent/progressive following multimodal therapy. Patients with untreated, symptomatic brain metastases are ineligible. Patients must have tolerated and completed ~4 months (16 ± 2 weeks) of treatment with nivolumab (3 mg/kg or 240 mg) IV Q2W and achieved a complete or partial response or stable disease. After this pre-study period, patients will be randomized 1:1 to receive IV nivolumab on one of two fixed-dose regimens: 240 mg Q2W or 480 mg Q4W. Randomization will be stratified by histology and response to pre-study nivolumab treatment at randomization (complete/partial response vs stable disease). The table shows primary/secondary endpoints; the objective is to establish that nivolumab 480 mg Q4W is not inferior to 240 mg Q2W. Planned enrollment is 620 patients.

Primary Endpoints	Secondary Endpoints
Progression-free survival rate at 6 months after randomization	Progression-free survival rate at 1 year after randomization by tumor histology and by response before randomization
Progression-free survival rate at 1 year after randomization	Progression-free survival rate at 2 years after randomization
	Overall survival rate (annually, up to 5 years after randomization)
	Safety and tolerability, as assessed by incidence and severity of adverse events

Results:
Not applicable

Conclusion:
Not applicable

Background:
Platinum-based chemotherapy with etoposide is the current first-line (1L) standard of care for the majority of patients with extensive-stage small cell lung cancer (ES-SCLC). Although initial response rates with chemotherapy range from 50% to 70%, survival outcomes remain poor (median overall survival [mOS] < 1 year), and new treatment approaches are needed. Atezolizumab is an anti–PDL1 monoclonal antibody that inhibits the binding of PD-L1 to its receptors PD-1 and B7.1, thereby restoring anti-tumor T-cell activity. In a Phase Ia study, single-agent atezolizumab demonstrated a tolerable safety profile and promising durability of response in patients with ES-SCLC: confirmed ORR was 6% (n = 1/17 [partial response]; DOR of 7 months) by RECIST v1.1 and 24% by immune-related response criteria (irRC) (n = 4/17, with 2 patients on atezolizumab for ≥ 12 months). In addition, pre-clinical and Phase I data suggest that atezolizumab plus platinum-based chemotherapy in NSCLC may be synergistic, resulting in durable responses that could potentially translate into improved survival over monotherapy alone. Taken together, these findings provide a rationale to investigate whether atezolizumab + carboplatin + etoposide can improve survival compared with carboplatin + etoposide in the 1L treatment of ES-SCLC.

Methods:
IMpower133 (NCT02763579) is a global, Phase I/III, randomized, multicenter, double-blinded, placebo-controlled trial comparing the efficacy and safety of atezolizumab + carboplatin + etoposide with that of placebo + carboplatin + etoposide in treatment-naive patients with ES-SCLC. Patients will be enrolled regardless of PD-L1 expression status. Exclusion criteria include untreated CNS metastases, autoimmune disease or prior anti-cancer therapy for ES-SCLC. The study stratification factors include sex, ECOG performance status and presence of CNS metastases. Eligible patients will be randomized 1:1 to receive four 21-day cycles of atezolizumab (1200 mg IV) or placebo in combination with carboplatin (AUC 5 mg/mL/min IV, day 1) and etoposide (100 mg/m[~2~], days 1-3), followed by maintenance with atezolizumab or placebo until PD per RECIST v1.1. Patients can continue with treatment until persistent radiographic PD, symptomatic deterioration or unacceptable toxicity. Co-primary endpoints of investigator-assessed progression-free survival per RECIST v1.1 and OS will be evaluated. Secondary efficacy endpoints include ORR and DOR. Safety and tolerability will also be assessed. Approximately 400 patients will be enrolled in this trial.

Results:
Section not applicable

Conclusion:
Section not applicable

Background:
For patients with advanced NSCLC without genetic driver alterations, cisplatin/carboplatin+pemetrexed is a standard-of-care first-line (1L) treatment for non-squamous histology; and cisplatin/carboplatin+gemcitabine for squamous histology. Although immunotherapies targeting PD-L1/PD-1 are currently available for 2L+ NSCLC, chemotherapy remains the main 1L option despite poor survival and toxicities. Atezolizumab, an anti–PDL1 mAb, prevents PD-L1 from interacting with its receptors PD-1 and B7.1, restoring tumor-specific T-cell immunity. Clinical efficacy was demonstrated with atezolizumab in non-squamous and squamous NSCLC, with Phase I and II studies exhibiting durable responses and survival benefit that increases with higher PD-L1 expression on tumor cells (TC) and/or tumor-infiltrating immune cells (IC). IMpower110, a global Phase III randomized, multicenter, open-label trial, will evaluate efficacy and safety of atezolizumab vs cisplatin/carboplatin+pemetrexed or gemcitabine as 1L therapy for PD-L1–selected chemotherapy-naive patients with advanced non-squamous or squamous NSCLC, respectively.

Methods:
Eligibility criteria include stage IV non-squamous or squamous NSCLC, measurable disease (RECIST v1.1), ECOG PS 0-1, no prior chemotherapy for advanced NSCLC and centrally-assessed PD-L1 expression ≥1% on TC or IC (TC1/2/3 or IC1/2/3 with VENTANA SP142 IHC assay; expected prevalence, ≈65%). Exclusion criteria include active or untreated CNS metastases, prior immune checkpoint blockade therapy or autoimmune disease. Patients will be randomized 1:1 to receive atezolizumab or cisplatin/carboplatin+pemetrexed (non-squamous)/gemcitabine (squamous) for 4 or 6 21-day cycles. Patients in comparator arms can receive pemetrexed (non-squamous)/best supportive care (squamous) until RECIST v1.1 disease progression. Patients receiving atezolizumab may continue until loss of clinical benefit. Co-primary endpoints are PFS and OS. Key secondary efficacy endpoints include ORR, DOR, IRF-assessed PFS (RECIST v1.1) and TTD. Safety and PK will also be evaluated. Tumor biopsies at RECIST v1.1 progression will be assessed for immunologic biomarkers associated with responses to atezolizumab and to differentiate non-conventional responses from radiographic progression.

Planned enrollment, N	570
Histology	Non-squamous	Squamous
Experimental arm	Atezolizumab (1200 mg q3w)
Comparator arm	Cisplatin (75 mg/m[2] IV q3w) + pemetrexed (500 mg/m[2] IV q3w) or Carboplatin (AUC 6 mg/mL/min IV q3w) + pemetrexed (500 mg/m[2] IV q3w)	Cisplatin (75 mg/m[2] IV q3w) + gemcitabine (1200 mg/m[2] IV days 1, 8) or Carboplatin (AUC 5 mg/mL/min IV q3w) + gemcitabine (1000 mg/m[2] IV days 1, 8)
Stratification factors	Sex ECOG Histology (non-squamous vs squamous) PD-L1 expression by IHC
ClinicalTrials.gov identifier	NCT02409342

Results:
Section not applicable

Conclusion:
Section not applicable

Background:
Effective options are limited for patients with non-small cell lung cancer (NSCLC) with progressive disease after first-line chemotherapy. In these patients, immune checkpoint modulators have recently proven to be successful targets, being nivolumab the first immune checkpoint inhibitor approved for NSCLC. In contrast to conventional chemotherapy, these agents appear to have potential for effecting durable responses and possibly long-term survival. Immune checkpoint inhibitors generate atypical types of tumour responses and have a specific toxicity profile which is challenging current practices. Objective: To investigate outcomes and adverse effects in patients treated with nivolumab.

Methods:
Stage IV NSCLC patients treated with nivolumab at our centre between 30th September 2015 and 30th June 2016 were retrospectively analysed. We describe clinical features, toxicity and outcomes in these patients.

Results:
Fifteen patients were included [mean age 62±8 years; mainly male (n=12)]. Almost all patients had a history of tobacco smoking (n=12; mean pack/year45). The observed histological type were adenocarcinoma (n=10) and squamous cell carcinoma (n=5). All patients received prior systemic therapy, mainly platinum based regimens. At time of the initiation of nivolumab most patients had an ECOG performance-status score of 1 (n=12) and stage IV cancer (n=10). Only one patient received subsequent cancer therapy and the remaining alive patients at time of the study was still under nivolumab treatment. Mean duration of treatment was 3.5months (median of 4 cycles). The median survival since the beginning of nivolumab was 2.3months (min 7days; max 8.7months). Treatment-related adverse events of grade 1 or 2 were reported in 20% of the patients: thyroid hormone alterations were present in 3 patients and 2 needed thyroid hormone replacement; 1 patient presented immune related eczema and another suspected myocarditis. Two patients suspended nivolumab temporarily and two patients died.

Conclusion:
Besides the efficacy profile of immune targeted agents it is important to be aware of possible immune-related adverse events. These toxicities remain largely unknown and will be more frequent in routine practice as the number of patients treated with nivolumab increases. Although severe adverse effects remain rare, they can become life-threatening if not anticipated and managed appropriately. Ongoing evaluation is needed to define the most appropriate timing and patient population that will benefit from therapy with an immune checkpoint inhibitors and to learn how to deal with its adverse effects.

Background:
Nivolumab is a human IgG4 monoclonal antibody that binds to the PD-1 receptor and blocks its interaction with PD-L1 and PD-L2, releasing PD-1 pathway-mediated inhibition of the immune response- including the anti-tumor immune response. Due to the novelty of the mechanism of action and the limited treatment experience all checkpoint inhibitors can potentially induce treatment related AEs in any organ system that have not been noticed until now. The Syndrome of Inappopriate Antidiuresis (SIAD)-leading to hyponatremia with variant symptoms of CNS affection- is a frequent paraneoplastic syndrom in patients with advanced cancer, especially small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), but can also be induced by a large number of anticancer drugs, however, not reported for Nivolumab until now.

Methods:
We report a 56year old heavily pretreated patient who was first diagnosed with squamous NSCLC in 2012. After symptomatic progression he started 4[th] line therapy on Nivolumab treatment (3mg/kg, q2w) in August 2015 after EMA licensing.

Results:
After 3 months of treatment we noted disease stabilization with a radiographic minor response together with a slight improvement of tumor symptoms. Thus, therapy was continued. After 6 months treatment CT scan showed for the first time a partial response, however patient reported a clinical deterioration with onset of new symptoms (headache, dizziness, fatigue, nausea, blurred vision). Lab results showed severe hyponatremia (nadir 116mmol/l) caused by SIAD (serum osmolality 254 mOsm/kg), possibly induced by Nivolumab as other potential causes were excluded. No signs of intracranial progression or hypophysitis (brain MRI, CSF cytology, endocrinology lab) were detected. In the phase III registration trial the median time to response was 2.2 months. In our patient hyponatremia occurred in a timely relationship with partial response of the disease (after 6 months of treatment). After 2 months Tolvaptan treatment (an oral ADH antagonist) sodium levels normalized, accompanied by improvement of hyponatremia-symptoms. The patient is still in PR on Nivolumab treatment without treatment interruptions with normal sodium levels.

Conclusion:
We report a patient case (squamous NSCLC) where chemotherapy was not able to induce long lasting radiographic remissions or clinical benefit. After switching to Nivolumab therapy the patient achieved a partial remission after 6 month of continous Nivolumab with a simultaneous onset of hyponatremia. To the best of our knowledge, this is the first report of a Nivolumab response occuring simultaneously with SIAD. Therefore we speculate that hyponatremia might be a predictor for Nivolumab treatment response.

Background:
Treatment options for patients with stage IIIB and IV NSCLC who progress to first line chemotherapy are limited. Immunotherapy represents a promising alternative for NSCLC patients. The aim of our study is to analyze nivolumab in compassionate use in our centre.

Methods:
A retrospective study of patients with stage IIIB and IV NSCLC that progress to chemotherapy and receive Nivolumab in compassionate use. A descriptive analysis using chi-square test and survival analysis using Kaplan-Meier estimates. The radiological response was assessed by RECIST 1.1 and immune-related adverse events (irAEs).

Results:
Thirty-two patients were included between July 2015 and March 2016, 87.5% men, 12.5%women, 75% adenocarcinoma and 25% squamous, from which 71% received nivolumab in second line and 29 % in third line or more; 12.5 % had PS 2 and 25 % brain metastases. The response rate observed was 17.4 % in the second line and 20 % in third line or more, with a progression-free survival (PFS) of 4 months (95% CI: 2.3-5.4) and 10 months (95% CI 2.9-18), respectively. The average number of administered cycles was 6: 1 in PS2 and 18 when there was pseudoprogression (n=5). The observed irAEs was: grade GI 11 (34 %), GII 4 (12 %) and GIII 1 (3 %), 6% pneumonitis (with previous radiotherapy), 3% autoimmune hepatopathy, 3% pemphigoid and 3% hypothryroidism. Global survival (GS) in PS0 was 6.5 months (95% CI: 4.6-8.3), PS1 of 7.4 (95% CI: 6-8.7) and PS2 of 1 (95% CI: 0, 67-0), with p0.001. The GS with brain metastasis was 3 months (95% CI: 1.7-4.2) vs. 7.9 months (CI 95%6-8.7), with p 0.001, in patients without brain metastasis.

Conclusion:
In our series, nivolumab was well tolerated and demonstrated clinical benefit both in second and in third line, except in patients with PS2 and/or brain metastases. Patients that present pseudoprogression obtain major benefit and more occurrence incidence of irAEs (possible indicator of response). External validity is limited by the small number of patients and this is not a randomized study

Background:
Targeted therapy with erlotinib is effective in reducing tumor burden in EGFR-mutant non-small cell lung cancer (NSCLC). However, resistance to therapy develops almost universally. Atezolizumab, an engineered mAb that inhibits binding of PD-L1 to its receptors, PD-1 and B7.1, has demonstrated promising monotherapy activity in NSCLC. Given that atezolizumab may enhance and perpetuate anti-tumor immunity, we hypothesized that combining atezolizumab with erlotinib may improve both clinical response and durability in EGFR-mutant NSCLC.

Methods:
This Phase Ib study consisted of a safety-evaluation stage in patients with NSCLC regardless of EGFR status followed by an expansion stage in TKI-naïve patients with tumors harboring activating EGFR mutations. Patients were enrolled regardless of PD-L1 status. After a 7-day run-in with 150mg erlotinib PO QD alone, patients received 150mg erlotinib PO QD and 1200mg atezolizumab IV q3w. To evaluate immune biology, biopsies were obtained in expansion-stage patients pre-treatment, after erlotinib run-in, at weeks 4-6, and at progression. The primary objective was to evaluate the safety and tolerability of the combination. Secondary objectives included evaluation of the clinical activity per RECIST v1.1. Data cutoff, 11 April 2016.

Results:
Twenty-eight patients (safety stage, n = 8; expansion stage, n = 20) who received ≥ 1 dose of erlotinib or atezolizumab were considered safety evaluable. Median age was 61y (range, 47-84); median survival follow-up was 11.2mo (range, 0.8-24.2). The incidence of either treatment-related G3-4 AEs was 39% and for serious AEs, 50%. The most common atezolizumab-related G3-4 AEs were pyrexia and increased ALT. No pneumonitis was reported. No treatment-related G5 AEs occurred. Five patients discontinued atezolizumab due to treatment-emergent AEs. No DLTs were observed. In the expansion-stage population, ORR was 75% (95% CI, 51-91). Disease control rate (CR + PR + SD ≥ 24 weeks) was 90% (95% CI, 68-99), median PFS was 11.3mo (95% CI, 8.4-NE) and median DOR was 9.7mo (range, 4.2-11.7). Increases in intratumoral CD8+ T cells post-erlotinib run-in were observed in 8/13 evaluable paired biopsies. Higher intratumoral CD8+ T-cell prevalence and immune gene expression signatures at baseline were associated with improved PFS.

Conclusion:
The combination of full dose erlotinib plus atezolizumab demonstrated a manageable safety profile. While response rates and median PFS for combination treatment appear similar to those observed with erlotinib monotherapy, the addition of atezolizumab to erlotinib may lead to more durable clinical responses in some patients. Additional follow-up is required to evaluate the full potential of this combination treatment. NCT02013219

Background:
Nivolumab is a new standard-of-care in platinum-refractory Non-small cell lung cancer (NSCLC), with significant survival increment in comparison to docetaxel shown in two phase 3 trials. Herein, we report the local experience in an expanded access program in Brazil.

Methods:
Patients with recurrent or metastatic NSCLC, treated with at least one prior chemotherapy regimen, were potentially eligible. Overall, three hundred twenty-one patients were screened in the country, and 287 were enrolled. Around 10% of these (N=29) were treated in a single cancer institution in Rio de Janeiro. The aim was to describe the early outcome in these 29 patients.

Results:
Median age was 64 years (range 37-83), most patients were male (62%), white (66%), smoker or former smoker (21% and 55%, respectively). Most cases (59%) were classified as non-squamous, and only 3 had a documented EGFR mutation. Sixty-two percent had received 2 or more prior chemotherapy lines. After a median follow-up of 4.9 months (95% CI, 4.2-5.5), 1 partial response (3%) was documented, and 13 patients (45%) presented with disease stabilization by RECIST 1.1. Median PFS and OS were not reached, and 6-month OS was 52%. Seventeen patients (59%) had at least 1 adverse event, the most common being asthenia (9 patients). Only 1 (3%) grade 3 event was documented.

Conclusion:
Nivolumab was well tolerated and led to disease control in 48% of patients in this early analysis, after a short follow-up. Survival data will be updated for presentation.

Background:
Immune checkpoint inhibitors are taking on a growing role in the treatment of patients with metastatic NSCLC. Pre-clinical evidence suggests that radiotherapy may increase the frequency, or enhance the strength of the host anti-cancer immune response. We report the preliminary results of an ongoing phase I/II trial combining stereotactic body radiotherapy (SBRT) and the anti-PD-1 antibody pembrolizumab in patients with metastatic NSCLC.

Methods:
Eligible patients are those with metastatic NSCLC who have received no prior immune-directed therapy, and have at least 2 sites of measurable disease as per RECIST 1.1. PD-L1 expression is not required for study entry. All patients are treated with pembrolizumab at 200 mg every 3 weeks until development of progressive disease by immune-related RECIST criteria (irPD). After irPD, patients receive SBRT to a single site of disease and continue pembrolizumab. The primary endpoint is safety and tolerability. Secondary endpoints include the pre- and post-SBRT overall response rate.

Results:
27 patients with advanced NSCLC have enrolled and started trial therapy. The overall response rate (irPR and irCR) to the initial course of pembrolizumab is 35%. To date, 13 patients have had irPD: 5 were not eligible for SBRT and stopped study treatment (2 developed new brain metastases, 3 had decline in PS), and 8 patients received SBRT to a single site of disease (6 thoracic, 1 adrenal, 1 vertebral) and continued pembrolizumab. 5 of these patients are evaluable for post-SBRT response: 1 patient had confirmed irPD, 4 have irSD and continue pembrolizumab post-SBRT at a median duration of 3 months (range 1 to 5 months). 2 of the 4 patients with irSD have had > 20% decrease in the sum of diameters of their unirradiated targets, since SBRT. Regarding adverse events, in the pre-SBRT phase 6 of 27 patients (22%) developed grade 3 treatment-related toxicity (2 colitis, hepatitis, pneumonitis, hypothyroidism, conjunctivitis). In the SBRT and post-SBRT phases, there have been no grade 2 or greater treatment-related events.

Conclusion:
The addition of SBRT to pembrolizumab has not resulted in an increase in treatment-related toxicity. Several patients who had serially confirmed irPD to pembrolizumab monotherapy underwent SBRT and now have irSD, with some evidence of tumor regression. Updated results will be presented.

Background:
Immunotherapy involving dendritic cells (DCs) vaccine has the potential to overcome the bottleneck of cancer therapy.

Methods:
Here, we engineered Lewis Lung cancer cells (LLC) and bone marrow derived DCs to express tumor-associated antigen (TAA), ovalbumin (OVA) via lentiviral vector plasmid encoding OVA gene. We then tested the anti-tumor effect of modified DCs both in vitro and in vivo.

Results:
The results demonstrated that in vitro modified DCs could dramatically enhance T cells proliferation (P < 0.01) and kill LLC significantly than control groups (P < 0.05). Moreover, modified DCs can reduce tumor size and prolong the survival of tumor-bearing mice than control groups (P < 0.01, P < 0.01; respectively). Modified DCs enhanced homing to T-cell-rich compartments and triggered naïve T cells to become cytotoxic T lymphocytes, which exhibited significant infiltration into the tumors. Interestingly, modified DCs also markedly reduced tumor cells harboring stem cell markers in mice (P < 0.05), suggesting the potential role of eliminating cancer stem-like cells in vivo. Figure 1



Conclusion:
These findings indicated that DCs bioengineered with TAA may enhance antitumor effect against murine lung cancer through novel mechanism that is worth further exploration.

Background:
Early-stage non-small cell lung cancer (NSCLC) is treated surgically, but 30%-70% of patients experience post-resection recurrence and succumb to disease. Adjuvant chemotherapy is the standard of care for fully resected NSCLC (stages IB [tumors ≥4 cm]-IIIA), and although cisplatin-based chemotherapy provides some benefit, the 5-year absolute survival benefit is ≈5%, underscoring the unmet need. Atezolizumab is an anti-PD-L1 monoclonal antibody that inhibits PD-L1 from binding to its receptors PD-1 and B7.1, thereby restoring anti-tumor immune response. Atezolizumab monotherapy has demonstrated promising efficacy and tolerable safety in patients with previously-treated advanced NSCLC, with a survival benefit observed across all PD-L1 expression levels. Given the need to improve survival for patients with early-stage NSCLC, IMpower010 (NCT02486718), a global Phase III randomized, open-label trial, has been initiated to compare the efficacy and safety of atezolizumab with best supportive care (BSC), following adjuvant cisplatin-based chemotherapy in patients with resected stage IB (tumors ≥4 cm)-IIIA NSCLC.

Methods:
Eligibility criteria include complete tumor resection 4-12 weeks prior to enrollment for pathologic stage IB (tumors ≥4 cm)–IIIA NSCLC. Patients must have adequately recovered from surgery, be eligible to receive cisplatin-based adjuvant chemotherapy and have an ECOG PS 0-1. Exclusion criteria include the presence of other malignancies, use of hormonal cancer or radiation therapy within 5 years, prior chemotherapy, autoimmune disease or exposure to prior immunotherapy. Approximately 1127 patients, regardless of PD-L1 expression status, will be enrolled. Eligible patients will receive up to four 21-day cycles of cisplatin-based chemotherapy (cisplatin [75 mg/m[2] IV, day 1] + either vinorelbine [30 mg/m[2] IV days 1, 8], docetaxel [75 mg/m[2] IV day 1] or gemcitabine [1250 mg/m[2] IV days 1, 8], or pemetrexed [500 mg/m[2] IV day 1; non-squamous NSCLC only]). Adjuvant radiation therapy is not permitted. Following adjuvant treatment, eligible patients will be randomized 1:1 to receive atezolizumab (1200 mg q3w, 16 cycles) or BSC. Stratification factors will include sex, histology (squamous vs non-squamous), extent of disease (stage IB vs II vs IIIA) and PD-L1 expression by IHC (TC, tumor cell; IC, tumor-infiltrating immune cell; TC2/3 [≥5% expressing PD-L1] and any IC vs TC0/1 [<5%] and IC2/3 [≥5%] vs TC0/1and IC0/1 [<5%]). The primary endpoint is disease-free survival, and secondary endpoints include overall survival and safety. Exploratory endpoints include PD-L1 status, immune and tumor related biomarkers before, during and after treatment with atezolizumab and at radiographic disease occurrence or confirmation of new primary NSCLC.

Results:
Section not applicable

Conclusion:
Section not applicable

Background:
SUNRISE, a global, double-bind, Phase III trial of docetaxel (D) plus bavituximab (B) or D plus placebo (P) in previously treated non-squamous non-small cell lung cancer, demonstrated similar overall survival (OS) in both treatment arms. Mass spectrometry and correlative analysis were used to create a test able to identify a subgroup of patients benefitting from the addition of B to D.

Methods:
Pre-treatment serum samples were available for 197 of the first 200 subjects enrolled in the trial. Mass spectra could be generated for 193 samples using the Deep MALDI method (Duncan et al, ASMS 2013), processed and features (peaks) identified. Mass spectral (MS) features associated with various biological functions were identified using a gene set enrichment analysis approach. Analysis of scores based on these MS feature, subsets indicated that in patients with high complement activation outcome depended on IL-10 activation in D+B but not in D+P. A test using the MS features associated with these functions was created to reliably identify a patient subgroup associated with clinical benefit using modern machine learning methods.

Results:
Complement activation, as assessed by a classifier trained using related MS features, was a prognostic factor in both treatment arms, with high activation associated with poorer clinical outcome (OS HR = 0.54, log-rank p = 0.013 for D+B; OS HR = 0.60, log-rank p = 0.040 for D+P). Within the subgroup with high complement activation [N=50 (D+B); N=54 (D+P)], a second classifier using features related to IL-10 activation was able to isolate a subgroup of patients showing numerical benefit from the addition of B [median OS 5.9 months (D+P), 12.5 months (D+B)]. The remaining subgroup showed no benefit from addition of B [median OS 10.4 months (D+P), 5.6 months (D+B)]. Blinded validation of the test in the remainder 397 patients randomized in SUNRISE is will be presented.

Conclusion:
Proteomic and correlative approaches identified complement activation and low IL-10 levels as important pathways for predicting improved outcomes of patient treatment with D+B, in line with preclinical work on B’s mechanism of action. The test resulting from this work will undergo blinded independent validation.

Background:
Multiple studies have shown the activity of the anti-PD(L)-1 agents in patients with advanced non-small-cell lung cancer (NSCLC). There is an urgent need to explore biomarkers that predict outcome to anti-PD-1 therapy. The electronic (e) Nose is used to analyse the exhaled gasses and is under development as a diagnostic tool for lung cancer. We aimed to determine the diagnostic accuracy of exhaled breath analysis for responders vs. non-responders to anti-PD-1 treatment in NSCLC patients.

Methods:
Patients with NSCLC who were about to receive Nivolumab, were asked to participate. At baseline and after 6 weeks of treatment exhaled breath analysis took place. Breathprints were collected in duplicate by an e-Nose positioned at the rear end of a pneumotachograph (SpiroNose) (de Vries J Breath Res 2015). RECIST 1.1 criteria were used for response evaluation at three months and six months and reported accordingly: Complete response (CR), Partial response (PR), stable disease (SD), and progressive disease (PD). Data-analysis involved signal processing, environment correction and statistics based on principal component analysis (PCA), followed by discriminant analysis (Matlab2014/SPSS20).

Results:
From August 2015 until April 2016, 56 patients participated in this trial. Forty-two patients had a response evaluation. Principal component 3 and 4 showed a significant difference (p=0.005 and p=0.001) between responders (PR and SD) and non-responders (PD) [Figure A]. Twenty-five patients had a second exhaled breath analysis after 6 weeks. Analysis showed significant differences in PC3 and PC4 between both SD vs. PR (p<0.001) and PD vs. PR (p=0.002) [Figure B].Figure 1



Conclusion:
E-Nose is able to discriminate between responders and non-responders to anti-PD-1 treatment at baseline and 6 weeks follow-up and may therefor be of great value to predict outcome.

Background:
Anti-PD1 antibodies have become the treatment of choice for most advanced NSCLC patients after failure of first line platinum-based chemotherapy. Responses are seen in roughly 20% of treated patients. PDL1 expression level and mutational burden might be predictive factors but are not always available and their predictive accuracy is limited. Predictive biomarkers are urgently needed. We hypothesized that clinical data and baseline blood tests might be predictive for benefit from nivolumab.

Methods:
A chart review was performed of patients with advanced NSCLC who received at least one cycle of nivolumab, at one of three cancer centers during 2015-2016. Additional inclusion criteria were: available baseline clinical data, evaluation of response and availability of blood test results. Blood test results collected were: Absolute Lymphocyte Count (ALC), Absolute Neutrophil Count (ANC), White Blood Cells (WBC), Hemoglobin (Hb), Platelets (PLT), Albumin (ALB), Lactate Dehydrogenase (LDH). Blood test results were collected at baseline and before the second and third treatment. Disease control (DC) was defined as any tumor shrinkage, or stable disease for at least 6 months, as assessed by the treating physician by computerized tomography scans. Patients with DC were compared with patients with progressive disease (PD, patients progressing within the first 6 months). Uni- and multivariate regression analyses were performed using Stata (version 11.2, StataCorp).

Results:
A total of 70 patients treated with nivolumab were included, median age 67 years, 66% males, 27% with DC. DC patients compared to PD patients were younger (61.6 vs 69.3 yr, p<0.001), more females (42% vs 27%, p<0.05), and had a lower baseline WBC (6.9 vs 9.2 K/microL, p<0.05). The difference in WBC between DC and PD patients increased during treatment (2.3 K/microL at baseline, 2.6 prior to third treatment). Lower baseline neutrophil count was associated with DC (p=0.02). Neither performance status nor LDH predicted outcome on uni-variate analysis. On multivariate analysis age (p=0.050) and baseline WBC (p=0.02) were associated with outcome. Patients less than 67 years of age with baseline WBC<8.04 K/microL (n=18) had DC rate of 50%, while DC rate was 4% in patients 67 years or more, with WBC >=8.04 K/microL (n=23).

Conclusion:
We have identified clinical biomarkers (age and baseline WBC) that are associated with DC under nivolumab treatment. Validation on an independent data set is warranted. The association of a low peripheral WBC/ANC with increased response rate raises the possibility that acute inflammatory responses are counteractive to anti-PD1 therapy.

Background:
Nivolumab is a programmed death-1 (PD-1) immune checkpoint inhibitor approved for previously treated advanced non-small cell lung cancer (NSCLC). Liquid biopsy is a non-invasive blood test that detects cell-free DNA (cfDNA) shed from the tumour into the bloodstream. Monitoring cfDNA in patients with NSCLC under treatment with Nivolumab may be helpful to assess efficacy of the therapy and may be related with patients’ survival.

Methods:
Peripheral blood samples were obtained from 74 patients with pretreated advanced NSCLC within a single-institutional translational research trial from May 2015 to April 2016. Patients received intravenous Nivolumab at 3 mg/kg every 2 weeks until progression or unacceptable toxicity. All the patients underwent CT-scan every 4 cycles and responses were classified according to immune related Response Criteria. CfDNA was extracted from plasma using the Circulating Nucleic Acid Kit (Qiagen). The quantification of cfDNA (ng/ml plasma) was performed by qPCR using hTERT single copy gene. Kaplan-Meier survival function was used to compare the survival curves from cfDNA at baseline and at the time of first evaluation (after 4 cycles of Nivolumab).

Results:
Among the 74 enrolled patients 72 were evaluable for cfDNA survival analyses; 14 experienced early death, 25 progressive disease (PD), nine partial response (PR),19 stable disease (SD) and five were not evaluable for response. 27 out of the 28 responsive patients (PR+SD) are still alive at the time of analysis. In 25 evaluable patients with PD after the first radiological evaluation, median cfDNA < 786 ng/ml was significantly associated with an improved median survival as compared to cfDNA ≥786 ng/ml (295 vs 96 days respectively, HR=0.09290, 95% CI 0.019987-0.4322, p-value: 0.0052); similar results have been obtained in the subset of 25 patients progressing at best response (p-value: 0.0042). Analyzing the OS of the 72 evaluable patients, median survival of those with cfDNA<786 ng/ml is still undetermined, while it is equal to 181 days for those with cfDNA>786 ng/ml (HR 0.3559, 95%CI 0.1674-0.7568, p-value 0.0035).

Conclusion:
Our preliminary data show a significantly improved survival for NSCLC patients treated with Nivolumab having cfDNA<786 ng/ml than those with higher cfDNA; the correlation with OS is observed in patients at the first radiological evaluation and in those with PD at best response.

Background:
Advanced non-small cell lung cancers (NSCLC) can be treated with anti-PD1 (programmed cell death 1) antibodies. Anti-PD-1 therapy can lead to immune mediated adverse events. This study examines the incidence of pneumonitis, a potentially fatal complication, in patients with advanced NSCLC treated with anti-PD-1 antibodies at 3 large hospitals in Sydney, Australia.

Methods:
NSCLC patients commenced on pembrolizumab (2 mg/kg or 10 mg/kg Q3W) or nivolumab (3 mg/kg Q2W) were assessed for adverse events including pneumonitis. Patient demographics, treatment history and immune mediated complications were collected. Pneumonitis was graded according to the Common Terminology Criteria for Adverse Events Version 4.0. Pneumonitis treatment and clinical outcomes were collected. Serial imaging was reviewed with a blinded radiologist.

Results:
A total of 104 patients between 2012 and 2016 were treated with anti-PD-1 therapy. Median age for included patients was 67. Fourteen (14%), 35 (34%), and 53 (51%) had anti-PD-1 as first, second, or third and subsequent line treatment respectively. Nine patients (9%) developed pneumonitis. Three patients (4%) developed grade 3 (G3) or higher pneumonitis including one patient (1%) that died due to pneumonitis. All patients with ≥G3 pneumonitis required hospital admission with one requiring admission to a high dependency unit. None of the patients with ≥G3 pneumonitis were retreated with anti-PD1 therapy. All patients with ≥G3 pneumonitis died within 5 weeks of their diagnosis of pneumonitis. Seven patients with pneumonitis were treated with steroids. The median length of treatment with steroid was 29 days. Pneumonitis involved both lungs in 3 patients. Of the remaining 6 patients – 2 had all right lung lobes involved, 2 had two lobes and 1 had one lobe. Fifteen (14%) patients had a history of receiving concurrent chemoradiotherapy prior to anti-PD-1 therapy. A further 6 (6%) had curative intent radiotherapy and 15 (14%) had palliative radiotherapy to the thorax prior to anti-PD1 therapy. One of the patient with G3 pneumonitis had previously received radiotherapy to the chest. No association between prior radiotherapy and pneumonitis was seen.

Conclusion:
The incidence of pneumonitis is rare but our real-life multi-institutional experience demonstrates an incidence higher than reported in the literature. This complication can be life threatening and onset of ≥G3 pneumonitis is associated with short survival.

Background:
Chemotherapy, including nab-paclitaxel, plus an immune checkpoint inhibitor has demonstrated antitumor activity in patients with metastatic breast cancer (mBC) and NSCLC. Here, results from the 2 lung cohorts of the phase I nivolumab + nab-paclitaxel in pancreatic cancer (± gemcitabine), NSCLC (+ carboplatin), and mBC safety trial are presented.

Methods:
Enrollment in the lung cohorts (C and D) was initiated in two sequential parts: Dose-limiting toxicity (DLT) evaluation was done in Part 1 prior to treatment arm expansion in Part 2. Chemotherapy-naive patients with stage IIIB/IV NSCLC received 4 cycles of nab-paclitaxel 100 mg/m[2] D 1, 8, 15 + carboplatin area under the curve (AUC) 6 D 1 + nivolumab 5 mg/kg D 15 (starting in cycle 1 [Arm C] or cycle 3 [Arm D]) of each 21-day cycle; nivolumab continued as monotherapy from cycle 5. Primary endpoints were DLTs (Part 1), and grade 3/4 treatment-emergent adverse events (TEAEs) and TEAEs leading to discontinuation (Parts 1 and 2). Patients who received ≥ 2 nivolumab cycles and remained on study for 14 days after the last nivolumab dose or discontinued due to DLT prior to completing 2 nivolumab cycles were considered DLT-evaluable. Key secondary endpoints include safety, PFS, OS, and ORR.

Results:
As of May 25, 2016, 21 patients have enrolled in Arm C (18 nivolumab-treated); most were aged ≥ 65 years (57.1%) and female (71.4%), 33.3% had ECOG PS 0, 42.9% and 33.3% had adenocarcinoma and squamous cell carcinoma, respectively. No DLTs were reported (5 DLT-evaluable patients). The most common grade 3/4 AEs in Arm C (all patients) were neutropenia (28.6%), anemia (19.0%), and hypokalemia (14.3%); gastrointestinal disorders (11.1%) were the most frequent grade 3/4 immune-related AE in nivolumab-treated pts. Seven patients (5 nivolumab-treated) discontinued treatment (majority due to progressive disease [PD]). Of the 18 nivolumab-treated patients, 9 had a PR, 8 had stable disease, and data is pending in 1 patient; tumor shrinkage (baseline to nadir) ranged from 3% to 83%. The median PFS (n = 4 with PD or death; nivolumab-treated) was 7.3 months (treatment duration, 0.7 - 9.4 months). Eight patients have enrolled in Arm D (4 nivolumab-treated); 1 DLT (pneumonitis) was reported in 4 DLT-evaluable patients.

Conclusion:
These results demonstrate that the combination of nivolumab with nab-paclitaxel/carboplatin is tolerable and has promising antitumor activity in patients with NSCLC. Updated results will be presented at the meeting. (NCT02309177)

Background:
VSV-IFNβ is a live, replicating oncolytic virus with activity against NSCLC. We have previously shown that VSV-IFNβ leads to an inflamed tumor microenvironment and enhances anti-tumor immunity in a syngeneic mouse model. Furthermore, we have observed increased PDL-1 expression on tumor cells after intratumoral injection with VSV-IFNβ. Here, we have further explored the mechanisms by which VSV-IFNβ exerts its immunologic effects and combined therapy with anti-PD1 and anti-PDL1 antibodies.

Methods:
VSV-human and murine IFNβ (hIFNβ and mIFNβ, respectively) and VSV-IFNβ-NIS are manufactured by the Imanis Life (Rochester, MN) and titered on Vero cells by limiting dilution method. H460, H2009, H838, H2030, and A549 human NSCLC cells were grown in RPMI with 10% serum. LM2 cells (murine lung adenocarcinoma cells) were grown in DMEM with 10% serum. For cytotoxicity assays, NSCLC cells are treated with VSV-IFNβ at varying MOI. CCK8 assay was used to estimate cell viability 72 hours later. For in vivo experiments, A/J mice are injected with 2x10[6] LM2 cells in the flank. After tumors form, unilateral intratumoral injections are given at varying doses on days 1,3, and 5. For combination experiments, VSV-IFNβ is given in combination with intraperitoneal anti-PD1 or PDL1 antibodies or Isotype IgG or with JAK inhibitor, ruxolitinib. Tumor infiltrating leukocytes (TIL) were analyzed by flow cytometry for presence of CD8/CD4 T cells, Tregs, and MDSCs after VSV-IFNβ infection.

Results:
VSV-IFNβ treatment on human NSCLC cells induced PDL-1 expression by Western blot and flow cytometry, but not VSV-GFP which is abrogated by pretreatment with ruxolitinib. Furthermore, viral replication was enhanced by pretreatment with ruxolitinib. In vivo immune effects of combination ruxolitinib and VSV-IFNβ are ongoing. TILs were examined by flow cytometry after intratumoral injection of VSV-mIFNβ or VSV-hIFNβ. There was increased T-cell infiltration, decreased Tregs and increased PDL-1 expression in both groups. Antitumor activity was similar between VSV-mIFNβ and VSV-hIFNβ suggesting that effects observed are mediated by the virus rather than exogenous IFNβ. CD4 T cell depletion had no effect on antitumor responses or on immune infiltration of CD8 T cells in the tumor microenvironment. CD8 T cell depletion experiments and combination treatments of VSV-IFNβ and VSV-IFNβ-NIS with Anti-PD1/PDL1 antibodies are ongoing.

Conclusion:
VSV-IFNβ is a promising oncolytic agent for non-small cell lung cancer and induces an inflamed tumor microenvironment in a process that is independent of exogenous IFNβ and CD4 T cells. Our data support clinical testing of VSV-IFNβ with checkpoint blockade for NSCLC.

Background:
Inhibitors of PD1/PD-L1 checkpoint have been shown to be active among a broad range of cancers including NSCLC. They induce proliferation of T cells within the tumor microenvironment (as revealed by IHC) leading to tumor eradication. There is however lack of detailed molecular characterization of these proliferating T cells including the dynamics of their clonalilty during treatment and its correlation with response, their antigen specificity and the molecular changes induced in the expanded clones at single cell level. Such understanding will serve as a biomarker to detect early response after one dose of therapy, ascertain efficacy (especially when radiological assessments are equivocal) and guide determination of optimal duration of therapy. Furthermore, insight into molecular changes in the proliferating T cell clones induced by these agents at single-cell level will identify the baseline unique characteristics of T cells clones that undergo rapid expansion upon exposure to anti-PD1 therapy, define the molecular mediators of tumor eradication in responders and serve as a foundation for the development of novel treatment strategies for non-responders.

Methods:
We performed next-generation T cell receptor alpha/beta chain sequencing on serially obtained tumor and PBMC samples from 54 NSCLC patients undergoing anti-PD1 therapy. We compared the dynamics of the T cell repertoire in responders versus non-responders within unsorted PBMC and in CD8 positive/negative T cells. We also assessed the expression of key mediators of cytotoxicity and T cell activation/dysfunction in these expanded CD8 T cell clones at single cell level among responders and non-responders.

Results:
We identified concordant early clonal T cell expansion after 1-4 doses of anti-PD1 therapy within the tumor and PBMC of responders. We confirmed these expanded T cell clones to be CD8 positive subgroup of CD3+ T cells in responders and CD8 negative subgroup of CD3+ T cells in non-responders. Furthermore, among responders we found that persistence of the expanded T cell clones for several months while on treatment is associated with durable response. Additional results on antigen specificity and gene expression of the expanded T cell clones in responders versus non-responders will be presented.

Conclusion:
Our results showed that early concordant clonal expansion of a defined population of CD8+ T cells detected both within the tumor and PBMC correlate with response to therapy. We also confirmed that the persistence of these unique T cell clones several months after their initial expansion correlates with durable response.

Background:
Recent advances in the modulation of immune checkpoints (ICPs) or their ligands (ICPLs) indicate their role in anti-tumor immunity and in mediating durable cancer regressions in NSCLC and other cancers. Epithelial-to-mesenchymal transition (EMT) enables the reprogramming of polarized epithelial cells towards a mesenchymal phenotype with migratory and invasive properties. EMT promotes cancer cell plasticity and favors tumor adaptability to encountered selective pressures. We hypothesize that EMT represents an escape mechanism to immune-surveillance.

Methods:
ICPLs gene expression patterns were analyzed in silico in 129 NSCLC cell lines (CCLE) in relation to their EMT status (Mak. CCR, 2015). These observations were validated using the TCGA RNAseq data available for lung adenocarcinomas (n=488) and squamous-cell carcinomas (n=501) and the GSE41271 dataset (n=275 NSCLC tumors). In vitro, CD70 expression was evaluated by FACS in (1) epithelial Vs mesenchymal lung cancer cell lines, (2) HCC44 cells (mesenchymal) that underwent CRISPR/Cas9-mediated knock out of ZEB1 and in (3) H3255 (epithelial) that overexpress SNAIL. The expression of CD70 and markers of immune infiltrate was assessed by immunohistochemistry in a cohort of 132 NSCLC.

Results:
Unsupervised hierarchical cluster analysis revealed that expression of CD70 was significantly associated with the mesenchymal status in NSCLC cell lines (p < 0.001). These results were confirmed in silico in all three datasets of NSCLC samples. We identified the E-box sequence CANNTG in the CD70 gene promoter, suggesting the possible regulation of CD70 by ZEB1 and/or SNAIL. CD70 expression was increased in mesenchymal Vs epithelial NSCLC cell lines. Overexpression of SNAIL in H3255 cells did not result in the acquisition of mesenchymal properties or in changes in CD70 expression. CRISPR/Cas9-mediated knock out of ZEB1 was successful in HCC44 cells and resulted in increased expression of E-cadherin and EpCAM when compared to the control. The analysis of CD70 expression in this model will be presented. We found increased CD70 positivity in sarcomatoid tumors compared to non-sarcomatoid NSCLC. Of note, the expression of CD70 in sarcomatoid tumors was limited to the mesenchymal compartment and co-localized with ZEB1. It is known that CD70 by tumor cells can facilitate evasion of the immune system. The analyses of tumor immune infiltrate markers and T-cell exhaustion in a cohort of 18 lung sarcomatoid tumors will be presented.

Conclusion:
The association of EMT and CD70 in lung tumors may play an important role of this interaction in immune scape. CD70 might represent a relevant target in sarcomatoid lung tumors.

Background:
Recent studies have identified that the degree of tumor infiltrating lymphocyte (TIL) infiltration and PD-L1 expression in the tumor microenvironment (TME) are significantly correlated with the clinical outcomes of anti-PD-1/PD-L1 therapies. Here we conducted this study to verify the distribution of PD-L1/CD8[+] TIL expression and its clinical significance in non-small cell carcinoma (NSCLC). Potential mechanism predicted for PD-1 blockade was explored in depth as well.

Methods:
Immunohistochemistry was performed to detect PD-L1 and CD8 expression in NSCLC. The Kaplan–Meier (KM) survival curve was used to estimate disease free survival (DFS) and overall survival (OS). Gene Set Enrichment Analysis (GSEA) was used to determine potentially relevant gene expression signatures.

Results:
288 cases with stage I-IIIA NSCLC were evaluated for PD-L1 and CD8+ TIL staining. Dual positive PD-L1 and CD8 (PD-L1+/CD8+) represents a predominant subtype in NSCLC, accounting for 36.5% (105/288), followed by PD-L1-/CD8- (24.3%, 70/288), PD-L1-/CD8+ (26.0%, 75/288) and PD-L1+/CD8- (13.2%, 38/288). Survival analysis of DFS (p=0.031) and OS (p=0.002) showed a significant difference between four subgroups. Furthermore, we analyzed the correlation between expression types of PDL1/CD8 and mutation burden and angtigen presentation. We can identified dual positive PD-L1 and CD8 was significant with increased mutation burden (p<0.001), high frequency of mismatch repair (MMR) related gene mutation. More interestingly, tumor with dual positive PD-L1 and CD8 manifested a remarkable activated angtigen presentation and T cell receptor signature compared with other subgroups. Figure 1



Conclusion:
Dual positive PD-L1 and CD8 was identified as a predominant subtype in NSCLC and correlates with increased immunogenicity. These findings provide the evidence that combined analysis of PD-L1 and CD8 in NSCLC may be a promising way to predict PD-1 blockade immunotherapy.

Background:
Therapeutic antibodies to programmed death receptor 1 (PD-1) have shown clinical activity in lung cancer. The aim of this study is to investigate the clinical factors, including inflammatory markers such as neutrophil/lymphocyte ratio (NLR), to predict response to anti-PD-1 antibody in advanced lung cancer patients.

Methods:
We retrospectively analyzed 51 patients who had advanced lung cancer and had been treated with anti-PD-1 antibodies between 2013 and 2015. The values of NLR were assessed at two time points: at baseline (pre-treatment) and at 6 week after the start of treatment (post-treatment). NLR of 5 was used as the cutoff value.

Results:
The median age of the patients was 68 years; 76.5% were male, and 27.5% were never smokers. Most patients had adenocarcinoma (n = 28); 17 had squamous cell carcinoma, and 6 had others. Eighteen of 51 patients (35.3%) had clinical objective response to anti-PD-1 antibody. Non-adenocarcinoma histology and low post-treatment NLR was significantly associated with clinical response, while gender, smoking history, line of treatment and pre-treatment NLR were not predictive of response. Liver metastasis, brain metastasis, and high post-treatment NLR were significantly associated with worse tumor response. Patients with a high post-treatment NLR had significantly shorter PFS (median 1.3 months vs. 6.1 months, p < 0.001). Multivariable analysis demonstrated that high post-treatment NLR (hazard ratio [HR] 20.1, 95% confidence interval [CI] 5.5 - 73.9, p < 0.001), presence of liver metastasis (HR 5.5, 95% CI 2.1 - 14.6, p = 0.001), and CNS metastasis (HR 2.9, 95% CI 1.1 - 7.4, p = 0.027) were independent predictive factors for short PFS. Figure 1



Conclusion:
Clinical factors including post-treatment NLR at 6 week might be predictive of clinical benefits from anti-PD-1 antibody therapy in lung cancer.

Background:
Using of genetically engineered T lymphocytes with tumor antigen-specific T-cell receptor for treatment of cancer has clinically proved promise, however, this approach is complicated by several potential problems: (1) on-target adverse events directed against normal tissues, especially when affinity-enhanced TCRs are used; (2) issues related to chain mispairing between the introduced and endogenous TCR genes; (3) off-target adverse events because of inherent cross-reactivity of the introduced TCR. In this study, we examined in detail the efficacy and safety of normal CD8[+] T lymphocytes transduced retroviral vector encoding siRNAs which specifically down-regulate endogenous TCR expression, and a siRNA-resistant WT1-specific TCR construct for adoptive immunotherapy against human lung cancer cells.

Methods:
A novel TCR vector system which simultaneously delivers shRNAs for endogenous TCR α/β genes and WT1-specific TCRs genes were transduced into normal peripheral CD8+ T cells. The safety and effectiveness of these transfectant against lung cancer cells was evaluated both in vitro and in vivo.

Results:
First, we confirmed the augmented and inhibitory efficacies of the WT1-specific TCRs with siRNAs targeting endogenous TCRs (WT1-siTCR) vector for expression of the respectively introduced and endogenous TCRs. The result indicating that sufficient functional suppression of endogenous TCR and enhanced expression of the introduced TCR are achievable using the WT1-siTCR vector. Secondly, The WT1-specific TCRs with siRNAs targeting endogenous TCRs double gene modified T cells were augmented cytotoxic activities compared with only WT1-TCR–transduced T cells against lung cancer cells by standard 5-hour 51Cr-release assays at various effector/target (E/T) ratios.

Conclusion:
These data revealed that WT1-siTCR vector system shows safety resulting from stronger expression of the introduced WT1-specific TCR with inhibition of endogenous TCRs. Results from this study also demonstrate that significant enhancement of anti-lung cancer cells reactivity of these WT1-siTCR gene-modified T cells. In summary, the present study shows considerable promise in terms of safety and efficacy for adoptive immunotherapy against lung cancer cells using WT1-specific TCRs with siRNAs targeting endogenous TCRs gene-engineered T cells.

Background:
Effectiveness of immunotherapy has been observed in around 20% of cases, nowadays there is no accurate biomarker to select those patients (pts) who will benefit the most.

Methods:
We evaluated retrospectively pretreatment (baseline) and post-treatment (every 2 months) serum-LDH in 94 pts with NSCLC treated with anti-PD1/PDL1. Repeated measures ANOVA, Kaplan-Meier and the proportional COX model were used to examine the association of LDH with overall survival (OS). The cutoff level of LDH was 400 based on the median of the sample. (normal range 105-333 UI/L).

Results:
From July 2013 to February 2016, 94 pts were treated with immunotherapy based in anti-PD1 (77,6%) and anti-PDL1 (22,4 %), in trials at VHIO. Median age was 62 (39-86). Histological subtypes were: adecocarcinoma 53.2%, squamous 42.5%, others 4.2 %. The OS was significantly different in pts treated with immunotherapy according to baseline LDH, if LDH ≥ 400 median OS was 8.2 months, while in pts with LDH <400 median survival was not reached (Figure 1) Figure 1 There were statistically significant differences in the evolution of LDH, with better responses if there was a downward trend in LDH levels. In long responding pts (45 of 94 pts), defined as ≥ 3 evaluations (6 months), a LDH level at the time of 6 months treatment below than the baseline LDH predicts better responses (22/45 pts): 68.2% partial response (RP); 18.2% stable disease (SD); 13.6% disease progression (PD). In contrast, when LDH at third evaluation was higher than baseline LDH (23/45 pts): 39.1% PR; 56.5% SD; 4.3% PD, (p=0.022). There were differences between the level of LDH pre and post-progression. 66.67% of patients who progressed had a higher level of LDH at the time of progression than at the previous assessment (p=0.03).



Conclusion:
LDH may be a potential predictive biomarker of survival benefit conferred by immunotherapy in patients with NSCLC.

Background:
Evidence of likely response to immunotherapy, including programmed death ligand-1 (PD-L1) expression, assumes more importance in selecting immunotherapy as a first line therapy over conventional therapy. However, PD-L1 expression is heterogeneous and known to be underestimated on small biopsies. Correlation of PD-L1 expression with clinicopathologic features may provide additional useful information in this setting. In phase II clinical trials (POPLAR study) survival benefit from atezolizumab correlated PD-L1 expression in either tumor cells or tumor-infiltrating immune cells assessed by immunohistochemistry with clone SP142 for patients with previously treated non-small cell lung carcinoma (NSCLC). To our knowledge the clinicopathologic features of NSCLC have not been reported for subsets defined by PD-L1 expression in either tumor cells or tumor-infiltrating immune cells.

Methods:
A total of 125 adenocarcinomas and 38 squamous cell carcinomas were included in the study. PD-L1 immunohistochemistry with the SP142 clone was performed on whole tissue sections and scored according to percent of PD-L1-positive tumor cells (TC0 for <1%, TC1 for 1-4%, TC2 for 5-49%, and TC3 for ≥50%) and percent tumor area with PD-L1-positive tumor-infiltrating immune cells (IC0 for <1%, IC1 for 1-4%, IC2 for 5-9%, IC3 for ≥10%).

Results:
Adenocarcinoma cases which scored either TC1/2/3 or IC1/2/3 include the majority (22 of 34; 65%) with high histologic grade, a minority (15 of 46; 33%) with low histologic grade, the majority (25 of 36; 69%) with solid subtype, and a minority for most other subtypes. Compared to the adenocarcinoma TC0 and IC0 subset, the TC1/2/3 or IC1/2/3 subset correlated with higher histologic grade (p = .0051, χ[2] test for trend) and solid subtype (p = .0006, Fisher’s exact test). Compared to the adenocarcinoma TC0/1 or IC0/1 subset, the TC2/3 or IC2/3 subset correlated with higher histologic grade (p = .0021, χ[2] test for trend), solid subtype (p = .0001, Fisher’s exact test), and higher smoking pack-years (p = .012, Mann-Whitney test). No other clinicopathologic variable, including survival, correlated with subsets for either adenocarcinoma or squamous cell carcinoma.

Conclusion:
Lung adenocarcinoma subsets defined by PD-L1 expression in either tumor cells or tumor-immune cells correlated with high histologic grade, solid subtype, and high smoking pack-years. Our results suggest that high histologic grade and solid subtype may provide useful supplementary information for the decision to treat with atezolizumab, particularly for first line therapy, when PD-L1 mmunohistochemistry is negative on small biopsies or samples are insufficient for testing.

Background:
The inflammatory status in advanced cancer patients before treatment have been asocciated with poor prognosis. Recently, neutrophil-to-lymphocyte ratio (NLR), derived NLR, platelet-to-lymphocyte ratio (PLR) and lymphocyte-to-monocyte ratio (LMR) have been proposed to mesure the inflammatory status at diagnosis. However their prognostic/predictive value for immune checkpoint inhibitors is unknown. The aim of this study is to evaluate the role of these parametres to predict outcomes to immune checkpoint inhibitors in NSCLC patients.

Methods:
We conducted a retrospective study of a cohort of advanced NSCLC patients (pts) treated with immune checkpoint inhibitors (nivolumab (nivo), pembrolizumab (pembro) or atezolizumab (atezo)) from Nov. 2013 to July 2016 in our institute. Clinical data were collected and complete blood count, lactate dehydrogenase (LDH), and albumin were also collected at baseline and at 2[nd] cycle (after 14 days in case of nivo and after 21 days for pembro and atezo) for monitoring the blood cells counts and the ratios. A statistical analysis was performed with R studio software.

Results:
65 NSCLC patients were included. Twenty-five patients (38%) were female, median age was 65 years (30-86); 55/65 patients were current/former smokers and 9 (14%) non-smokers; 50 patients (76%) had performance status 1. Forty-two (64.6%) had an adenocarcinoma, seventeen (26%) squamous cell carcinoma, three (5%) large cells, and three others histologies. Fifty-seven patients (88%) had stage IV (25% had M1a, 75% M1b), and eight (12%) locally advanced disease. The median of immunotherapy line was 2 (2-9). Seventeen patients (26%) received another line of treatment after immunotherapy. Absolut leucocyte count (WBC) and absolut neutrophil count (ANC) at immunotherapy beginning (baseline) were correlated with prognosis (p<0.0001 and p<0.0001). NLR, dNLR [ANC/(WBC-ANC)] and PLR were significantly correlated with survival from immunotherapy beginning (p<0.001, p=0.021 and p=0.003, respectively). An early increase of NLR and dNLR at 2[nd] cycle were prognostic for shorter survival (p<0.0001 and p=0.0011). Increases of ANC and absolut eosinophils count (AEC), and decreases of absolut lymphocytes count (ALC) at 2[nd] cycle were also associated with poor prognosis (p=0.002, p=0.002 and p=0.048 respectively). Lactate dehydrogenase (LDH) and albumin at baseline were significant prognostic factors to immunotherapy (p<0.0001 and p=0.001).

Conclusion:
Our preliminary results suggest that “low cost” and routine blood markers and their early changes could be associated with outcomes to immunotherapy in advanced NSCLC. A multivariate analysis on 130 patients will be presented.

Background:
The class I human leucocyte antigen (HLA) molecules play a critical role in tumor recognition by T cells and the loss of expression seems to be an escape mechanism of antitumoral immunity. Novel immune-targeting cancer vaccines are currently developped in HLA-A2 positive patients for modulating the T cells immune response. We hypothesized that HLA-A2 status could influence the prognosis and response to immune checkpoints (IC) inhibitors.

Methods:
Advanced NSCLC patients treated with nivolumab or within clinical trials (with pembrolizumab or atezolizumab) were prospectively included from November 2013 to July 2016 in our institute. HLA-A2 status was analysed by flow cytometry; PDL1 was analysed by immunohistochemistry. Clinical and biological data were collected at baseline and after cycle 1. Statistical analysis was performed with R studio.

Results:
Out of 160 patients treated, HLA-A2 status was available for 65 patients. 40 patients (61.54%) were male, median age was 65 years (30-86); 55 (84.6%) were smokers and 57 (72.3%) had performance status 0-1. 42 patients (64.6%) had an adenocarcinoma, 17 (26.1%) squamous cell carcinoma and 6 (9.2%) others histologies. Molecular status was available in 55 patients: 6 (10.9%) were EGFRmut, 3 (5.4%) ALK positive, 13 (23.6%) KRASmut, 3 (5.4%) BRAFmut and 12 (21.8%) wildtype. PDL1 expression was positive in 13 patients (20%), negative in 5 (7.7%) and unknown in 47 (72.3%). The median of previous lines of treatment was 1 (1-8). HLA-A2 status was positive in 32 patients (49.23%) and negative in 33 (50.76%). HLA-A2 positivity was associated with higher number of metastases at baseline and the presence of liver metastasis (p=0.04 and p= 0.016, respectively). Other patient and tumor characteristics were well balanced between HLA-A2 + and - groups. The median progression free survival to immunotherapy (iPFS) was 4 months [0-40]. In HLA-A2 positive the median iPFS was 4 months vs 4 months in HLA-A2 negative (p=0.63). The median overall survival (OS) was 21 months [0-121]. The median OS was 18.5 months in HLA positive vs. 24 months in HLA-A2 negative patients (p=0.25). No significant difference between both HLA-A2 groups was identified.

Conclusion:
Our preliminary results suggest that HLA-A2 status has no predictive or prognostic value in NSCLC patients treated with immune checkpoint inhibitors. An updated analysis on 78 patients will be presented.

Background:
The human immune system recognizes and eliminates certain types of tumor cells, whereas other malignancies are capable of suppressing immune function. For example, a number of cancers cell types express programmed cell death ligand 1 (PD-L1), which binds to its receptor PD-1 on T cells to prevent their activation. High levels of PD-L1 expression are typically associated with poor patient prognosis. Based on these results, researchers have developed immunotherapies (e.g., inhibitors of the PD-1/PD-L1 pathway) to stimulate the immune system, allowing the body's natural defenses to combat the tumor. To determine which patients are suitable candidates for receiving immunotherapy, levels of PD-L1 expression are often determined from tumor biopsies, but tumor heterogeneity can confound these results and obtaining tumor tissue is often not feasible. To enable non-invasive detection and sequential monitoring of tumor-associated PD-L1 expression we have developed a highly sensitive method of detecting PD-L1 levels in circulating tumor cells (CTCs). Here we sought to analytically validate the PD-L1 assay by introducing PD-L1-positive (H358) and PD-L1-negative (BT474) cells into control blood samples, and measuring detection accuracy.

Methods:
PD-L1 expression levels on carcinoma cell lines were identified by flow cytometry. For analytical validation, H727, BT474 H358, HCC78 and H820 cells were spiked into CEE-SureTM blood collection tubes, in replicates and on different days, incubated overnight and thereafter processed. The leukocyte fraction was incubated with our pan-CTC antibody capture cocktail, labeled with biotinylated secondary antibody, followed by enrichment in our streptavidin coated microfluidic channels. Enriched cells were stained for DAPI, cytokeratin, CD45, PD-L1 (clone 28-8) and CEE-Enhanced (pan-CTC stain). After automated fluorescence scanning, 400 spiked tumor cells per microfluidic channel were identified and average PD-L1 intensities were quantified for each cell and cut-off criteria were determined.

Results:
In our microfluidic PD-L1 assay we demonstrate H727 and BT474 cells to be negative for PD-L1, while H358 cells have low-medium and HCC78 and H820 cells high PD-L1 expression. We determined a cut-off value (average fluorescence intensity value) that yielded 100% concordance between the result of the PD-L1 test and the identity of the introduced cell lines, based on a 95% confidence level and a 3.9% negative cut-off.

Conclusion:
The Biocept PD-L1 assay can accurately detect added CTCs that express PD-L1 in blood samples. This ability affords a way to identify patients likely to benefit from immune therapy as well as monitor the efficacy of such treatments.

Background:
The use of immunotherapy for the whole Non Small Cell Lung Cancer (NSCLC) population, is like an untargeted shooting. So trying to discover predicitve factors to response still represents the key to the problem. We retrospectively analyzed a cohort of patients (pts) treated with Nivolumab, in the attempt to correlate clinical and molecular features with response.

Methods:
69 heavily pretreated advanced NSCLCs (16 squamous/ 53 adenocarcinomas) were retrospectively evaluated for response to Nivolumab. Pts’ samples from a subgroup of responders (14/17 pts, 82%), were further analyzed for PD-L1/PD-1 expression by immunoistochemistry (IHC), and for TILs density. We used rabbit monoclonal antibodies anti PD-L1 [clone E1L3N] for tumor cell expression (0-3, negative-intense) and mouse monoclonal antibody anti PD-1 [clone EH33] for TILs.

Results:
Clinico-pathologic characteristics: mostly smoker males (81%), PS 0-1 (85%), EGFR+ 7%, K-RAS+ 23%. Overall response rate was 25% (2% complete response and 23% partial response), stable disease 30%, progressive disease 41%. Median progression free survival (PFS) and overall survival (OS) for the entire cohort were 2.9 and 8.3 months (mo) respectively. 1 and 2-y OS rates were both 44% (95% CI, 29-58). Pts with EGFR + NSCLC showed a significantly lower median OS with respect to the wild type cohort (4.5 vs NR; p < 0.005) as well as pts with brain metastases (4.1 vs NR), while a trend toward improvement in PFS for K-RAS+ was seen. A subgroup analysis according to the time to progression to prior chemotherapy regimen (< 3 mo versus > 6 mo), confirmed a poorer survival for those with rapid spread of disease. Among laboratory tests, a better outcome for those who developed G2 leucopenia was demostrated (OS 8.3 vs 5.0 mo). Severe drug-related adverse events occurred in only 5.7% of pts. PD-L1, PD-1, TIL expression for 14/17 pts with OR, were as follows: PD-L1 > 5% 6/14 pts (43%); PD-1 2/14 (14%); focal TILs presence 7/14 (50%).

Conclusion:
Nivolumab confirms activity in NSCLC with durable responses and accettable safety profile. Of note, 44% of our patients were alive at 2 years. No predictive role emerged in our small cohort, according PD-L1, PD-1 and TILs expression, for those obtaining a tumor response. Interactions among alternative factors such as smoking habit, mutational status, time to progression, bone marrow toxicities (ie leucopenia), may have more powerful association with response and clinical outcome. Updated clinical activity and biomarker analysis will be presented.

Background:
The NLR, a marker of systemic inflammation, has been associated with outcomes in multiple cancers. In patients (pts) with metastatic melanoma treated with ipilimumab, pre-therapy NLR < 5 has been associated with improved progression-free survival (PFS) and overall survival (OS). However, the utility of NLR as a marker of outcomes in pts with NSCLC treated with programmed-death 1 (PD-1) inhibitors is not known.

Methods:
We conducted a retrospective cohort study of pts with advanced NSCLC treated with nivolumab off clinical trials at the University of Pennsylvania between March 2015 and March 2016. NLR was calculated from complete blood counts obtained within two weeks of starting nivolumab. Pts were dichotomized based on a NLR <5 or ≥ 5. We calculated PFS and OS using the Kaplan-Meier method and used multivariate Cox proportional hazards models to adjust for sex, age, histology (squamous vs. non-squamous), Eastern Cooperative Oncology Group Performance Status (ECOG PS) (0-1 vs. ≥ 2), smoking history [heavy (≥10 pack-years) vs. light/never (<10 pack-years)], and number of prior systemic therapies (1 vs. ≥ 2).

Results:
175 pts received a median of 5 cycles of nivolumab (range, 1-24; IQR 3-9). Median age was 68 years (range 33-88, IQR 60-74), 46% of pts were male, 75% were white, 25% had an ECOG PS ≥ 2, and 46% had ≥ 2 prior systemic therapies. Eighty-four percent of pts had a ≥10 pack-year smoking history, and 76% had non-squamous histology. Median baseline NLR was 5.5 (IQR, 3.1 – 9.4), with NLR < 5 in 73 pts (42%) and NLR ≥ 5 in 102 patients (58%). Through the date of this analysis (June 1, 2016), disease progression had occurred in 124 pts (71%), and 92 pts (53%) had died, resulting in median PFS and OS of 2.1 and 6.5 months, respectively. After controlling for the aforementioned clinical and demographic factors, pts with baseline NLR<5 had significantly improved PFS (median 2.8 vs. 1.9 months; adjusted HR=0.70, 95% CI: 0.50-0.99; p = 0.04) and OS (median 8.4 vs. 5.5 months; adjusted HR=0.54, 95% CI: 0.34-0.84; p = 0.007) compared to pts with NLR ≥ 5.

Conclusion:
Pre-therapy NLR is independently associated with PFS and OS in advanced NSCLC pts treated with nivolumab. It is unclear whether this marker is predictive or prognostic. Prospective studies are warranted to determine the utility of NLR in predicting outcome in the context of other biomarkers of PD-1 therapy.

Background:
Lung cancer remains a major cause of cancer mortality. Malignant lesions are normally endogenously corrected by the immune surveillance system. However, tumors evade this immunity by inducing immunosuppressive microenvironments during cancer progression. Recent studies demonstrate that multiple cancer types, including melanoma, lung, kidney, bladder, and stomach, respond to immune checkpoint inhibitors, such as PD-L1 and PD-1 with 11-30% response rates and durable responses. However, a substantial number of patients still fail to respond to immunotherapy and the refractory mechanisms are largely unknown. In this study, we focus on KRas-driven lung cancers, as there are no clinically effective targeted drugs available for treating this type of lung cancer.

Methods:
We examined tumor infiltrated immune cells using FACS, CyTOF2, and Immunostaining of lung sections during the progression of lung tumors in KRas mutation and p53 knockout-driven lung cancer mouse models; KRas[G12D/+];p53[-/-] (KP). Using this mouse model, we determined the anti-cancer efficacy of combined inhibition of MEK and immune checkpoint molecules.

Results:
We demonstrate that there is a gradual increase in the number of myeloid derived suppressor cells (MDSC) and that the combination of either anti-PD-1 or anti-PD-L1 antibody along with a MEK inhibitor shows anticancer efficacy in these animal models. These combinations, in comparison to either single agent alone, effectively blocks the growth of subcutaneously injected syngeneic mouse lung cancer cells in immune competent transgenic KP mice, significantly increasing the survival rates: 37.5% (for anti-PD-1 antibody and MEK inhibitor), 62.5% (for anti-PD-L1 antibody and MEK inhibitor) vs. 0% single agents or control at the end of treatment. We find that the tumors in the control treated group harbor a substantial number of immune cells, including PD-L1 expressing MDSC.

Conclusion:
The combination treatment with either an anti-PD-1 or anti-PD-L1 antibody along with a MEK inhibitor dramatically modulates the composition and the activity of tumor infiltrated immune cells. Tumors in the combination treatment group show a significant decrease in PD-L1 expressing MDSC in comparison with control tumors. Additionally, combination treatment blocks PD-L1 activity of the infiltrated PD-L1 expressing MDSC in malignant tumors and thus lead to improved survival. These results point to a potential therapeutic opportunity for currently untargetable KRas-driven lung cancers.

Background:
CD4[+]CD25[+]Foxp3[+] regulatory T-cells (Tregs) are known to suppress immune responses. Treg-mediated immunosuppression is a key mechanism for tumor immune-evasion, which could lead cancer immunotherapies to failures. Systemic depletion of Tregs has been tried; however, intravenously delivering antibodies against Tregs might not sufficiently deplete Tregs in tumor-microenvironment or could deplete effector cells. Moreover, auto-immune responses could cause potential side effects. To overcome these problems, we exploited near infrared photoimmunotherapy (NIR-PIT) at the tumor to deplete only intratumoral-Tregs.

Methods:
F(ab’)~2~ fragments of an anti-mouse CD25 antibody, PC61.3, were generated and conjugated with a phthalocyanine dye, IRDye-700DX (nti-CD25-F(ab’)~2~-IR700). In vitro NIR-PIT effect was examined against CD25-expressing-T-lymphocytes, HT2-clone-A5E-cells. In vivo CD25-target-NIR-PIT was performed after an intravenous injection of the conjugate to mice bearing subcutaneous, luciferase transfected, LL/2 (Lewis lung carcinoma, LL/2-luc). Tumor-volume, bioluminescence signals (BLI), and Immune responses following the therapy were examined

Results:
In vitro NIR-PIT-induced cytotoxicity was light-dose-dependent. Local CD25-target-NIR-PIT selectively depleted intratumoral-Tregs; yet, Tregs in any other organs were not affected. The local CD25-target-NIR-PIT induced a intratumoral rapid activation and cytotoxic action of CD8-T cells and NK-cells. This led to significant reductions of tumor-volume (p < 0.0001) and BLI (p < 0.05) and prolonged survival (p < 0.0001) compared to non-treated controls. Intriguingly, this local CD25-target-NIR-PIT induced a transient systemic cytokine storm and antitumor-effects on distant non-irradiated specific tumors. Effects of local CD25-target-NIR-PIT were significantly (p < 0.0001) inhibited by a CD8-, NK-, or INFg-depletion, suggesting the anti-tumor roles of CD8 T-cells and NK-cells.

Conclusion:
Depletion of intratumoral-Tregs with a local CD25-target-NIR-PIT rapidly induced CD8 T- and NK-cell activation, thereby restoring local anti-tumor immunity. Consequently, activated immunity led to regression of not only NIR-PIT-treated-tumors but also non-NIR-light-exposed-tumors in separate parts of the body (Fig). These observations suggest that local CD25-target-NIR-PIT may be a promising new strategy for cancer immunotherapy.Figure 1

Background:
Nivolumab has become a consolidated therapeutic approach for previously treated non-small cell lung cancer (NSCLC); however, consistent prognostic and predictive factors are still lacking. Since these agents act by enhancing the immune response against tumor cells, it is possible that distinctive patterns in the circulating T cell sub-populations might be associated with different responsiveness. The aim of this study is to determine whether variations in these sub-populations might predict objective response to nivolumab in NSCLC.

Methods:
Blood samples were collected and stored from patients receiving nivolumab (3 mg/Kg every 14 days) for advanced NSCLC within a single-institutional translational research study conducted in the San Martino Hospital – National Institute for Cancer Research, Genova, Italy (approved by the local ethical committee). Sample collection was performed before each administration for 4 consecutive cycles, followed by computed tomography (CT)-scan. Response assessment was performed with the response evaluation criteria in solid tumors (RECIST) v. 1.1 and the immune-related response criteria (irRC); responses were defined as partial response (PR), stable disease (SD), and progressive disease (PD). Additional CT-scans were performed at 4 cycles intervals. Peripheral blood mononuclear cells (PBMC) were analyzed for the frequency of the major adaptive cell subsets, including B cells, natural killer (NK) cells, and T-cells; the latter were divided into CD8+ T cells, exhausted CD8+ T cells, CD4+ cells, and regulatory T cells (Tregs); the relative frequencies and the ratios between the sub-populations at each sample collection were compared with radiological response.

Results:
Fifty-four patients were considered eligible: median age= 70 (44-85); male/female: 70%/30%; current or former smokers= 87%; non-squamous/squamous histology= 80%/20%. Patients achieving PR at the first RECIST assessment had a significant upregulation of Tregs (CD4+ Foxp3+ CD39+ cells; p= 0.021), as well as a decreased CD8+/Treg ratio (p= 0.033) at the baseline sample. Conversely, patients experiencing PD at the first RECIST assessment had a significantly upregulated CD8+/Treg ratio at cycle 2 (p= 0.029). Finally, patients experiencing PD at irRC had a higher proportion of activated T cells (PD1+ CD56+ CD3+) compared to the other patients (P= 0.018) at cycle 2.

Conclusion:
The proportions of Tregs and activated T cells appear to be correlated with different responses to nivolumab according to RECIST and irRC. While the immunologic mechanism at the basis of these findings has to be defined, further studies involving PBMC as predictors of response to immunotherapy for NSCLC are highly advised.

Background:
NSCLC survival and progression-free survival (PFS) curves often follow first-order kinetics (Stewart, Lung Cancer 2011;71:217). We hypothesized that the number of curve decay phases reflects number of biologically distinct subpopulations with distinct rates of progression or death that are determined by dichotomous (present vs absent) factors. In NSCLC, some PDL1-negative patients respond to PDL1 inhibitors, while some PDL1 strongly-positive patients do not.

Methods:
We used “arohatgi.info/WebPlotDigitizer/app/” to digitize published NSCLC PDL1-inhibitor PFS curves and used GraphPad Prism5 for nonlinear regression analyses (one-phase and two-phase decay; constraints:Y~0~=100, plateau=0).

Results:
26 of 28 curves fit 2-phase decay models with R[2]>0.90, with distinct “fast progression” (FP) and “slow progression” (SP) subgroups. In studies with PFS curves for different PDL1-expression groups, patients with higher PDL1 tended to have a larger SP subgroup, although some with low PDL1 had slow progression and some with high PDL1 had fast progression (see Table). Figure 1



Conclusion:
1) PFS-curve nonlinear regression analysis identified 2 distinct subgroups (FP/SP) with differing degrees of benefit from PDL1 inhibitors. 2) Some PDL1-low patients had SP while some PDL1-high patients had FP, although there was a trend to more SP with PDL1-high than PDL1-low. 3) The observation of 2 distinct subpopulations leads us to hypothesize that there is a dichotomous variable (eg, gene mutation, deletion, amplification or silencing) driving PDL1 inhibitor benefit. The trend to higher benefit with high PDL1 expression, but inexact prediction of benefit by PDL1 expression is similar to the published trend seen with response vs PDL1 expression, and suggests that this dichotomous variable is linked to but distinct from PDL1 expression. 4) The published observation that high mutation burden is associated with PDL1 inhibitor benefit suggests that high mutation burden increases the probability of presence of a putative dichotomous favorable factor. We observed similar outcomes in other tumor types.

Background:
Anti-PD1 inhibitors are becoming the treatment of choice for 2[nd] line non-small cell lung cancer (NSCLC). While existing testing for PDL-1 expression may correlate with anti-PD1 benefit, current data do not support these tests to be sufficient to guide therapy. We evaluated the utility of a serum-based pre-treatment test first developed to identify patients benefitting from anti-PD1 therapy in metastatic melanoma[1] in patients with NSCLC. These results were compared to the data obtained from application of the established VeriStrat[2] test to the same samples.

Methods:
60 advanced NSCLC patients treated with nivolumab in an observational study were included. Pretreatment serum samples were classified using the fully locked mass spectrometry-based multivariate tests BDX008 and VeriStrat. BDX008 generates a classification of positive (BDX008+, good outcomes) or negative (BDX008-, poor outcomes); VeriStrat classifies samples as Good and Poor. The association of test classifications with overall survival (OS), progression-free survival (PFS), and time-to-failure (TTF) were assessed using Kaplan-Meier method and Cox proportional hazards model.

Results:
37% of patients were classified as BDX008+ and 63% as BDX008-; 62% were classified as VeriStrat Good and 38% as Poor. Both tests significantly stratified OS (Table), but not PFS or TTF, and remained significant for OS in multivariate analyses (p=0.0167 and 0.0184, for BDX008 and VeriStrat, respectively). Out of 11 patients who died before the first radiological evaluation, 10 were classified as BDX008-, 9 as VeriStrat Poor.

Test	Log-rank p value	CPH HR [95% CI]	Median Survival (months) [95% CI]
BDX0008 (+ vs -)	0.0026	0.189 [0.056-0.637]	BDX0008+: Not reached BDX0008-: 5.5 [2.6-11.9]
VeriStrat (Good vs Poor)	0.0186	0.390 [0.173-0.880]	VS Good: Not reached VS Poor: 4.1 [1.0-Undef.]

Conclusion:
BDX008 developed for immunotherapy of patients with melanoma can be applied to NSCLC patients, and shows a significant separation for OS. Clinical utility of BDX008 will need to be further evaluated. VeriStrat is also prognostic for the same patients. 1. J. Weber et al, “Pre-treatment patient selection for nivolumab benefit based on serum mass spectra”, SITC 2015. 2. V. Gregorc et al, The Lancet Oncology, p713, 15(7), 2014.

Background:
Immune checkpoint blockade have demonstrated durable responses and improvement in overall survival (OS) in approximately 20% of unselected patients with advanced non-small cell lung cancer (NSCLC). To develop reliable, validated biomarkers to identify those subgroups of patients most likely to benefit remains a challenge.

Methods:
We retrospectively analyzed pretreated advanced NSCLC patients (p) receiving anti-PD1 or anti-PDL-1 inhibitors at our institution. The immune-related response Criteria (IRC) was used for response evaluation. Basal and at 6 weeks lymphocytes and eosinophils counts were correlated with efficacy and toxicity.

Results:
44 p were treated between April 2014 and December 2015. We realized evaluation of response in 40 p. 13 p received Atezolizumab, 23 p Nivolumab and 4 p Pembrolizumab. Patients characteristics: 68% males, median age 63 years, 62 % non-squamous histology, 3 % never-smoker, PS 0/1/2 in 43/50/7%. The molecular status assessed in 32 p (KAS mutation 10p). Immunotherapy was the second-line treatment in 26p with a median number of doses received of 5 (range 2-27). Median OS was 12 months (95%CI 3–21) and median PFS was 4 months (95%CI 0.7–7) from the start of checkpoints inhibitors. Overall survival rate at 12 months was 40%. Immune-related adverse events were observed in 15p (37.5%), including 7 grade 3–4 events (17.5%). No drug-related death occurred. We found that there was a significant association between the risk of toxicity and the increased number of eosinophils between the first reassessment and the baseline measurement OR 6, p 0.014. At the time of the first reevaluation, 28p had progression disease (PD), 6 partial response (PR) and 6 stable disease(SD). In 22/28p with progression at first evaluation, CT was realized ≥4 weeks. Pseudoprogression was confirmed in 6/22p (21%). No differences were found in response by gender, ECOG, histology, KRAS status, smoking status, antiPD-1 vs antiPD-L1, or drug used. 15/18p with clinical benefit (PR+SD+pseudoprogression) had basal level of eosinophils ≥100 mm3, (p 0.003 OR 0.114) whilst correlation between response and basal lymphocytes was not statistically significant (p 0.35 OR 0.58). One of 18p with clinical benefit had levels of lymphocytes at the first evaluation <1000 mm3 versus 10/22p with PD(p 0.013, HR 0.13) whilst levels of eosinophils ≥100 mm3 at the first evaluation was not statistically significant (p 0.052 HR 0.26).

Conclusion:
Our very preliminary results suggest that lymphocytes and eosinophils could help us to characterize activity and toxicity of immunotherapy treatments. Further studies are guaranteed.

Background:
Immune checkpoint inhibitors (ICI) have become standard therapy after platinum failure in advanced non-small cell lung cancer. ICI response patterns differ from chemotherapy with the potential for delayed regression and pseudo-progression in patients benefiting from treatment. Additional markers beyond PDL-1 expression are needed to assist in patient selection, response evaluation and treatment decision-making.

Methods:
The relationship between clinical outcome (response, treatment duration, survival) and hematologic parameters (absolute neutrophil count [ANC], neutrophil to lymphocyte ratio [NLR]) was explored in a cohort of patients treated with ICIs at a major cancer centre from 05/2013 to 05/2016. Clinical benefit was defined as achievement of complete or partial response (CR, PR) or stable disease (SD) at 8 weeks. Hematologic parameters at baseline (T0) and on treatment (T1=2 or 3 weeks, T2=8 weeks) were included.

Results:
Of 101 Non-SCLC patients treated with ICIs, 84 (83%) had documented response assessment. All received PD-1 axis inhibitors, (71 anti-PD-1, 12 anti-PDL-1, 1 anti-PDL-1 plus -CTLA-4); tumour PDL-1 expression was +/-/unknown in 32/12/40; median follow-up was 5.1 months (range 0.4 – 36.8) from treatment start. Clinical benefit was seen in 62% (20 PR, 32 SD). Baseline NLR≤4 was associated with greater clinical benefit (75% vs 51%, p=0.025) and median survival (21.7 vs 6.9 months) compared with NLR>4; (HR=0.45, 95% CI: 0.22-0.90, p=0.03). This appears independent of tumour PDL-1 expression. Lower on-treatment (T1, T2) ANC values (trend analysis p=0.0037) and NLR≤4 (T2) were also associated with PR/SD (Table 1). Longer treatment duration was associated with on-treatment NLR≤4 (T1 6.2 vs 3.4 months, p=0.037; T2 6.2 vs 2.7 months, p=0.0075) and lower ANC (T1 p=0.014, T2 p=0.012). Figure 1



Conclusion:
Pretreatment NLR≤4 may be a potential predictor of clinical response in patients receiving ICIs, as well as lower on-treatment ANC and NLR.

Background:
Immune check-point inhibitors are effective for the treatment of advanced non-small cell lung cancer (NSCLC); however, their mechanism of action is associated with peculiar immune-related adverse events (irAEs). While cardiac irAEs are seldom reported, animal data suggest that the myocardium might be sensitive to PD1/PD-L1 impairment. Minimal alterations of Cardiac Troponin-I (CTnI) can identify subclinical cardio-toxicity induced by antineoplastic agents like anthracyclines. The aim of this study is to determine whether CTnI might be used as a biomarker of cardiologic irAEs during treatment with nivolumab in advanced NSCLC.

Methods:
Serum samples were collected and stored from 61 patients receiving nivolumab (3 mg/Kg every 14 days) for advanced NSCLC within a single-institutional translational research study conducted in the San Martino Hospital – National Institute for Cancer Research, Genova, Italy (approved by the local ethical committee); samples were collected at baseline and at each cycle up to 5 cycles, and then every 2 cycles. Cardiac Troponin-I was retrospectively quantified with the luminescent oxygen channeling immunoassay (LOCI™) optimized on the Dimension Vista[®] analytical platform (Siemens Healthcare, Milan, Italy); and defined as undetectable (<0.015 μg/L) or detectable (>0.015 μg/L); a value of 0.045 μg/L was considered significant. Cardiologic anamnesis of the patients with detectable CTnI was collected from clinical documentation; additionally, patients alive at the time of the analysis underwent cardiologic evaluation.

Results:
Fifty-nine patients were evaluable: median age= 69 years (44-81); male/female: 69%/31%; current or former smokers= 86.4%; non-squamous/squamous histology= 80%/20%; median number of cycles= 6 (1-29). Twenty-six out of 351 collected samples had detectable CTnI levels. Thirteen patients (22%) had detectable CTnI levels in at least one sample; among these, 6 (10%) patients had significant alterations in at least one sample, and in 3 cases (5%) this alteration was reported in multiple samples. No specific time-related pattern was identifiable for CTnI alterations. Five patients with detectable CTnI, of which 2 with significant alterations (0.292 μg/L and 0.285 μg/L), had neither evident cardiovascular disease, nor cancer-related para-cardiac infiltration. Two patients had pericardial effusion, while two other had concurrent irAEs (hyperthyroidism and hepatitis).

Conclusion:
Troponin-I was altered in a considerable number of patients receiving nivolumab, in some cases with no evident concurrent cardiovascular disease or manifest indirect noxae. Although a rationale for immunotherapy-related myocardial inflammation is acknowledged, further investigations on the cardiovascular effects of PD1/PDL1 inhibitors are required to draw meaningful conclusions, such as studies involving prospective cardiovascular assessments of patients receiving these agents.

Background:
Recently, the tumor immune environment has been found to play an intricate role in lung cancer progression. Studies have demonstrated that cancer cells can attract neutrophils into the tumor stroma through precise chemokine signaling pathways. Consequently, neutrophils promote angiogenesis, metastasis and inhibit apoptosis, whereas lymphocytes assist with tumor defense. Increased peritumoral neutrophil-to-lymphocyte ratio (NLR) has been shown to mediate T cell anergy and tumor immune evasion. Elevated blood NLR has been shown to correlate with increased tumor neutrophil infiltration and decreased CD3(+) T-cell infiltration in various solid tumor pre-clinical models. Immune checkpoint inhibitors, which harness endogenous lymphocytic response to fight cancer, have recently emerged as the most promising anticancer therapies in Non-Small Cell Lung Cancer (NSCLC). We hypothesized that peripheral blood NLR could be a predictive marker for clinical benefit from immune checkpoint inhibition.

Methods:
We performed a single institution retrospective analysis of 50 patients with NSCLC treated with Nivolumab from July 2015 to April of 2016. Each patient’s NLR was evaluated prior to starting therapy with Nivolumab. A cut-off of 5 was used to classify NLR as high or low based on prior studies. The presence or absence of clinical benefit to therapy was determined. Clinical benefit was defined as objective response or stable disease as per RECIST criteria after at least 3 months of therapy. Descriptive statistics were utilized to summarize the data. Chi-square test was used to compare clinical benefit from Nivolumab in low and high NLR populations.

Results:
There were 48 evaluable patients. 32 patients (66.7%) had a low pre-Nivolumab NLR. 24 (75%) of these patients were noted to have a clinical benefit at 3 months of Nivolumab treatment.16 patients (33.3%) of patients had a high NLR, of whom only 3 patients experienced a clinical benefit (18%). This difference was statistically significant (P=0.05). 2 patients with low NLR were lost to follow up and were not included in the analysis.

Conclusion:
The immune response to cancer is lymphocyte dependent. Lymphopenia, or neutrophilia, resulting in a high NLR may predict for a lack of clinical benefit from Nivolumab. In our study, the majority of patients with low pre-treatment NLR experienced clinical benefit from Nivolumab whereas most of the patients with elevated NLR did not achieve a clinical benefit. Our data suggests that NLR as a predictive biomarker for clinical benefit from immune checkpoint inhibitors should be further investigated in large prospective studies.

Background:
Inmune checkpoint blockade monoclonal antibodies have demonstrated an improvement in the overall survival (OS) of patients with NSCLC. The aim of our research was to explore biomarkers of early death among the routinely used biological parameters in the population diagnosed with advanced lung cancer treated with immunotherapy.

Methods:
In this retrospective study, we collected blood levels of lymphocytes, eosinophils and lactate dehydrogenase (LDH) before inmunotherapy infusion. Inclusion criteria were a diagnosis of stage IV NSCLC, at least one previous line of chemotherapy.

Results:
44 patients (p) were treated between April 2014 and December 2015. Thirteen p received atezolizumab,27 p nivolumab and 4 p pembrolizumab. 70.5% males with a median age of 63 (range 42-82), 29.5% squamous and 2.3% never-smoker patients. Eighteen p had PS 0 (41%), 22p PS1 (50%) and 4p PS 2 (9%). Thirty-one p had ≥ 2 metastatic sites. Immunotherapy was the second-line treatment in 27p. The median number of doses received was 4.5 (range 1-27). Median OS was 12 months (95% CI 3–21) from the start of checkpoints inhibitors. Median PFS was 4 months (95% CI 0.7–7) from the start of checkpoints inhibitors. At 1 year, the overall survival rate was 40%. Eighteen p (41%) died within the first 3 months. The risk of early death based in terms of PS (15p PS0-1), smoking status (1p never-smoker), histology (14p non-squamous), molecular status (5p KRAS), metastases (3p brain metastases), response of previous line and line of treatment (second line in 8/14 p (55 %), was not statistically significant. The risk of early death was higher in 14/18p (78%) had basal LDH levels above the normal range with a difference statistically significant, p 0.036; OR =4.6. However, 11/18 p (62%) had baseline eosinophils <100 mm3, the risk of early death was higher compared to the patients with eosinophils >100 mm3, p 0.04; OR 0.28. The risk of early death based on basal lymphocytes level ≥1000 mm3 or lymphocytes level ≥1500 was not statistically significant. In the multivariate analysis, baseline LDH levels remained as an independent predictor of overall survival but not of early death, p 0.055; HR 4.3 (95% IC 0.9-19).

Conclusion:
Our study suggests that baseline serum determination of LDH and eosinophils might help us to identify patients with NSCLC who achieve more benefit from immunotherapy. Futher studies are guaranteed.

Background:
Nivolumab is an immune checkpoint inhibitor that reactivates cytotoxic T cells against tumor cells in non-small cell lung cancer (NSCLC) patients (pts). There are many ongoing efforts to find predictive biomarkers for immune checkpoint inhibitors. We have previously reported that platelet can selectively bind to leukocytes and, as a consequence, modify their function. To evaluate whether this modification is relevant for the response to checkpoint inhibitors, we determined the percentage of different subsets of leukocytes with bound platelets in the peripheral blood of pts before starting treatment with Nivolumab.

Methods:
Peripheral blood samples were collected at baseline from patients with NSCLC candidates for receiving Nivolumab. After labeling cells with antibodies specific for lymphocytes, monocytes and neutrophils, we added anti-CD41a mAbs (specific for platelets). We next determined the percentage of PDL1+ cells and CD41+ cells in each leukocyte subset by flow cytometry. These results were compared in patients with different clinical response by one-way ANOVA. The clinical response was determined by RECIST v1.1 criteria.

Results:
From January 2015 to February 2016, we collected peripheral blood from 12 pts (4 females and 8 males). Mean age at time to starting Nivolumab was 73 (range 53-86). 4 pts were smokers and 8 former smokers. 5 pts were had squamous cell carcinoma and 6 non-squamous cell carcinoma. 6 Pts received Nivolumab in second line and 6 patients in third line. Response after 3 months with Nivolumab: 4 patients with partial or complete response, 4 patients with stable disease and 4 with progressive disease. There were no differences in the baseline percentages of CD4+, CD8+, Natural Killer cells, B lymphocytes, monocytes and neutrophils among the three groups of response. By contrary, the proportion of monocytes with bound platelets (CD14+CD41+/ total monocytes) was significantly higher in patients with response to nivolumab than those with stable or progressive disease (90.28+7.63%, 33.92+7.02 and 61.44+19.5% respectively, p=0.002). And Also, the PDL1 expression on monocytes was different between the three groups (1.9+0.16, 4.37+1.05 and 2.39+0.73 respectively, p=0.003).

Conclusion:
The functional modification induced by the platelet binding to the monocytes seems to be beneficial for the clinical response to checkpoint inhibitors.

Background:
Checkpoint inhibitors such as nivolumab (anti-PD1) represent a recent breakthrough in the management of patients with advanced non-small cell lung cancer (NSCLC) after disease progression following initial platinum-based therapy. Prospective identification of likely responders remains a challenge as PDL-1 testing, while helpful, is imperfect. Identifying additional indicators is warranted. Studies in melanoma patients demonstrated that analyzing some baseline clinical laboratory parameters had predictive value in the setting of immunotherapy (Weide et al Clin Can Res 2016, and Ferrucci et al BJC 2015). We attempted the same in our lung cancer population.

Methods:
We performed a retrospective analysis in a patient population with biopsy proven advanced NSCLC who received nivolumab. Patient charts were reviewed to obtain data on demographics, ECOG performance status, stage, number of previous therapies, and baseline complete blood count (CBC), from which the ratio of absolute neutrophil count (ANC) to absolute lymphocyte count (ALC) was calculated. Imaging data for response assessment were available. Univariate analysis was performed to study the association between clinical and demographic parameters and progression-free survival (PFS) using SAS software.

Results:
In our cohort of 114 patients treated during 2015-2016, the median follow-up was 5.4 months (range 0-15.8), median age was 67 years (range 40-91), and median number of prior therapies was 2. There were 52% males, 60% Caucasians, 32% Hispanics, 8% African Americans, and 75% had ECOG performance status of 0-1. Our univariate analysis showed the following: Figure 1



Conclusion:
Our data indicates that low baseline ANC/ALC (<5), female gender, and ECOG 0-1 are independent factors associated with significantly favorable PFS in patients with advanced NSCLC treated with nivolumab. A more detailed analysis of a larger cohort, including data on mutational burden, will be presented.

Background:
Programmed death-ligand 1 (PD-L1) is known to be over-expressed in non-small cell lung cancer (NSCLC). However, the impact of chemotherapy on the altered status of PD-L1 expression has not been examined for NSCLC. The present study was intended to examine the impact of neoadjuvant chemotherapy on PD-L1 expression and its prognostic significance in lung squamous cell carcinoma (SCC).

Methods:
Matched tumor samples were obtained from SCC patients prior to and after neoadjuvant chemotherapy. The expression of PD-L1 was evaluated by immunohistochemistry. Survival analysis was performed by the Kaplan-Meier method.

Results:
A total of 76 eligible SCC patients were recruited. There were 51 males and 25 females with a median age of 60 (39-72) years. The smoking status was former (n=46) and never (n=34). Prior to neoadjuvant chemotherapy, PD-L1 expression was identified in 52.6% (40/76) of SCC patients while 61.8% (47/76) were positive for PD-L1 expression after neoadjuvant chemotherapy . Nine patients switched from negative to positive while another two patients’ samples showed the reverse of the above result. Multivariate analysis demonstrated that postoperative expression of PD-L1 was an independent prognostic factor for overall survival (HR=0.50, P=0.003), but not for PD-L1 expression prior to neoadjuvant chemotherapy.

Conclusion:
Neoadjuvant chemotherapy may up-regulate the expression of PD-L1. As compared with the status of PD-L1 expression prior to chemotherapy, the postoperative expression of PD-L1 is a better prognostic factor for overall survival in SCC.

Background:
It has been suggested that certain patients could be primed to respond to anti-programmed cell death-1 (PD-1) therapy due to heightened baseline “immunocompetence,” but data supporting this is limited as is our ability to measure it. The experience with ipilumumab suggests that immune related adverse events (irAEs) experienced by melanoma patients may predict improved clinical outcomes (Weber et al, J Clin Oncol 2012). We retrospectively analyzed NSCLC patients from a single center on the KEYNOTE-001 trial and evaluated the association between treatment related adverse events (trAE) and clinical outcomes.

Methods:
We performed a retrospective analysis of the 97 NSCLC patients treated on KEYNOTE-001 at UCLA with either 2 mg/kg Q3W or 10 mg/kg Q2/3W of pembrolizumab (data cut-off 3/2016). Investigators reported AEs and graded according to CTCAE v4.0, labeling them as unlikely, possibly, or probably treatment related. AEs labeled as possibly/probably related were considered trAEs. The initial scan was at 9 weeks and subsequent scans were every 9 weeks. Investigator assessed irRC was the radiographic assessment used for clinical decisions at individual sites. Progression-free survival (PFS) and overall survival (OS) were calculated using the Kaplan-Meier method and compared using the log-rank test.

Results:
10% (85/826) of AEs reported on trial were considered trAEs. The most frequent trAEs were rash (29%), fatigue (9%), and pneumonitis (8%). The occurrence of a trAE was associated with higher objective response rate (ORR) (OR=0.1509, P=0.0009), PFS (HR=0.3004, P<0.0001) and OS (HR=0.4391, P<0.0001). To assess whether the shorter duration of follow-up in those progressing earlier biased this analysis, additional analyses were performed. The relationship remained, particularly for longitudinal outcomes, when assessed only in patients that continued on trial >9 weeks. This was true both when including trAEs over the entire trial duration (ORR: OR=0.1839, P=0.005; PFS: HR=0.3525, P<0.0001; OS: HR=0.4526, P=0.0008) or when including only trAEs occurring within the first 9 weeks (ORR: OR=0.4063, P=0.1047; PFS: HR=0.5568, P=0.0211; OS: HR=0.6404, P=0.0465). Neither number of prior lines of therapy nor age, gender, or smoking history predicted frequency of trAE occurrence.

Conclusion:
This single center, retrospective analysis, revealed that a trAE predicted for improved clinical outcome with pembrolizumab. When controlling for the inherent bias of asymmetric follow-up, these associations remained. Although this analysis has the weakness of being conducted at a single center representing less than 20% of patients on trial, the strength is that a limited number of investigators assessed if an event was an AE and was treatment related.

Background:
Immunotherapy with anti-PD1 and anti-PDL1 monoclonal antibodies significantly increases overall response rate (ORR) and overall survival (OS) of patients with advanced NSCLC in comparison with second line conventional chemotherapy. Prognostic and predictive factors able to distinguish those patients with a higher benefit from immunotherapy are warranted. Our work retrospectively analyses several clinical, pathological and analytical variables with an eventual potential prognostic and predictive value in daily patients with advanced NSCLC receiving Nivolumab.

Methods:
A retrospective review of clinical charts of patients with advanced NSCLC from fourteen centres of the GIDO group receiving Nivolumab between May-2015 and May-2016 was performed. Age, sex, stage, Performance Status (PS), location of metastases, presence of tumour-related symptoms and comorbidities, number of organs with metastasis, previous chemotherapy, antiangiogenic and radiotherapy treatments, and analytical data from standard blood count and biochemistry were collected and statistically analyzed.

Results:
A total of 175 patients fulfilled inclusion criteria. Median age was 61.5 years. One hundred and twenty-eight male (73.1%), 136 ECOG-PS 0-1 (77.7%), 150 stage IV (86,7%) and 135 had non-squamous carcinoma histology (77.1%). Sixty-five received Nivolumab in second line (37.1%), 66 as third line (37.1%) and 44 as forth or further line of treatment (25.1%). Seventeen patients (9.7%) received antiangiogenic drug in previous line of treatment, and 30 were treated with radiotherapy within 30 days before Nivolumab. Thirty-eight patients had brain metastasis (22%), 39 liver metastasis (22.3%) and 126 had more than one metastatic location (72%). 140 patients were evaluable for response, the ORR was 15.7%, median Progression Free Survival was 2.8 months, and median OS 5.81 months. Stage III (OR 3.57) and time since the beginning of previous line of treatment longer than 6 months (OR 2.52) were associated in multivariable analysis with higher probability of response to Nivolumab. PS 2 vs 0-1 (HR 1.83), time since the beginning of previous line of treatment <6 vs >6 months (HR 1,70) and more than one metastatic location vs one location (HR 1.79) were independently associated with shorter overall OS in multivariable analysis.

Conclusion:
Poor Performance Status, short period of time since the beginning of previous treatment and more than one metastatic location are the clinical-pathological features associated with poorer prognostic in patients with advanced NSCLC treated with Nivolumab. Limitations of the study are the small numbers and the retrospective nature

Background:
The Neutrophil/Lymphocyte ratio (NLR), calculated from peripheral blood tests, represents an independent and easily available prognostic biomarker in numerous cancers, including lung cancer. This study aimed to investigate the prognostic role and the correlation with the therapeutical response of baseline NLR in patients with advanced Non-small cell lung cancer (a-NSCLC) submitted to anti-PD1 therapy.

Methods:
Nivolumab (3 mg/kg intravenously by rapid injection every 14 days) was administered to 47 patients (6 women , 41 men) with a-NSCLC. The mean age of patients was 47 years (range 40-83, SD 9.07), while the histotype was: 28 adenocarcinoma, 18 squamous, 1 adenosquamous. 68% of patients were current/former smokers. 25/47 patients (53%) received more than 2 previous lines of therapy. The baseline absolute neutrophil and lymphocytes count and the Neutrophil /Lymphocytes ratio were recorded. Time to progression (TTP) was statistically evaluated by Kaplan-Meyer method; univariate analysis was conducted by Cox regression method .

Results:
A median of 7.8 (range 1-20) cycles of therapy was administered . A better TTP (3 months vs 1,5 months; Cox regression rate (Hazard Ratio ) 1.000118, p= 0.003 (CI 1.000041-1.000195) was observed in patients with a lower absolute baseline Neutrophil count (Less than 7500); conversely, a higher absolute Lymphocyte baseline count was linked with a longer TTP (3.7 months vs 1.8; Cox regression rate (HR) 0.9994947, p= 0.055 (CI 0.9989785-1.000011). NLR was correlated with Time to Progression (TTP), that was longer in patients with NLR less than 4 (3.71 months vs 1.87 ; Cox regression rate (HR) 1.144335, p= 0.001 (CI 1.068327-1.22575) (Figure) Figure 1



Conclusion:
These preliminary findings highlight the correlation between the NLR and clinical outcome of a-NSCLC patients treated with anti-PD1. Further investigation in this setting is warranted, both to confirm the prognostic role and to investigate if NLR and the microenviromental inflammatory alterations could predict the response to immune-checkpoint inhibitors.

Background:
Mixed response (MR) to cancer therapy is a common, but poorly described phenomenon. MR represents a therapeutic dilemma and is defined by these criteria: 1. At least one tumor has increased in size while another has decreased. 2. One or more tumors have remained stable while another has increased 3. One or more tumors have remained stable while another has decreased 4. A new tumor has developed while other tumors either decrease or remain stable There is a paucity of data regarding both the incidence of this problem and potential mechanisms as to why it happens. Potential etiologies include: tumor heterogeneity; differences in tumor microenvironment; and discrepancies in drug delivery to different tumor deposits. It is also possible that MR simply reflects differences in the rate of resistance emerging. We sought to gain insights into MR in a patient with advanced SCC of the lung.

Methods:
A 65 yo female with squamous cell carcinoma of the lung received 1st-line therapy with cisplatin and gemcitabine followed by vinorelbine. She received 3rd-line therapy with nivolumab. Slight, generalized progression was observed after 4 cycles; however, the patient improved clinically, so treatment was continued. The MR was observed after cycle 8. Both a nonresponding and a responding tumor were biopsied. SCC was confirmed in both samples. NGS was performed.

Results:
Genomic Profiles for Discordant SCC Lung TumorsFigure 1



Conclusion:
This is the first study known to the authors to genomically profile tumors demonstrating a mixed response to systemic therapy. The clinical significance of these aberrations and discrepancies is uncertain. Nevertheless, genomic discrepancies may provide some insight into why MR occurs and this may provide evidence for rational combinatorial therapeutics in patients in who experience a MR. More importantly, it may provide insights into developing novel combinations that may prevent the occurrence of MR.

Background:
Culminating evidence shows the importance of the immune response in NSCLC and other cancer types. TILs seem to be a marker of good prognosis in many different tumor types, including NSCLC. The prognostic importance of PD-L1 expression in NSCLC remains less clear. This study will contribute more information to this topic in NSCLC and will verify the influence of driver mutations on TILs levels and PD-L1 expression. In addition, the predictive role of TILs and PD-L1 expression in EGFR mutants, who received erlotinib, will be evaluated.

Methods:
Clinical data, genetic analysis and tumor biopsies of the FIELT-1 cohort (stage IIIb or IV NSCLC patients with little or non-smoking history) were retrospectively evaluated. PD-L1 expression was evaluated with a PD-1/PD-L1 IHC double staining. TILs were evaluated on H&E slides, using the method developed by an international working group under direction of R. Salgado.

Results:
Measuring stromal TILs on H&E slides proved to be reproducible (ICC=0.74). The measurement of intratumor TILs (ICC=0.16) did not reach the cut-off ICC of 0.70. There was no difference in stromal TILs counts in KRAS (p=0.454) and EGFR (p=0.962) mutant tumors compared to their respective wild type tumors, nor was there any difference in sTILs counts between KRAS and EGFR mutants (p=0.605). The median OS in the general population was 49 weeks. There was a significant difference in median OS between the stromal TILs high tumors and the stromal TILs low tumors (68 weeks vs. 35 weeks respectively; p=0.003). A similar observation was made in the KRAS mutant tumors (95 weeks vs. 12 weeks; p=0.003). In the EGFR mutants no significant difference in median OS could be found according to the stromal TILs counts (p=0.65). There was no difference in the stromal TILs counts of EGFR mutants who responded (p=0.160) or showed clinical benefit (p=0.621) after receiving erlotinib, compared to those who did not. The analysis of the PD-1/PD-L1 double staining has been postponed. Results will be available by the end of August 2016

Conclusion:
The results of the current study reinforce the prognostic role of TILs in NSCLC. Furthermore, this is the first study to confirm that the used scoring method on H&E slides is reproducible in NSCLC. This study is also the first to report about the relation between driver mutation and TILs, with results suggesting that the immune system plays a more crucial role in KRAS mutants than in EGFR mutants.

Background:
Programmed death-1 (PD-1) axis inhibitors are increasingly being used to treat patients with advanced NSCLC. Despite durable responses relative to chemotherapy, resistance to such therapy develops in the majority of responders, with median duration of response from 12-17 months. Mechanisms of acquired resistance (AR) to PD-1 axis inhibitors are poorly understood.

Methods:
Patients with advanced NSCLC and acquired resistance (AR) to PD-1 axis inhibitor therapy were enrolled to an IRB approved repeat biopsy protocol allowing collection of clinical data, archived and fresh tumor tissue, and blood for analysis. Molecular analyses including whole exome sequencing of pre- and post-treatment tumor specimens were performed.

Results:
Twelve cases were available for analysis (table 1). Eight and two patients developed resistance limited to lymph nodes (LNs) and adrenal gland respectively. The two remaining patients experienced tumor progression in LNs with other sites of tumor growth (one in liver, one in lung). Nine patients had sufficient archived pre- PD-1 axis inhibitor tumor tissue for analysis/ comparison, leaving three unpaired cases. Genomic analysis of tumor specimens identified two patients with acquired tumor beta-2-microglobulin (B2M) defects at resistance. A patient derived xenograft generated from one of the resistance samples (patient #6) lacked production of B2M protein and did not express surface MHC-1. Additional analyses including immunophenotyping with multiplexed quantitative immunofluorescence on these and other patient samples are ongoing. Figure 1



Conclusion:
Lymph nodes may be a particularly susceptible area to AR to PD-1 axis inhibitors. Defects in B2M leading to loss of tumor MHC-1 presentation may represent a unique mechanism of AR to immune checkpoint inhibitors. Further studies to determine the frequency of defects in antigen presentation machinery in tumors with resistance to PD1 axis inhibitors are warranted.

Background:
in France, in May 2015, Nivolumab early access program was established for patients with advanced NSCLC progressing during or after platinum-based chemotherapy. There is little evidence of Nivolumab use out of clinical trials. We report here one year of Nivolumab use in a French Universitary hospital.

Methods:
Observational prospective review of patients with advanced NSCLC treated with Nivolumab monotherapy (3 mg/kg/2weeks) in our center, in routine clinical practice. Patients in clinical trial were excluded. Analyze was done on clinico-pathological features, tolerance and outcomes.

Results:
63 patients were included (men: 76.1%, age: 65 (range: 40–78), squamous: 33.3%; smoker: 93.7%%, EGFR/ALK negatives: 98.4%, unknown PDL1: 70%; at least one significant comorbidity: 54%; performans status 0/1/2: 34%/49%/17%; cerebral metastasis: 38%; nivolumab as second, third and more than third lines: 38%/38%/24%. Median number of nivolumab cycles: 6 (1-24), more than 12 cycles: 20.6% Disease control rate : 59% (3 complete responses) ; Clinically significant adverse event: 13 (20%) patients (asthenia: 4 patients, grade 2 to 4 colitis: 3 patients, pneumoniae: 3 patients, nephritis: 1 patient). After Nivolumab, 50% of the patients received an another systemic therapy. Two patients were able to go back to work.

Conclusion:
In real life setting, nivolumab had the efficacy level reported by pivotal clinical trial but with a higher rate of clinically significant adverse events, particularly colitis.

Background:
Programmed death1 (PD-1) pathway is a negative feedback system limiting T cell activity in normal tissues,frequently upregulated by tumors to escape from immune destruction. Blockade of this pathway with anti PD-1 antibodies has shown significant clinical activity in different cancer types; nevertheless it is still unclear why some patients respond to immunotherapies while others do not. Recently it was observed that cancers with higher somatic mutation burden, as tumors with genome instability due to DNA repair defects, develop more elevated anti PD-1 induced neoantigen specific T cell response which results into increased susceptibility to PD-1 blockade. We hypothesize that NSCLCs with polymorphisms of ERCC-1 gene (encoding for a key enzyme of DNA nucleotide excision repair pathway) may be more responsive to PD-1 blockade than ERCC-1 proficient NSCLCs as result of higher rates of mutation due to their genetic instability.

Methods:
We evalueted the rs11615, rs3212986 and rs2298881 ERCC-1 polymorphisms by pyrosequencing analysis on tumor DNA of stage IV previously treated NSCLC patients receiving nivolumab 3 mg/kg q2w.

Results:
Between Jul 8, 2015 and Jan 19, 2016, we enrolled 24 NSCLC patients to receive nivolumab. Patient characteristics were as follows: M/F =18/6; median age (range) = 65 (49-80); ECOG PS, 0/1 = 22/2; sqNSCLC/non sq NSCLC = 6/18; smokers/nonsmokers/former smokers = 10/2/12; EGFR status, mutant/wildtype/unknown = 2/11/11; median nivolumab cycles delivered (range) = 9 (1-22). No patients presented rs11615 and rs2298881 polymorphisms. 8 patients were positive for the rs3212986 polymorphism. The rate of objective response for the entire population was 25% (95% CI, 10 to 47). The ORR was significantly higher in NSCLC patients positive for rs3212986 polymorphism than wild-type NSCLC patients (62.5% [95% CI, 25 to 92] vs. 6% [95% CI, 0 to 30], P=0.006). Among patients positive for rs3212986 polymorphism, median PFS was not reached. In contrast wild-type patients presented a median PFS of 2.0 months (0.21; 95% CI, 0.07 to 0.58; P= 0.004).

Conclusion:
This study suggested that rs3212986 ERCC-1 polymorphism is associated with a higher RR and PFS in advanced NSCLC patients treated with nivolumab. Confirmation of these results in a validation set is ongoing.

Background:
Preclinical studies demonstrated that local radiotherapy (RT) acts synergistically with RNActive[® ]vaccines to increase tumor-infiltrating immune cells and enhance anti-tumor effects. BI 1361849 (CV9202) is an immunotherapeutic cancer vaccine comprising optimized mRNA constituents (RNActive[®]) encoding six NSCLC-associated antigens. Here we report clinical outcomes and immune response data of a phase Ib study, employing local RT and BI 1361849 in advanced NSCLC.

Methods:
Patients (Pts) with stage IV NSCLC and a response or stable disease after first-line chemotherapy or therapy with an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) were enrolled in three cohorts based on histological and molecular NSCLC subtypes (non-squamous vs. squamous vs. EGFR-mutated NSCLC). Pts received two initial vaccinations with BI 1361849 prior to local RT to the primary tumor or a metastatic lesion (four consecutive daily fractions of 5 Gy), followed by further vaccinations until start of another treatment. Maintenance Pemetrexed (mP) and EGFR-TKIs were continued according to the label. Primary endpoint was safety; secondary endpoints included objective response, PFS and OS. Cellular and humoral immune responses were measured ex vivo by multifunctional intracellular cytokine staining, IFN-γ ELISpot, and ELISA in pre- and post-treatment blood samples.

Results:
26 pts were enrolled. 15 pts received mP, two received EGFR TKIs. Most frequent AEs were mild to moderate injection-site reactions and flu-like symptoms. Two pts experienced BI 1361849-related grade 3 AEs (fatigue, pyrexia). No BI 1361849-related SAE or grade 4 AE was reported. Interim results indicate one confirmed PR in a patient receiving mP and SD in 13/25 evaluable pts (52%, 8 pts on mP, 3 pts without maintenance therapy, 2 pts on EGFR-TKI), with two pts showing remarkably long-lasting disease stabilization of up to 72 and 54 weeks, respectively. Shrinkage of lesions outside the irradiated field of ≥15% occurred in 7 pts, all but one receiving mP. Longitudinal assessment of tumor response allows for further insight into patterns of progression. BI 1361849 was capable of eliciting antigen-specific immune responses in the majority of the patients including both cellular and humoral immune responses.

Conclusion:
BI 1361849 elicits antigen-specific immune responses and can be safely combined with local RT and mP treatment. Shrinkage of non-irradiated lesions and prolonged disease stabilization was observed in a subset of pts, mainly in combination with mP. Final clinical outcomes and analyses of cellular and humoral immune responses will be presented.

Background:
The prognosis of patients with advanced Sq-NSCLC worsens with the increase of the number of treatment linesand no effective therapeutic options were available for those refractory patients so far.Nivolumab demonstrated significant benefits against the SoC docetaxel in 2[nd] line treatment of advanced sq-NSCLC. In the real life experience of the EAP we could assess the clinical activity and tolerability of nivolumab not only in patients treated in 2[nd] line but also in patients who had received at least 2 lines of therapy prior than nivolumab.

Methods:
Nivolumab was provided upon physician request for patients aged ≥18 years who had relapsed after a minimum of 1 prior systemic treatment for stage IIIB/stage IV Sq-NSCLC. Nivolumab 3 mg/kg was administered intravenously every 2 weeks for <24 months. Pts included in the analysis had received ≥1 dose of nivolumab and were monitored for adverse events (AEs) using Common Terminology Criteria for Adverse Events (version 4.03).

Results:
210 patients, corresponding to 56.4% of the entire Italian cohort (n=372), received nivolumab after at least 2 prior lines of chemotherapy in the EAP: 120 (57.1%), 69 (32.9%) and 21 (10%) had received 2, 3 and > 3 prior lines of therapy, respectively. Response was evaluable in 204 patients: with a median number of 8 doses (range, 1–24) and a median follow-up of 5.1 months, the disease control rate was 47%, with 3 patients (1%) in complete response, 30 patients (14%) in partial response and 66 patients (32%) in stable disease. 36 patients (17%) were treated beyond RECIST-defined progression, with 11 of them achieving disease control. As of April 2016, median progression-free survival and median overall survival were respectively 3.8 and 11.2 months. 117/210 patients (55.7%) discontinued treatment for any reason except toxicity; 11 out of 210 (5.2%) discontinued due to AEs.

Conclusion:
These findings showed that nivolumab provided clinical activity with a manageable safety profile in patients with advanced, refractory Sq-NSCLC. These data suggest that nivolumab can be a treatment option for patients failing more than one line of chemotherapy.

Background:
Chemotherapy-induced thrombocytopenia (CIT) is a common dose limiting toxicity of clinical chemotherapy drugs, which may lead to reduced dose chemotherapy or delay chemotherapy time, and even terminate chemotherapy treatment. This is the first randomized, open-label, multicenter, phase Ⅲ study to compare the efficacy and safety of prophylactic treatment for thrombocytopenia in China. We tried to investigate the efficacy and safety of preventive application with rhTPO or rhIL-11 to protect against CIT in advanced non-small-cell lung cancer (NSCLC) patients.

Methods:
From June 2009 to July 2016, 108 patients with NSCLC who were receiving the first-line platinum-based chemotherapy suffered from severe thrombocytopenia(the nadir of platelet counts＜50×10[9]/L) during the prior chemotherapy cycle. They were randomized to rhTPO arm or rhIL-11 arm in the following chemotherapy cycle, and the chemotherapy regimens and drug doses were consistent with that in the prior and following cycle (GC Gemcitabine 1000-1250 mg/m[2], D1 and D8; Carboplatin dosing by AUC value=5, D1; Q3W) or GP (Gemcitabine 1000-1250 mg/m[2], D1 and D8; Cisplatin 75 mg/m[2], D1; Q3W). 77 patients (56males, 21 females) were enrolled in rhTPO arm and 31 patients (18 males, 13 females) were enrolled in rhIL-11 arm. There were no statistical difference between two arms in terms of gender, age and the nadir of platelet counts during prior chemotherapy cycle(P＞0.05). rhTPO (15000U/d) was injected subcutaneously on the 2[nd], 4[th], 6[th], 9[th ]Day after the initiation of chemotherapy, and IL-11(3mg/d) was injected subcutaneously per day from Day 9 to Day15 after the initiation of chemotherapy. Blood routines were conducted to test before chemotherapy initiation and the 3[th], 5[th], 7[th], 9[th], 11[th], 13[th], 15[th], 17[th], and 21[th] day after chemotherapy. Toxicity and efficacy were monitored.

Results:
In the following chemotherapy cycle there were no statistical difference between rhTPO arm and rhIL-11 arm on the following indexes: the nadir of platelet counts(62.6±39.4×10[9]/L vs. 52.8±36.8×10[9]/L, P＞0.05) , the maximum platelet counts (223.5±127.3×10[9]/L vs. 245.8±158.7×10[9]/L, P＞0.05) , duration of platelet counts less than 50×10[9]/L[Median (95%CI): 4.0(3.0-5.0) days vs. 4.5(3.0-9.0) days, P＞0.05], time of platelet count recovered to 75×10[9]/L [Median(95%CI): 5(3-7) days vs. 6(4-8) days, P＞0.05] and to 100×10[9]/L[median(95%CI): 6(6-8) days vs. 6(5-9) days, P＞0.05]. Drug-related adverse events in rhTPO arm were less than those of rhIL-11 arm (5 cases (6.49%) in rhTPO arm, 8 cases (25.8%) in rhIL-11 arm, P<0.05).

Conclusion:
Although there is no statistical difference on efficacies, prophylactic administration of rhTPO is safer and more convenient than that of rhIL-11 in advanced NSCLC patients.

Background:
The prognosis of NSCLC patients (pts) with brain metastases is still quite poor. These pts usually do not meet the inclusion criteria to be enrolled in clinical trials. Nivolumab Italian Expanded Access Program (EAP) allowed this subpopulation of pts to be included, providing the opportunity to evaluate safety and efficacy of nivolumab treatment in pts with brain metastases.

Methods:
upon physician written request, nivolumab was provided to pts who met the following inclusion criteria: aged ≥18 years, who had received a diagnosis of squamous NSCLC, and who had relapsed after a minimum of one prior systemic treatment for stage IIIB/stage IV squamous NSCLC. Nivolumab is administered intravenously at the dose of 3 mg/kg every 2 weeks for a maximum duration of 24 months. We describe efficacy and safety of nivolumab in pts who received at least one dose. Adverse events were monitored using Common Terminology Criteria for Adverse Events.

Results:
from our cohort of 372 patients diagnosed with squamous NSCLC, we report the results of 38 (10.2%) pts with treated and asymptomatic brain metastases. In these pts, with median follow-up of 4.5 months and median number of doses of 6 (range, 1–18), disease control rate was 47.3%, including 1 complete response, 6 partial responses and 11 stable diseases. Treatment beyond RECIST defined progression was allowed, under protocol defined circumstances, in 4 pts. Median progression-free survival was 5.5 months, and overall survival was 6.5 months (data lock of April 2016). Out of the 38 pts included, only 1 discontinued treatment due to AE (2.6%), whereas 21 pts (55.3%) discontinued treatment for non-toxicity related reasons.

Conclusion:
although preliminary, these results demonstrate efficacy of nivolumab in squamous NSCLC pts with brain metastases. Safety of nivolumab in these pts is consistent with previously reported data from clinical trials. These results suggest nivolumab could be beneficial in this subpopulation of pts with unfavourable prognosis.

Background:
Nivolumab monotherapy has shown survival benefit in patients (pts) with melanoma, lung cancer, renal cell carcinoma and head and neck cancer. The experience of pts and physicians in routine clinical practice is often different from those in a controlled clinical trial setting. Here, we report efficacy and safety of nivolumab monotherapy in pts with squamous non small cell lung cancer (Sq-NCSLC) treated in the nivolumab Expanded Access Programme in Italy.

Methods:
Nivolumab was available upon physician request for pts aged ≥18 years who had relapsed after a minimum of one prior systemic treatment for stage IIIB/stage IV Sq-NSCLC. Nivolumab 3 mg/kg wass administered intravenously every 2 weeks to a maximum of 24 months. Pts included in the analysis had received at least 1 dose of nivolumab and were monitored for adverse events (AE) using Common Terminology Criteria for Adverse Events.

Results:
In total, 371 Italian pts participated in the EAP across 96 centres and 363 patients were evaluable for response. With a median follow-up of 5.2 months (range 0-12.9) and a median of 7 doses, the best overall response rate (BORR) was 18%, with 3 complete responses (CR) and 62 partial responses (PR), and the disease control rate (DCR) was 47%. DCR was comparable among pts regardless previous lines of therapy, brain metastasis, age and smoking habits. A non-conventional benefit was observed in 23 (17 SD and 6 PR) out of 66 pts treated beyond RECIST defined progression. As of April 2016, median progression-free survival and median overall survival were 3.9 (95% CI: 3.2-4.6) and 9.1 (95% CI: 6.7-11.5) months, respectively. Regarding the safety profile, 267 out of 371 pts (72%) had at least one AE of any grade, considered to be drug-related in 106 pts (29%). Grade 3/4 AE were reported in 66 pts and considered to be drug-related in 20 pts (5%). AE were generally manageable following the specific guidelines.

Conclusion:
To date, this is the largest clinical experience with nivolumab in a real-world setting. These preliminary EAP data seems to confirm the efficacy and safety data of nivolumab from registrational trials, supporting its use in current clinical practice for pre-treated pts with Sq-NCSLC.

Background:
The efficacy and safety of nivolumab in patients with squamous NSCLC (sq-NSCLC) have been demonstrated in several trials including the phase 3, randomized, controlled CheckMate 017 study whose results led to the approval of the product for this indication. However, data on the use of nivolumab in the real world setting is still limited and collecting it is paramount. The Italian nivolumab EAP for sq-NSCLC represents an important source of information in that respect. The current analysis describes results of the use of nivolumab in the group of EAP patients aged >75 years.

Methods:
Nivolumab was provided upon physicians’ request for patients aged ≥18 years who had relapsed after a minimum of one prior systemic treatment for stage IIIB/stage IV Sq-NSCLC. Nivolumab 3 mg/kg was administered intravenously every 2 weeks for <24 months. Patients included in the analysis received ≥1 dose of nivolumab and were monitored for adverse events (AEs) using Common Terminology Criteria for Adverse Events.

Results:
70 out of 372 (18.8%) patients with advanced Sq-NSCLC participating in the EAP in Italy were ≥75 years old and 68 of them were evaluable for response. With a median number of doses of 7 (range, 1–20) and a median follow-up of 4.7 months, the disease control rate was 42.9%, including 13 patients with a partial response and 17 with stable disease. 16 pts were treated beyond RECIST-defined progression and 5 of them achieved disease control. As of April 2016, the median progression-free survival and median overall survival among those elderly patients were 3.2 and 7.6 months, respectively. Among 70 pts, 41 pts (58.6%) discontinued treatment for any reason except toxicity; 8 out of 70 discontinued due to AE (11.4%).

Conclusion:
This analysis, conducted on elderly patients with sq-NSCLC in a real life setting, suggests that nivolumab is an effective and well tolerated treatment for this special population.

Background:
Nivolumab showed durable antitumor activity and survival benefit in previously treated patients with advanced non-small cell lung cancer (NSCLC). The aim of this study was to evaluate the efficacy and safety of nivolumab in elderly (≥75 year old) or poor performance status (PS; ≥2) patients with NSCLC, most of who have been excluded from clinical trials.

Methods:
This was a retrospective cohort study investigating the outcome of patients with advanced or post-operative recurrence NSCLC who received nivolumab from January to April 2016 in the National Cancer Center Hospital. Patient characteristics, the efficacy of nivolumab, survival, and adverse events were analyzed. Immunohistochemical (IHC) expression of programmed cell death ligand 1 (PD-L1) in pretreatment tissue (10 biopsy and 3 operation specimens) was assessed using the rabbit monoclonal PD-L1 antibody (clone 28-8; Abcam). Staining of ≥1% of tumor cells was considered as the cut-off value of positive PD-L1 expression.

Results:
A total of 20 patients, including 10 elderly patients with non-squamous NSCLC and 10 poor PS patients (7 with non-squamous and 3 with squamous NSCLC), received nivolumab. Objective responses were observed in 6 patients: 4 (40%) elderly and 2 (20%) poor PS patients. The median progression-free survival was 2.8 months and 1.7 months in the elderly and poor PS groups, respectively. Of 13 cases with quantifiable IHC results, 10 cases were positive for PD-L1. PD-L1 expression was not predictive of a response, which occurred in 4 of 10 (40%) patients with PD-L1-postive tumors, 1 of 3 (33%) patients with PD-L1-negative tumors, and 1 of 7 (14%) patients with an unknown PD-L1 status (p = 0.52). Treatment-related adverse events led to discontinuation of nivolumab in 6 patients (1 elderly and 5 poor PS patients). Figure 1



Conclusion:
Nivolumab had a clinically meaningful response for elderly or poor PS advanced NSCLC patients, but toxicity led to the discontinuation of nivolumab.

Background:
Nivolumab is an immune checkpoint inhibitor antibody and it has demonstrated durable responses and tolerability in pretreated patients with advanced NSCLC. This is an observational study to evaluate the efficacy and safety of nivolumab in previously treated patients with advanced NSCLC in the expanded access programme.

Methods:
Elegibility criteria included, histologically confirmed NSCLC clinical stage IIIB vs IV, evaluable disease, at least one prior therapy, performance status of 0/1 and an adequate organ function. Exclusion criteria included, positive test for hepatitis B, C or human immunodeficiency virus, severe autoinmune disease and patients with systemic corticosteroids or immunosuppressive medications. Patients received nivolumab 3 mg/kg IV (60 min) every 2 weeks until progressive disease (PD) or unacceptable toxicity. The aim of the study was to report the efficacy and safety profile of Nivolumab in pretreated patients with advanced NSCLC of our everyday clinical practice. The exploratory assessments include response rate (RR), progression-free survival (PFS), overall survival (OS) and treatment related adverse events (AEs).

Results:
From August of 2015 to February of 2016, with a median follow time of 7 months, 66 patients were enrolled from 7 different centers. The patients demographics were: median age 60 years, 19 female and 47 male; 7 never smoked and 59 former or current smoker; 45 patients adenocarcinoma, 4 large-cell carcinoma, 12 squamous-cell carcinoma and 4 NSCLC; 20 stage IIIB and 46 stage IV; 17 with central nervous system metastasis; 30 received 2 or more prior therapy lines and 62 had PS 1. Among 48 patients evaluated, 3% had complete response, 21% partial response, 27% disease stabilization and 21% disease progression. At the time of database lock, the median of PFS was 2.03 IC 95% (1.2-2.7) and OS was not reached. Grade 1-2 treatment related adverse events (AEs) occurred in 38 patients and the most common ones were asthenia (25), rash/pruritis (12), anorexia (7), endocrine (5) and diarrhea (4). Each of the toxicities were manageable and there were no grade 3-4 AEs or treatment-related deaths.

Conclusion:
Early data from this study suggests that Nivolumab is effective and well tolerated in patients with pretreated advanced NSCLC.

Background:
Nivolumab is a fully humanized, IgG4 antibody that inhibits the programmed cell death protein 1 (PD-1) immune checkpoint. It has demonstrated durable responses and tolerability in patients with treatment resistant, advanced non-small-cell lung cancer (NSCLC). This retrospective study evaluates the efficacy and safety of nivolumab, which was approved for the treatment of advanced NSCLC in December 2015 in Japan.

Methods:
This study comprised 50 patients with advanced or recurrent NSCLC who were administered with nivolumab 3mg/kg IV every 2 weeks from December 2015 through April 2016 at Matsusaka Municipal Hospital.

Results:
Patient demographics were as follows: a median age of 69 years (range: 53–86); 17 females and 33 males; 12 non-smokers and 38 former or current smokers; 47 patients with ECOG performance status (PS) of 0 or 1 and three with a PS of 2; seven patients with postoperative recurrence, nine with post-definitive chemoradiotherapy, 31 with stage IV disease, and three with others; 14 patients with squamous cell carcinoma, 33 with adenocarcinoma, two with pleomorphic carcinoma, and one with NSCLC NOS; 17 patients received nivolumab as second-line and 33 patients as third-line therapy or later; and six patients with EGFR mutation and one with ALK rearrangement. Among 50 patients, nine showed partial response, 17 showed stable disease, and 22 showed progressive disease, 2 were not evaluated yet. Five patients experienced an initial increase in the size of their tumor lesions, but with a subsequent decrease in tumor burden. At the time of submission, the median PFS was 3.8 months, with OS yet to be evaluated. Grade 3–4 AEs occurred in seven patients, with Grade 5 AEs occurring in only one patient.

Conclusion:
Early data from this study suggests that nivolumab is effective and well tolerated in patients with advanced or recurrent NSCLC in a real clinical setting.

Background:
Patients with metastatic NSCLC progressing to 1L have a poor prognosis. Nivolumab is an anti-PD-1 monoclonal antibody, which has shown to prolong overall survival (OS) in patients who have progressed to platinum-based chemotherapy. We report our experience with nivolumab in pretreated metastatic NSCLC patients.

Methods:
Retrospective study of patients with metastatic NSCLC treated with nivolumab (3 mg/kg every 2 weeks) in second line (2L) and subsequent lines (SL). We evaluate response rate (RR), progression-free survival (PFS), OS and toxicity.

Results:
Twenty patients were included (2L: 17, SL: 13). Median age: 68 years. Histology: Adenocarcinoma (60%), Squamous cell (33%), Large cell (7%). ECOG PS: ECOG 0-1 (83%), ECOG 2 (17%). Median number of cycles: 8. RR (Twenty-three patients evaluable for response): Complete response (9%), partial response (35%), stable disease (26%), disease progression (30%). Objetive response rate (ORR) in 2L vs SL: 55% vs 33%, p=0.30. ORR in squamous vs non-squamous: 25% vs 47%, p=0.40. Median follow-up: 6 months. PFS and OS events at the time of analysis: 43% and 33%, respectively. Median PFS and OS: 7 months and not reached (NR), respectively. PFS in 2L vs SL: NR vs 5 months, HR 0.81, p=0.71. PFS in squamous vs non-squamous: 5 months vs NR, HR 1.39, p=0.58. OS in 2L vs SL: NR vs NR, HR 1.53, p=0.50. OS in squamous vs non-squamous: 7 months vs NR, HR 2.61, p=0.14. The incidence of adverse events was low. The most frequent toxicity (any grade) was asthenia (67%). A patient with chronic liver disease had hepatotoxicity grade 1 and continued treatment. Three patients discontinued treatment due to toxicity: pneumonitis grade 3 (1), rash grade 3 (1), impaired renal function grade 3 (1). There were no toxic deaths.

Conclusion:
In clinical practice, nivolumab is effective and safe in 2L and SL in patients with metastatic NSCLC.

Background:
Nivolumab is an IgG4 monoclonal antagonist antibody to PD-1 that is approved for the treatment of patients with advanced squamous and non-squamous NSCLC with progression of disease on or after standard platinum-based chemotherapy, regardless of tumor PD-L1 protein expression. The aim of our study is to evaluate the efficacy and safety of nivolumab in this group of patients.

Methods:
We enrolled 23 patients with squamous and non-squamous NSCLC, stage IIIB-IV,19 males and 4 females, with median age 68 years who had failed two or more lines of systemic platinum based chemotherapy. All patients received at least 4 doses of nivolumab as monotherapy, at a dose of 3 mg/kg once every 2 weeks intravenously, until disease progression or unacceptable toxicity.

Results:
3 (13%) of 23 patients had an objective response as assessed by RECIST criteria and all of the responses were ongoing at the time of analysis. 19 (82.6%) of 23 patients had stable disease and one experienced progression of the disease. 2 (9%) of 23 patients reported treatment-related adverse events, including peripheral edema ,one (4%) with pleural effusion and one (4%) with pericardial effusion, which all were well tolerated and treated. No deaths were attributed to nivolumab.

Conclusion:
Nivolumab has clinically meaningful activity and a manageable safety profile in previously treated patients with advanced, resistant, squamous and non squamous non-small cell lung cancer.

Background:
Currently, treatment options are limited for patients with advanced and/or metastatic NSCLC particularly after initial treatment. In a prior phase 1 study, abemaciclib, a CDK4 & 6 inhibitor, demonstrated single-agent anti-tumor activity when dosed orally on a continuous schedule, with an acceptable safety profile in patients with previously treated metastatic NSCLC (NCT01394016). PI3kinase is an escape pathway after CDK inhibition in tumor models and aberrant immunity is a hallmark of cancer, providing the rationales to combine abemaciclib with PI3K and with checkpoint inhibitors. An ongoing Phase 1b multicenter, open-label, 3+3 dose-escalation trial with an expansion phase is investigating abemaciclib in combination with multiple single-agent options in metastatic NSCLC (NCT02079636). Here we report preliminary results for two arms of the study.

Methods:
In Part D, abemaciclib was administered orally on a continuous schedule every 12 hours (q12h) in combination with the PI3K/mTOR inhibitor, LY3023414, at 100, 150, or 200 mg q12h. In Part E, abemaciclib was administered in combination with the anti-PD-1 antibody, pembrolizumab (200 mg I.V. infusion q3 weeks). Patients with late stage NSCLC and 1-3 prior therapies without central nervous system metastasis were treated until disease progression or other discontinuation criteria were met. Primary endpoints for each cohort included safety/tolerability and identification of the recommended phase 2 dose. Safety assessments followed the Common Terminology Criteria for Adverse Events (NCI-CTCAE v4.0). Parts D and E began enrolling patients on April 13, 2015 and April 29, 2016, respectively.

Results:
As of August 24, 2016, Parts D and E escalation included, respectively, 22 [male (64%)/Caucasian (77%)/stage IV (91%)/adenocarcinoma (91%)] and 6 patients [male (33%)/Caucasian (100%)/stage IV (67%)/adenocarcinoma (100%)]. ECOG PS was ≤1 in both cohorts. In Part D, 1 patient on dose level-2 (DL-2) experienced a dose limiting toxicity (DLT) (G4 thrombocytopenia). Evaluation of additional dose levels is ongoing. Seventeen patients (77%) experienced ≥1 treatment-related emergent adverse event (TRAE). Common TRAEs were nausea (50%), diarrhea (50%), vomiting (36%), fatigue (32%), and decreased appetite (27%). In Part E, no DLTs or deaths occurred in the two dosing cohorts evaluated. Four patients (67%) experienced ≥1 TRAE with 75% G1/2. Common TRAEs included fatigue (50%), diarrhea and proteinuria, (33%, each).

Conclusion:
The majority of previously treated advanced/metastatic NSCLC patients administered abemaciclib with LY3023414 or with pembrolizumab had manageable and tolerable adverse events, similar to those of the single agents.

Background:
Programmed cell death-1 (PD-1), plays a pivotal role in tumor immune escape. Recently, antibodies targeting PD-1 and PD-L-1 have been approved for treatment of advanced Non Small Cell Lung Cancer (NSCLC). In this pilot study, we aimed to investigate the effect of anti-PD1 treatment or chemotherapy on the frequencies of circulating PD-1[+] T cells and PD-L1[+] immunosuppressive cells in NSCLC patients.

Methods:
Peripheral blood samples were collected from 35 advanced NSCLC patients before initiation of treatment and after 3 cycles. Twelve treatment-naïve patients received front-line chemotherapy, whereas 23 patients received anti-PD1 treatment in the second-line setting. Flow cytometry was used to quantify PD-1- and PD-L1-expressing immune cells. Changes in the frequencies of these cells were compared between the two settings and correlated with the clinical outcome.

Results:
Chemotherapy had no effect on the percentages of PD-1[+]CD4[+] and PD-1[+]CD8[+] T cells after 3 cycles, whereas there was a significant decrease in PD-1[+]CD4[+] and PD-1[+]CD8[+] T cells in patients who received 3 administrations of anti-PD1 antibody (p=0.007 and p=0.05, respectively). Moreover, the levels of PD-1[-]CD4[+] (p=0.009) and PD-1[-]CD8[+] (p=0.009) were increased in response to anti-PD-1 therapy. The frequencies of both peripheral CD4[+] Treg (CD3[+]CD4[+]CD25[high]CD127[-/low]CD152[+]FoxP3[+]) and granulocytic MDSCs (G-MDSC; CD14[-]CD15[+]CD33[+]CD11b[+]HLA-DR[-]Lin[-]) expressing PD-L1 were decreased following anti-PD1 therapy (p=0.01 and p=0.02, respectively). In contrast, after 3 cycles of chemotherapy, the levels of PD-L1[+]CD4[+] Treg were increased, but not of the PD-L1[+]G-MDSC (p=0.04). Anti-PD-1 treatment significantly reduced the percentages of PD-1[+]CD4[+], PD-1[+]CD8[+] T cells, PD-L1[+]CD4[+] Treg and PD-L1[+]G-MDSCs when compared to the effect of first line chemotherapy (p=0.04, p=0.05, p=0.002 and p=0.01, respectively). Furthermore, a significant decrease of PD-1[+]CD8[+] T cells, PD-L1[+]CD4[+] Treg and PD-L1[+]G-MDSCs after 3 doses of anti-PD-1 was observed in patients who experienced stable disease compared to baseline (p=0.006, p=0.05 and p=0.03, respectively). At the time of response evaluation to chemotherapy, the percentage of the PD-L1[+]CD4[+] Treg after 3 cycles was significantly decreased compared to baseline, in disease progressors (p=0.04).

Conclusion:
Treatment with anti PD-1 antibodies significantly reduces the levels of PD-1+ effector cells, as well as the PD-L1+ suppressive Treg and G-MDSCs. In contrast chemotherapy led to an increase of PD-L1+ Treg. These data indicate that treatment with anti-PD1 agents have an overall positive effect on immune system by reducing the immunosuppressive cells and increasing the effector cells. Additional studies are needed in a larger cohort in order to document its impact on their clinical relevance in NSCLC patients.

Background:
Mutation of the tumor suppressor gene NF2 was detected in 30-40% of malignant pleural mesothelioma (MPM) patients. NF2 suppresses tumorigenesis in part by inhibiting Cullin4 ubiquitin ligase. Cullin4A (CUL4A) gene amplification and its’ overexpression has been detected in MPM cell lines and tumors. We hypothesized that cullin4 is a potential treatment target for MPM. Cullins’ activity can be blocked by the inhibition of neddylation, a post-translational modification for cullins. In this study we assessed the efficacy of pevonedistat, an inhibitor of protein neddylation.

Methods:
Thirteen MPM cell lines and 3 MPM primary cells grown in monolayer (2D) were employed to assess the efficacy of pevonedistat in vitro compared to normal mesothelial cells, using MTT assay. The expression of cullins was assessed by quantitative real time PCR and western blot. Cell cycle was analyzed by flow cytometry. Four cell lines were cultured in multicellular spheroid (3D) format and measured for viability by acid phosphatase assay.

Results:
Five MPM cell lines overexpressing CUL4A are highly sensitive to pevonedistat (figure1). The treatment induced G2 cell cycle arrest and accumulation of cells containing >4N DNA content, representing cells undergoing DNA re-replication. DNA re-replication is known to be mediated by the accumulation of a DNA replication licensing factor, CDT1. Indeed, higher CDT1 accumulation was detected in the sensitive compared to the resistant cell lines. All primary cells showed no CUL4A overexpression compared to normal mesothelial cells, nonetheless 2 of them were sensitive to pevonedistat. Interestingly, these cells exhibited higher levels of neddylated (activated) CUL4A and higher CTD1 accumulation following the treatment. Cells lines overexpressing CUL4A remained sensitive to pevonedistat when grown in 3D spheroids. Figure 1



Conclusion:
Inhibition of cullins by pevonedistat induced growth arrest preferentially in MPM cells overexpressing CUL4A in 2D and 3D cultures. The major mechanism seems to be mediated by DNA re-replication induced by CDT1 accumulation.

Background:
Malignant pleural mesothelioma (MPM) represents an aggressive malignancy with dismal prognosis and limited therapeutic options. MPM occurs in three main histological subtypes: epithelioid, sarcomatoid and biphasic, which are characterized by differences in morphological growth pattern, aggressiveness and patient prognosis. However, the mechanisms and causes responsible for the different cell morphologies are poorly understood. Epithelial-mesenchymal transition (EMT) has been implicated in cancer progression and chemoresistance, but its role in MPM is not well understood. Fibroblast growth factor (FGF) signals promote cell growth, survival and aggressiveness in several tumors including mesothelioma. Aim of this study was to characterize growth factor-induced, EMT-like changes with respect to the MPM histological subtypes.

Methods:
Morphological and behavioral changes of treated cell models were analyzed by morphometry, immunoblotting and functional assays. Alterations in gene or microRNA expression were evaluated via qPCR and array hybridization. Pathway enrichment analysis was based on KEGG.

Results:
In several cell lines established from biphasic MPM, treatment with FGF2 and EGF induced morphological changes reminiscent of EMT and aggressive behavior such as scattering, increased migration, proliferation and invasiveness. Inhibition of the fibroblast growth factor receptors (FGFR) or the MAPK axis via small-molecule inhibitors could prevent these changes and, in cell lines with sarcomatoid-like shape, reverse scattering and induce a more epithelioid morphology. Comparable results were obtained using an engineered FGFR1 enabling contactless activation via blue light. Analyses of genes and microRNAs regulated by FGF2 or EGF showed an overlap with previously established EMT markers but also identified several novel potential markers such as MMP1, ESM1, ETV4, PDL1, ITGA6 or BDKRB2. Blocking the FGFR or MAPK pathways resulted in the opposite regulation of these genes. Inhibition of MMP1 via siRNAs or pharmacological inhibitors prevented FGF2-induced scattering and invasiveness. In unsupervised clustering, the gene expression profiles of solvent- or cytokine-treated cells were associated with those of epithelioid and sarcomatoid MPM, respectively. Immunohistochemistry showed an association of MMP1 as well as phospho-ERK with the sarcomatoid part of tissue specimens from biphasic tumors. Pathway enrichment analysis of differentially expressed genes as well as the targets of altered microRNAs after FGF2 treatment showed that the regulated genes are assigned to categories important for cell growth and aggressive behavior.

Conclusion:
Our data characterize FGFR-mediated signals as important players in MPM aggressiveness and the morphological and behavioral plasticity of mesothelioma cells, leading to a better understanding of the link between the MPM histological subtypes and their influence on patient outcome.

Background:
Malignant pleural mesothelioma (MPM) initially arises not from the visceral pleura but parietal pleural mesothelial cells in the thoracic cavity. MPM has a close relationship to asbestos exposure in etiology. The carcinogenic potential of asbestos fibers has been linked to their geometry, size, and chemical composition. Long respirable fibers(length>5μm, diameter<3μm) have an increased potential to cause mesothelioma. Asbestos also induces non-neoplastic diseases of the pleura. Pleural plaques are thought to be formed by lymphatic transport of short asbestos fibers from lung parenchyma to lymphatic stomata in the parietal pleura, with the fibers undergoing phagocytosis by macrophages in the submesothelial layer to synthesize collagen. Long fibers are lodged and retained at these stomata orifices to lead to asbestos carcinogenesis. Plaques, almost always, are produced in the parietal pleura, of which surface is covered with a single mesothelial cell layer. In this study, we evaluated whether mesothelium covering pleural plaque was primarily involved in asbestos-induced mesothelial carcinogenesis in human.

Methods:
40 patients with MPM were received a medical thoracoscopy with narrow band imaging(NBI) and autofluorescence imaging(AFI), in addition to white light under local anaesthesia. 10 patients were T1, and 8 were T2 clinical stage. All patients had a free thoracic cavity with pleural effusion. 40/32（80％）patients had a history of asbestos exposure(ex. 20 occupational exposure, 7 environmental exposure, 5 none).

Results:
Small nodules of mesothelioma and plaques were present simultaneously on the parietal pleura in 15 MPM patients. NBI could depict the blood vessels on parietal pleural surface more clearly than white light. T1 tumors changed in color to Brown with NBI, and to magenta with AFI. Plaques were usually sharply demarcated from surrounding the parietal pleura, and were avascular and raised hard yellow to white lesions. Individual plaques were smooth surfaced or composed of small rounded knobs. Small nodules of T1 tumors were visualized on the parietal pleura except for the surface of pleural plaques, where neo-vascularization was clearly demonstrated with NBI. With progress of the clinical stage of MPM(T1⇒T2), implanted small nodule came to be seen on the surface of pleural plaque with AFI and NBI.

Conclusion:
Thoracoscopical examination for early clinical stage of MPM shows that the origin of MPM is the mesothelial cells in the parietal pleura, and that mesothelium covering on the surface of pleural plaque was not primarily involved in asbestos-induced mesothelial carcinogenesis in human.

Background:
Recent clinical studies have demonstrated positive correlation between tumour mutational burden and response to immune checkpoint inhibitors (CPI) in several malignancies. Although initial reports of some CPI in Malignant Mesothelioma (MM) have shown promise, the rate of somatic mutations in MM is known to be low and copy number aberrations (CNA) are the prominent genetic alterations. Using a large cohort of MM patients, we investigated CNA, PD-L1 expression and the surrounding immune infiltrates and correlated these parameters to clinicopathological features.

Methods:
Tissue microarrays (TMA) were constructed and stained with PD-L1 (E1L3N,CST, Massachusetts), CD4, CD8 and Foxp3 antibodies. PD-L1 positivity (PD-L1+) was defined as >5% membranous staining regardless of intensity and high positive as >50%. Genomic DNA was obtained from tumour cores of a representative subset (100 patients) and used for genome-wide copy number analysis. Percent genome aberrated (PGA) was computed for each sample as the total number of base pairs within altered regions, divided by the total number of base pairs in each region included in the array. Correlations of PGA, CNA profile and individual aberration (loss/gain) frequency with parameters including PD-L1 expression and survival were explored.

Results:
Amongst 329 patients evaluated, the median age was 67 years and most were male 274(83.2%). Epithelioid histology (N=203; 62.9%) was the commonest. PD-L1 positivity was seen in 41.7% with high positivity in 9.6%. PD-L1+ correlated with non–epitheloid histology (P=<0.0001) and increased infiltration with CD4, CD8 and FOXP3 lymphocytes. High PD-L1 expression correlated with worse prognosis (HR=2.37; 95%CI: 1.57-3.56; P=<0.0001) on univariate analysis but the effect was found to be time dependent. Neither PGA (P=0.57) nor CNA profile (P=0.76) were found to be associated with PD-L1 expression. After correction for multiple testing, no individual CNA count was significantly associated with PD-L1 status. Although epithelioid histology had higher PGA (P=0.04), high PGA was associated with poorer survival (HR=2.01; 95% CI: 1.24-3.26; P=0.004). This was also true when only epithelioid tumours (n=63) were considered.

Conclusion:
Increased genomic alterations in MM did not correlate with PD-L1 expression but was associated with poorer survival. High PD-L1 expression was associated with non-epithelioid MM, poor clinical outcome and increased immunological infiltrates.

Background:
Mesothelioma remains an incurable cancer with limited therapy. Genetically targeted personalised treatment strategies are currently lacking. Mesotheliomas harbor frequent loss of chromosome 9p21.3 locus encoding for CDKN2A and frequently encompassing methyladenosine phosphoryl transferase (MTAP). Loss of MTAP has recently been shown to be associated with dependency on the symmetrical demethylation of arginine-4 on histone H4 methyltransferase PRMT5. We sought to determine whether mesothelioma cells with MTAP HD would be vulnerable to inhibition of PRMT5, and to explore the pharmacodynamics associated with its suppression.

Methods:
Genome-wide copy number variation (CNV) analysis was undertaken in 94 patients. CNVs were determined using the array based platform, Affymetrix Oncoscan v3. Multiregional whole exome sequencing was also performed on samples from 6 patients. The expression of MTAP and the effect of the drugs tested on H4R3Me2s was evaluated by western blot. Cell growth was analysed by clonogenic assay after focused RNAi targeting MTAP and/or PRMT5, or treatment with a PRMT5 inhibitor

Results:
Oncoscan analysis identified homozygous loss of CDKN2A in 50%, and heterozygous loss in 20.2% of patients. Homozygous loss of MTAP was seen in 39.3% and heterozygous loss in 28.7%, 76.6% of patients had concurrent loss of CDKN2A and MTAP. Homozygous MTAP deletion was found to be present in all regions of tumour ie a truncal deletion, in 3 out of 6 patients. In 65 patients treated with surgery only, homozygous loss of CDKN2A or MTAP was prognostic for progression free survival (CDKN2A: 7.5 vs. 32.9 months, HR 4.536 95%CI 1.765-11.659, p=0.002; MTAP: 7.5 vs. 18.4 months, HR 3.289 95%CI 1.314-8.237, p=0.007). To determine the dependency of MTAP negative cells on PRMT5, we used either siRNA targeting PRMT5 or a PRMT5 inhibitor. PRMT5 silencing did not induce measurable apoptosis however a significant suppression of clonogenic activity was observed after 10 days in MTAP negative cells(H2591). The same effect was observed after concurrent silencing of PRMT5 and MTAP in MTAP positive mesothelioma cells(MPP89). We then utilised a PRMT5 small molecule inhibitor, EPZ015666, to recapitulate RNAi knockdown. Although a clear suppression of H4R3Me2s was observed after 96 hours treatment, the relative potency on clonogenic assay was less than RNAi.

Conclusion:
Homozygous deletion of MTAP is a common genetic event in mesothelioma. Suppression of PRMT5 represents a novel potential approach for targeting mesotheliomas with 9p21.3 loss. The discrepancy between small molecule inhibitors and RNAi, suggests that PRMT5 dependence may require functions that extend beyond its methyltransferase function.

Background:
Growth factor receptors are central elements of signal transduction pathways and increasingly important targets for anticancer drugs. In recent years naturally occurring light sensitive protein domains (LSPDs) from different kingdoms of life have been used to generate genetically encoded chimeric signalling molecules that can be activated reversibly and with spatiotemporal precision by light. The development of such optogenetic tools has led to a plethora of new discoveries in the neurosciences but has received comparably little attention in cancer research - partly due to a lack of appropriate tools. Our aim was therefore to generate synthetic growth factor receptors that can be activated with light and allow fine-tuned control of growth factor-associated signal transduction pathways.

Methods:
To generate receptor tyrosine kinases (RTKs) that can be optically activated (Opto-RTKs), intracellular domains of RTKs were fused to LSPDs of the light-oxygen voltage (LOV) family from various species. The resulting chimeric receptors were tested for light-dependent activation of signal transduction by reporter gene assays, immunoblotting and various cell biological tests assessing DNA synthesis, epithelial mesenchymal transition (EMT) and angiogenesis.

Results:
Three of the tested LOV domains enabled light-dependent receptor dimerisation and activation of the corresponding signal transduction pathways when fused to the intracellular domains of FGFR1, EGFR, RET, c-Met or ROS1. Opto-RTKs enabled stringent control of the MAPK, PI3K and PLCγ pathways. Signal activation could be spatially confined to illuminated regions of culture plates and signals rapidly subsided after cessation of illumination. Light was able to replace FGF2 for the induction of cell proliferation and EMT in mesothelioma cells and VEGF for the stimulation of angiogenic sprouting in endothelial cells. Moreover, Opto-RTKs enabled light-assisted screening for small molecule inhibitors of EGFR, FGFR1 and the orphan RTK ROS1.

Conclusion:
Our optogenetic approach allows light-mediated control of growth factor receptors representing clinically relevant drug targets. Opto-RTKs enable dissection of dynamic signals with increased spatiotemporal resolution and open new possibilities for drug screening. Transfer of the design principle to additional membrane receptors is ongoing.

Background:
Malignant pleural mesothelioma (MPM) continues to increase in incidence worldwide and has limited therapeutic options. MPM displays characteristic changes in gene expression, including noncoding RNAs such as microRNAs, which have potential therapeutic relevance. One such miRNA is miR-137, a tumour suppressor whose promoter region is frequently methylated in other cancers and lies in in a commonly deleted chromosomal region in MPM (1p21-23). A potential role for miR-137 has yet to be investigated in MPM. One known target of miR-137 is YB-1, a multifunctional protein often up-regulated in other aggressive cancers, where elevated YB-1 levels are linked to poor clinical outcomes. This study investigates the causes of miR-137 suppression, the relationship between miR-137 and YB-1, one of its targets, as well as their roles in MPM cell growth and malignant behaviour.

Methods:
Basal expression of miR-137 and YB-1 was determined in 13 MPM cell lines by RT-qPCR and immunoblotting. Cells were treated with 5’Aza-cytidine and RT-qPCR was conducted to link methylation with miR-137 suppression. Copy number variation (CNV) was investigated by ddPCR. Cells were transfected with miR-137 mimic and subsequent YB-1 expression was investigated using RT-qPCR. Proliferation, colony formation and wound-healing assays were conducted after transfection with miR-137 mimics or YB-1-specific siRNAs.

Results:
miR-137 was absent in 4 MPM cell lines (p<0.01) and was up-regulated in response to 5’Aza-cytidine treatment in these lines, as well as other lines with low basal expression. Copy-number loss was evident in 5 cell lines and gain was present in 2. Increasing levels of miR-137 generally inhibited MPM cell migration, proliferation and colony formation. miR-137 mimics significantly down-regulated YB-1 expression, while YB-1 protein was overexpressed in the majority of MPM cell lines, compared to MeT-5A. YB-1 knock-down resulted in dose-dependent growth inhibition over 120 hours, reduced colony formation and also decreased cell migration. Effects were more pronounced in those cell lines showing high YB-1 protein levels.

Conclusion:
Our results show that methylation and CNV are likely to play a role in miR-137 down-regulation in MPM and that miR-137 acts as a tumour suppressor in MPM through at least in part the down-regulation of YB-1. We also demonstrated that YB-1 is commonly overexpressed and plays a role in proliferation and migration. These results imply a direct relationship between miR-137 and YB-1 expression, a biological interaction that may prove a useful target in developing future therapeutic approaches in MPM.

Background:
Malignant pleural mesothelioma (MPM) is an aggressive asbestos-related thoracic cancer. Chemotherapy is the most frequent treatment option but almost every patient will be confronted with recurrence of disease and drug resistance. Previous studies have used 3D spheroid cultures to investigate drug response in MPM. We showed that microRNAs are important players in MPM biology and that they contribute to the response of MPM cells to some chemotherapy drugs. In the current study we aimed to investigate the role of microRNAs in the drug resistance of a 3D spheroid model of MPM.

Methods:
MPM cells were grown in standard 2D culture or as 3D spheroids in low adherence round bottom multi-well plates. The structure of the spheroids was confirmed by conventional and scanning electron microscopy. MicroRNA expression was profiled using TaqMan Low Density Arrays. RT-qPCR and droplet digital PCR were used to validate candidate microRNAs. HIF1a expression was examined in MPM spheroids using immunofluorescence staining. Drug cytotoxicity was investigated in both 2D and 3D cultures using standard proliferation assays, and the effect of drugs on gene expression was analysed. MicroRNA mimics and siRNAs were used to determine the influence of microRNA and HIF1a expression on drug resistance.

Results:
In our adapted model of 3D cell growth, MPM cell lines formed spherical 3D structures, in contrast to the donut shapes reported with other models. MPM cells in these spheroids were more resistant to cisplatin and gemcitabine when compared to cells grown in 2D cultures. Immunofluorescence revealed a hypoxic gradient with high HIF1a expression observed in the centre of the spheroids. Spheroids also exhibited a significant up-regulation of miR-210, miR-21, miR-378a, miR-195 and miR-146b, and down-regulation of miR-320b and miR-1225b. Transfecting MPM cells in 2D culture with miR-210 or miR-21 mimics resulted in increased drug resistance, whereas HIF1a knockdown inhibited spheroid formation and decreased drug resistance. Spheroids displayed higher expression of the ABCG2 drug pump, and ABCG2 was also up-regulated in cisplatin and gemcitabine treated MPM cells.

Conclusion:
Our spheroid model revealed a clear impact of hypoxia on gene expression in MPM cells. Hif1a was highly expressed in the hypoxic centre of the spheroids and is an upstream regulator of the microRNAs we found to be differentially expressed. Pharmacologic and genetic modulation of microRNA and HIF1a levels altered drug resistance in MPM cells, suggesting a link between hypoxia, microRNAs and drug resistance in MPM.

Background:
Pleural effusion (PE) is a common clinical presentation of a large number of different diseases including malignant pleural mesothelioma (MPM). The approach to patients with PE is not simple and pre-operative cytological examinations or even intra-operative frozen sections are often unhelpful in the differential diagnosis. This fact makes critical the management of PE, especially when MPM is suspected. Changes in the expression of microRNAs (miRNAs) have been implicated in several diseases, making miRNAs attractive biomarkers. Our aim was to determine whether a miRNA signature in plasma or in Exhaled Breath Condensate (EBC) may help to discriminate between PE related to MPM and PE of patients affected by other pleural diseases (NM).

Methods:
We prospectively enrolled consecutive patients with PE from suspected MPM, scheduled for thoracoscopic pleural biopsy. We recorded clinical data and definitive histological diagnosis. Exclusion criteria were age< 18 years, history of previous tumor or immunological disorder. Ethics committee approval was achieved. Written informed consent was obtained from all participants. We collected a sample of plasma and EBC from each patient before the biopsy. We screened 733 miRNAs in blood and EBC by high-throughput Open Array and compared their expression in the two groups. We used a multiple linear regression model adjusted for four principal variables (age, sex, BMI and smoking habits) to compare MPM and NM.

Results:
We enrolled 32 patients; the figure below shows main clinical data. Figure 1 After miRNA screening, we identified 3 miRNAs which were upregulated in EBC and 44 in plasma. In particular, in EBC: miR-378, miR-206, miR-9 with a fold-change (FC) of 1.54, 1.4, 1.23 respectively (p-value= 0.019, 0.04 and 0.04 respectively). The most significant in plasma was miR-489 (FC= 1.35, p-value= 0.0001).



Conclusion:
We identified a miRNAs panel that might be useful for developing a non-invasive procedure for MPM diagnosis in patients with PE.

Background:
Certain microRNA (miRNA)-mRNA interactions are associated with critical biologic processes in malignant pleural mesothelioma (MPM). We wondered how miRNA interact with p53 in MPM since this tumor characteristically retains the wild-type allele which is frequently bypassed by deletion of CDKN2A. This interaction among miRNA and p53 in MPM is poorly understood. Study of this interaction could provide insights on disease mechanisms and/ or novel therapeutic strategies.

Methods:
We retrieved several public miRNA expression MPM data sets to perform a broad survey. We combined two meta-analyses approaches to the normalized data to maximize identifying altered miRNA specific to MPM. Then miRNAs were fit into a network where they inhibited MDM2, releasing inhibition of p53, and in turn were themselves induced by p53 (p53 regulation via a reinforcing loop). Significant miRNA of this screening algorithm were confirmed by qPCR analysis in MPM tissues and cell lines. Specific miRNAs were re-expressed in MPM cells by a lentivirus system or by mimic transfection. p53-luciferase reporter system was used to assess p53 activity. MTS assay assessed cell proliferation. Apoptotic cells were detected by Annexin-V assay. Tumorigenic characteristics of MPM cells were evaluated by clonogenicity, soft agar colony formation and 3D sphere assays. Clinical relevance of these miRNA were assessed in the TCGA MPM cohort (cancergenome.nih.gov).

Results:
Our meta-analysis, revealed significant changes in several p53-regulated miRNAs. For example, miR-145 expression is repressed by 40% at steady state in MPM specimens (n=38) compared to non-malignant controls (n=21). We directly confirmed in MPM tissues a similar trend of this miRNA, while MDM2 mRNA levels were inversely higher. Next, we assessed the functional role of miR-145 in MPM cell lines with wild-type p53. Using a lentiviral expression system to sustain elevations in miR-145 levels, MDM2 transcript and protein levels were repressed, leading to increases in p53 protein and its transcriptional activity. We observed in miR-145-overexpressed MPM cell lines more apoptosis by Annexin V assay, loss of clonogenicity, growth inhibition, and attenuated tumorigenicity. To confirm that p53 can perpetuate a positive reinforcing loop inducing miR-145, we treated a panel of MPM cells with Nutlin-3a and observed coordinated increases in p53 associated with a rise in miR-145 levels. Interestingly, at least one of these miRNA was prognostic in Kaplan-Meier modeling of overall survival.

Conclusion:
We have identified candidate miRNAs that, in part, regulate p53 activity in MPM cells. These miRNAs function as tumor suppressors. They are candidates for therapeutic validation.

Background:
Pleural malignant mesothelioma(MM) is a highly aggressive tumor that mainly related to asbestos exposure. Some microRNAs (miRNAs) contribute to MM initiation and progression, but the exact mechanism remains largely unknown.

Methods:
The expression of miR-30d in mesothelioma cell line NCI-H2452, chrysotile exposed MeT-5A cells and plasm of 78 human subjects with asbestos exposure was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). After NCI-H2452 cells were transfected with mimics miR-30d to overexpress mir-30d, cell viability was measured by MTS method; in vitro cell migration and invasion was measured by transwell assay and scratch assay; cell cycle and apoptosis was examined by flow cytometry; the mRNA and protein expressions of VIM, TWIST1 and CDH1 were detected by qPCR and western blotting, respectively.

Results:
miR-30d was significantly downregulated not only in NCI-H2452 cells compared with MeT-5A cells, but also in chrysotile exposed MeT-5A cells compared with the negative control and in plasm of asbestos exposed subjects compared with unexposed subjects. After transfected by miR-30d, the proliferation rate, migration rate and invasion cells of NCI-H2452 cells decreased, early apoptosis rate and total apoptosis rate increased in miR-30d transfected NCI-H2452 cells. The mRNA and protein expression of VIM and TWIST1 decreased and the mRNA expression of CDH1 increased by overexpressed miR-30d in NCI-H2452.Figure 1



Conclusion:
Downregulation of miR-30d is related to malignant mesothelioma and asbestos exposure. miR-30d might be a tumor-suppressor miRNA, and its down-regulation might contribute to pleural MM progression and asbestos carcinogenesis.

Background:
to investigate whether there was any relationship between survival and the expression of tumor infiltrating lymphocytes (TILs), programmed cell-death-ligand-1 (PDL-1), BAP-1 (BRCA1-Associated Protein 1), VEGFR-2 (vascular endothelial growth factor receptor 2) and IGF-1R (Insulin-Like Growth Factor 1 Receptor) in malignant pleural mesothelioma (MPM).

Methods:
63 cases of MPM were identified. All tissues were obtained at the time of diagnosis. There were 40 males; mean age was 70.4 years. 34 patients were smokers and 40 had a certain history of asbestos exposure. All histological slides were revised; there were 30 epithelioid subtypes, 20 biphasics and 13 sarcomatoids. The presence of TILs was scored as absent, weak, moderate and strong according to a quantitative assessment on hematoxylin and eosin slides. The expression of BAP-1, VEGFR-2, PDL-1 and IGF-1R was analyzed by immunohistochemistry. The impact of asbestos exposure, tobacco consumption and histological subtypes on survival were also assessed. The survival analysis was analyzed by Kaplan Meier curve.

Results:
TILs were present in 89% of cases and were found to be a favorable prognostic factor (p=0.009) although related with histological subtypes (p=0.008). The absence of TILs was higher in biphasic and sarcomatoid subtypes (90.9%, 30/33) compared to epithelioid MPM (53.3%, 16/30 p<0.001). Median survival in TILs and non-TILs patients was 28 months and 11 months, respectively. The expression of PDL-1 in tumor cells (cut-off: 10%, p=0.028) and VEGFR-2 in TILs (p=0.003) were related with survival, but they were differently expressed in histological subtypes. Using a logistic regression model, TILs, PDL-1 and VEGFR-2 in TILs correctly classified 21/30 epithelioid subtypes (70%) and 29/33 biphasic and sarcomatoid subtypes (87.9%). IGF-1R was overexpressed in 82% of the tumors (21 epitheliods and 31 sarcomatoids) and in 25% of TILs (7 epitheliods and 3 sarcomatoids) and was a favorable prognostic factor (p=0.023) independently of the histological subtype. Median survival was 4 and 13 months in patients not overexpressing and overexpressing IGF-1R, respectively. In a Cox regression model including both IGF-1R and histological subtype, IGF-1R remained significant [p=0.006, HR=0.41 (0.20-0.84)]. Tobacco, asbestos exposure, age and BAP-1 expression were not significantly related with survival.

Conclusion:
the histological subtype is an important prognostic factor in MPM and it’s related to different histological markers: the presence of TILs, PDL-1 and VEGFR-2 in TILs. Moreover, the overexpression of IGF-1R is an independent favorable prognostic factor. Therefore, histological markers may improve the prognostic assessment of MPM and provide mechanistic clues for new therapeutic strategies.

Background:
Distinction between mesothelioma and reactive mesothelial proliferation is difficult because of morphological overlap between mesothelioma cells and reactive mesothelial cells. It is often difficult to draw the line between epithelioid mesothelioma and biphasic mesothelioma with atypical stromal proliferation. However, separation of biphasic mesothelioma from epithelioid mesothelioma is important because therapeutic option and prognosis is different between two subtypes of mesothelioma.

Methods:
We collected 143 cases with malignant mesotheliomas (83 epithelioid, 22 biphasic and 38 sarcomatoid) and 33 cases with reactive mesothelial proliferation. Immunostaining was performed with anti-BAP1 antibody. Fluorescence in situ hybridization (FISH) analysis was performed with BAP1 probe and with p16 probe. BAP1 loss and deletion of p16 was separately analyzed in 19 biphasic mesotheliomas.

Results:
We analyzed 76 cases with BAP1 immunostaining, 87 cases with p16 FISH, and 37 cases with BAP1 FISH. BAP1 loss with immunohistochemistry was observed in 55% of epithelioid and 37% of biphasic mesotheliomas, but not in sarcomatoid mesotheliomas. Homozygous deletion (HD) of BAP1 was observed in 50% of epithelioid and 11% of biphasic mesotheliomas, but not in sarcomatoid mesotheliomas. HD of p16 was observed in 64% of epithelioid, 91% of biphasic, and 100% of sarcomatoid mesotheliomas. Concordance of BAP1 loss and HD of p16 between epithelioid and sarcomatoid components of 19 biphasic mesotheliomas was 100%. Four of epithelioid mesotheliomas were difficult to differentiate from biphasic mesothelioma with histology alone because of florid proliferation of atypical stromal cells; however, BAP1 loss and HD of p16 were not observed in atypical stromal proliferation and diagnosis of epithelioid mesothelioma could be made. There was a significant difference in overall survival according to histologic subtype (epithelioid 24M, biphasic 15M, sarcomatoid 4.5M). Mesotheliomas with loss of BAP1 expression showed increased survival (20M vs 8M) and HD of p16 showed poor survival (9.5M vs 28M). However, if only epithelioid cases were analyzed, there was no trend toward increased survival with BAP1 loss (24M vs 22M) while HD of p16 still showed poor survival (17M vs 28M).

Conclusion:
Most of biphasic mesotheliomas and all sarcomatoid mesotheliomas harbor HD of p16. BAP1 loss and HD of BAP1 are observed in epithelioid and biphasic mesotheliomas, but not in sarcomatoid mesotheliomas. BAP1 immunostaining and FISH analysis of p16 help making distinction between epithelioid and biphasic mesothelioma as well as between benign and malignant mesothelial proliferation. p16 is a prognostic factor for patients with epithelioid mesothelioma, but BAP1 is not.

Background:
Malignant pleural Mesothelioma (MPM) belongs to the most deadly cancers and is closely associated with asbestos exposure. Tumors arise typically after a long latency period (20-50 years) and three different tumor subtypes have been identified: epithelial, sarcomatoid and biphasic. Chirurgical resection can expand lifespan but is not curative and unfortunately, no effective chemotherapy or targeted therapies are currently available to effectively treat MPM. A better understanding of the molecular basis of the disease is urgently needed and therefore we have assessed what the cells-of-origin are in MPM using a versatile in vivo – in vitro system and how this influences tumor characteristics.

Methods:
Primary mesothelial cells isolated from Cdkn2a deficient mice carrying conditional alleles of Nf2 and Trp53 were transduced in vitro by lentiviruses expressing Cre-recombinase driven from a general promoter. After culturing these cells for a few passages to maintain the cellular heterogeneity, clonal cell lines were selected and analysed for their protein expression marker profile related to subtype-specific expression of markers used for MPM diagnosis. Histopathological analysis of MPM tumors that developed in recipient mice after grafting of primary cell populations and clonal cell lines was performed as well as the effect of the extra-cellular matrix (ECM) on the differentiation of the target cell for transformation in vitro. RNAseq profiles of our mice derived cell lines were compared to a large data set of human primary MPMs.

Results:
Transplantation of recombined primary mesothelial cell populations in immuun proficient recipient mice resulted in efficient MPM development of all tumor subtypes. Clonal sarcomatoid cells accelerated tumor development significantly compared to clonal epithelial clonal cells after grafting and both cell types retain their phenotype during tumorigenesis. Clonal biphasic cells are less tumorigenic and require an immuun deficient background to develop into biphasic tumors with varying contributions of epithelial and sarcomatoid tumor cells. In vitro we show that biphasic cell differentiation (into either epithelial or sarcomatoid tumor cells) is jointly regulated by the composition and stiffness of the cellular matrix. RNAseq performed on tumor subtype-specific clonal cell lines shows that epithelial cells are highly similar to biphasic cells while sarcomatoid cells show a different profile.

Conclusion:
Our results indicate that multiple cell-types sharing the same mutations are present in the mesothelial lining, each prone to serve as a cell-of-origin of one of the distinct MPM subtypes illustrating that the cell-of-origin plays a pivotal role in determining the tumor subtype of MPM.[.]

Background:
Breast cancer type 1 susceptibility associated protein (BAP1) is a deubiquitinating hydrolase that plays a key role in various cellular processes and acts as a tumor suppressor gene. Malignant mesothelioma is a deadly disease strongly associated with asbestos exposure. Most of the cases (70%) are pleural mesotheliomas while peritoneal and pericardial mesotheliomas account for 25% and 5%, respectively. Germ-line and somatic inactivation of BAP1 has been recurrently reported in pleural mesothelioma. However, due to its rarity and challenging diagnosis, little is known about the BAP1 status in peritoneal mesothelioma, with the largest series so far sequenced including only 12 tumors.

Methods:
Taking advantage of the extensive French national networks MESOPATH and RENAPE, we collected biological material and clinical and epidemiological data for 46 peritoneal mesothelioma patients. In order to determine the status of BAP1 in these samples, three different levels were evaluated: copy number changes by comparative genomic hybridization arrays (Chirac et al., Human Path 2016), mutations by next-generation sequencing, and protein expression by immunohistochemistry.

Results:
We detected copy number losses, mutations, and/or loss of expression of BAP1 in 42.2%, 33.3%, and 56.8% of the malignant peritoneal mesotheliomas analyzed, respectively. In most of the cases with additional data available (13/16), the loss of BAP1 expression was explained by co-occurring copy number loss and/or mutation. Overall, 73.2% of the malignant peritoneal mesotheliomas analyzed carried an inactivated BAP1 gene. In addition, BAP1 mutations were exclusively detected in males and a better overall survival was observed for patients with loss of BAP1 expression independently of age, sex, smoking and asbestos exposures (p=0.03).

Conclusion:
Inactivation of BAP1 seems to have a key role in the development of both pleural and peritoneal mesotheliomas. In addition, we found that loss of BAP1 expression in peritoneal mesotheliomas was mostly explained by copy number losses and mutations, and was associated with a better overall survival.

Background:
Mutations in the genes cyclin-dependent kinase inhibitor 2A (CDKN2A), BRCA-1 associated protein 1 (BAP1), and neurofibromatosis type 2 (NF2) are observed in malignant pleural mesothelioma (MPM). We observed biallelic BAP1 alterations in 61% of MPMs and found that mutations are particularly frequent in epithelioid-type malignant mesotheliomas (Cancer Sci, 103:868-74, 2012). In addition, Loss-of-function mutations in NF2 are relatively frequent (40%) in MPMs have indicated. Merlin is a tumor suppressor protein coded by NF2; both merlin and the ezrin/radixin/moesin proteins are associated with suppression of invasion and metastasis of tumor cells. In this study, we examined the association between stainability of merlin and the tumor properties of MPM.

Methods:
Following definitive histological diagnoses of 35 cases of MPM (epithelial: n=31, biphasic: n=2, desmoplastic: n=2), we conducted immunohistochemical staining for merlin in thin sections of paraffin-embedded tumor tissue. Stainability was assessed with H-scores. The clinical stage of MPM was defined at the time near the tumor tissue harvesting time; the association between the clinical stage and therapeutic outcomes was assessed based on the outcomes of first-line chemotherapy with cisplatin (CDDP) or carboplatin(CBDCA) plus pemetrexed (PEM). The anti-merlin antibody used was LS-B394 (LSBio, Seattle, Washington, USA).

Results:
1) Seven MPMs (20%) were negative or weakly positive for merlin (H-score 0-30); of these seven MPMs, one was desmoplastic, while six were epithelial. Six MPMs were strongly positive for merlin (H-score ≥250); all six of these MPMs were epithelial. 2) No difference in merlin stainability was observed between epithelial (n=31) and non-epithelial (n=4) MPMs. Similarly, on examination of the association between stainability and IMIG staging, no differences in stainability were observed between stages 1 to 4. 3) No differences in stainability were observed between the first-line chemotherapy partial response group and progressive disease group.

Conclusion:
Loss-of-function mutations in the tumor suppressor gene NF2 lead to enhanced expression of focal adhesion kinase; although these mutations are demonstrated in decreased normal expression of the tumor suppressor protein merlin, no trend was observed in the morphological differentiation patterns of MPM. In addition, although the ratio of cells with high aldehyde dehydrogenase enzymatic activity is increased by CDDP/PEM treatment, we observed no association between CDDP/PEM sensitivity and merlin stainability. The association between NF2/merlin and tumor properties must be studied in more cases.

Background:
Malignant mesothelioma (MM) is an aggressive tumor, with poor prognosis and limited possibility of treatment. MMNG HOS Transforming gene (MET) is a proto-oncogene located in the 7q31 that encodes the high-affinity receptor for hepatocyte growth factor (HGF). MET tyrosine-kinase was recently proposed for a targeted therapy and clinical trials are in progress in many tumors. MET amplification identifies a subgroup of patients potentially able to respond to HGF/MET inhibitors and may represent an element of resistance for anti-EGFR inhibitor therapy. The aim of this study was to evaluate MET amplification and expression in MM.

Methods:
The protocol of this study was approved by the Liguria Region Ethics Committee (P.R. 207REG2014) and the written informed consent was obtained from all the patients. We analysed 109 MM (67 male; 65 epithelioid, 26 sarcomatoid, 14 biphasic, 2 desmoplastic, 2 papillary). Seventy-nine MM were from a tissue microarray (MS801 and MS 1001, US Biomax Inc, Rockville, MD, USA), 12 cases of formalin-fixed paraffin-embedded tissues were from, IRCCS AOU San Martino-IST (Genova) and 18 tissues from ASL N°5 (La Spezia). MET gene amplification was investigated by FISH using MET/CEP7 probe cocktail (Vysis MET Spectrum Red FISH Probe Kit reagent/Vysis CEP 7 (D7Z1) SpectrumGreen Probe, both reagents from Abbott Molecular, Des Plaines, IL USA). Immunohistochemistry was performed by Anti-c-Met Antibody IHC-plus™ LS-B2812 (LSBio, Seattle, WA).

Results:
By using the UCCC-scored system we found one epithelioid MET amplification (MET to CEP7 ratio ≥2 or at least 15 copies of MET signals in ≥10% of the tumour cells). In contrast, 8/109 (7.3%) MM (6 epithelioid, 1 sarcomatoid, 1 biphasic) showed high MET polysomy (according to mean ≥4 copies/cells in ≥40% of tumour cells) in a range of 4-10 spots of MET gene in about 60-80% of tumour cells. (Table 1). Immunohistochemistry showed that amplification was associated with moderate expression of MET protein in cytoplasm and membrane of MM cells. In contrast, high gene polisomy resulted always associated with low staining of MET protein.

Conclusion:
Amplification and high polysomy of MET, associated with c-MET receptor expression may be present in MM. These preliminary observations might represent the basis for designing new clinical trials assessing MET targeting agents in MM. Moreover, the possibility of MET amplification should be considered before starting MM patient treatment with the anti-EGFR targeted inhibitors.

Background:
Malignant pleural mesothelioma (MPM) is a global health issue. Pegylated arginase (PEG-BCT-100) has shown anti-tumor effects in hepatocellular carcinoma, acute myeloid leukemia and human melanoma. We aimed to study the preclinical anticancer effects of BCT-100 in MPM.

Methods:
A panel of 5 mesothelioma cell lines (from ATCC) was used to study the in vitro effect of BCT-100 by crystal violet staining. The in vivo effects of BCT-100 (± chemotherapy) were studied using two nude mice xenograft models. Protein expression and arginine concentration were evaluated by Western Blot and ELISA respectively. Cellular location of BCT-100 was detected by immunohistochemistry and immunoflorescence staining. TUNEL assay was used to identify cellular apoptotic events.

Results:
BCT-100 reduced in vitro cell viability (IC~50~: 13-24 mU/ml) across different cell lines and suppressed tumor growth in both 211H and H226 xenograft models. Argininosuccinate synthetase was expressed in H28, H226, H2452 cells as well as 211H and H266 xenografts. Ornithine transcarbamylase was undetectable in all cell lines and xenograft models. BCT-100 (60 mg/kg) significantly suppressed tumor growth with increased median survival in both xenograft models. No beneficial effect was observed when combining BCT-100 with pemetrexed or cisplatin. BCT-100 decreased serum and intratumoral arginine level. BCT-100 was mainly located in cytosol of tumor cells. Apoptosis (PARP cleavage in 211H xenograft, Bcl-2 downregulation and cleavage of PARP and caspase 3 in H226 xenograft as well as TUNEL-positive staining in both xenografts) and G1 arrest (downregulation of cyclin A2, D3, E1 and CDK4 in 211H xenograft and suppression of cyclin A2, E1, H and CDK4 in H226 xenograft) were evident with BCT-100 treatment. Furthermore, proliferative factor Ki67 was downregulated in BCT-100 treatments arms.

Conclusion:
MPM tumor growth was suppressed by BCT-100 via apoptosis and G1 arrest in vivo. This provides scientific evidence to support further clinical exploration of BCT-100 in treatment of MPM. (Acknowledgment: This research was supported by Hong Kong Pneumoconiosis Compensation Fund Board, HKSAR.)

Background:
Malignant pleural mesothelioma (MPM) is a highly aggressive pleural tumor associated with asbestos exposure and with limited survival despite treatment. Chromosomal abnormalities are abundant in MPM and the most frequently mutated genes are BAP1, NF2 and CDKN2A. Expanded molecular profile in MPM may provide targetable molecular aberrations and improve treatment options for these patients (p).

Methods:
Thirty two MPM patients who progressed to standard chemotherapy underwent genetic tumor profiling in a molecular prescreening program at our institution between 2006 and 2015. Paraffin-embedded biopsies were used for the analysis. Mass detection (MassARRAY, Sequenom) including analysis of mutations in 25 oncogenes was used and since June 2014 multiplexed amplicon sequencing (AmpliSeq, Illumina) was implemented assessing mutations in 59 oncogenes. No patients received systemic treatment prior to obtain the tumor sample.

Results:
Demographics: male/female (22/10); median age 60.5 (range 32-83 years); histology epithelial/no epithelial (24/8); PS 0/1 (15/17); stage III/IV (14/13). All patients were treated with chemotherapy. Sequenom was performed in 21 p and AmpliSeq in 11 p. Median follow up was 23.3 months and median overall survival (OS) for entire cohort was 30.2 months. The median OS for patients with epitheliod and no-epithelioid histology was 31.5 vs 21.4 months (p=0.033). Genetic alterations were detected in 5 patients (4 p with AmpliSeq and 1 p with Sequenom).The mutations detected were RNF43/ZNRF3 (2p), PI3KCA (1p), APC (1p) and P53 (1p). We did not identify significant association between mutations with histology, gender and clinical stage (p>0.05). Median survival for patients with mutations was not reached and for patients without mutations was 30.2 m (p=0.462)

Conclusion:
This study shows genetic alterations are not frequent in MPM and AmpliSeq detected more genetic alterations than Sequenom. With a limited number of patients, we suggest further investigations about the role of mutations in Wnt pathway in MPM

Background:
Muti-walled carbon nanotube (MWCNT) is widely used worldwide, but reports already show MWCNT is toxic to experimental animals and cell lines, even causes severe caner, such as mesothelioma. The mechanism of MWCNT toxicities is not very clearly. So, in this study, some toxic effects of MWCNT on MeT-5A cells are evaluated, to offer inclues for futher study.

Methods:
Cell survival rate was detected by LDH, and cell cycle/ cell apoptosis were measured by flow cytometry. Cell scratch assay was used to evaluate the cell migration capacity.

Results:
The cytotoxicity of MWCNT on MeT-5A cells are dose- and time-dependent. Cell cycle are blocked in G1/G2 phase, cells in S phase are reduced. The same as cell proliferation, the apoptosis of MeT-5A cells is also time-dependent. MeT-5A cells display a significant reduction of cell migration after MWCNT exposure for 24h and 48h.

Conclusion:
The cytotoxicity of MWCNT on MeT-5A cells are dose- and time-dependent and the cellular behavior are perturbed by MWCNT treatments.

Background:
Malignant pleural mesothelioma (MPM) is an aggressive inflammatory cancer associated with exposure to asbestos. Untreated, MPM has a median survival time of 6 months, and most patients die within 24 months of diagnosis. Therefore an urgent need exists to identify new therapies for treating MPM patients. The potential for therapeutically targeting receptor tyrosine kinase (RTK) signalling networks is emerging as a critical mechanism in ‘oncogene addicted’ cancer, with RTK inhibitors evolving as areas of considerable importance in cancer therapy. Furthermore, RTK hetero-dimerization has emerged as a key element in the development of resistance to cancer therapy. As such TKIs which target several RTKs may have superior efficacy compared with TKIs targeting individual RTKs. We and others have identified c-MET, RON, Axl and Tyro3 as RTKs frequently overexpressed and activated in MPM, making these attractive candidate therapeutic targets. A number of orally bioavailable small molecule inhibitors have been developed which can target these receptors. LCRF0004 specifically targets RON, whereas ASLAN002 (BMS-777607) or Merestinib (LY2801653) are orally bioavailable small molecule inhibitors which inhibit c-MET, RON, Axl and Tyro3 at nanomolar concentrations. These drugs may therefore have applicability in the treatment/management of MPM.

Methods:
A panel of MPM and normal pleural cell lines were screened for expression of Tyro3, c-MET, RON and Axl by RT-PCR, and subsequently examined in a cohort of patient samples comprising benign, epithelial, biphasic, and sarcomatoid histologies by qPCR. The effects of two small molecule inhibitors LCRF0004, ASLAN002 on MPM cellular health were assessed in vitro. The effects of LCRF0004 and ASLAN002 were subsequently examined in an in vivo SQ xenograft tumour model.

Results:
Expression of various RON isoforms, c-MET, Tyro3 and Axl were observed in all cell lines. Significantly higher expression of all genes were found in the malignant tumour material versus benign pleura and this was validated in other datasets. Both LCRF0004 and ASLAN002 demonstrated significant anti-tumour efficacy in vitro. In xenograft models ASLAN002 was far superior to LCRF0004.

Conclusion:
Our results suggest that a multi-TKI, targeting the RON/MET/TAM signalling pathways, may be a more effective therapeutic strategy for the treatment of MPM as opposed to targeting RON alone.

Background:
Several tumor markers have been proposed in differentiating between benign and malignant pleural effusions (PE). The aim of this prospective study was to evaluate the usefulness of serum carcinoembryonic antigen (CEA), vascular endothelial growth factor (VEGF), and matrix metalloproteinase-7 (MMP-7) assay in patients with PE of uncertain origin.

Methods:
A series of 36 consecutive patients with suspicious PE requiring VATS-guided biopsy underwent serum CEA, VEGF, MMP-7 measurement before PC and biopsy. There were 20 (55.6%) males and 16 (44.4%) females, with an overall median age of 67 (range 40-82 years). According to the receiver operating characteristic (ROC) curve, the optimum cutoff levels were 5 ng/mL, 7.5 ng/mL, and 250 pg/mL for CEA, VEGF and MMP-7, respectively.

Results:
Final pathology showed 10 (27.8%) patients with NSCLC, 13 (36.1%) with LMs, and 13 (36.1%) with benign PE. The age did not differ between groups (p=0.59). The sensitivity, specificity and accuracy of PC were 56.5%, 92.3%, and 69.4%, respectively. The results of serum markers measurement are reported in the Table (95% CI). The logistic regression excluded CEA from the model, and thus we calculated the area under the curve (AUC) of the combination VEGF+MMP-7. The AUC was 0.681 (95% CI: 0.413-0.743) and the diagnostic accuracy was 77.8%, which was superior than that of MMP-7 alone (72.2%, p=0.41). Figure 1



Conclusion:
In patients with PEs, the measurement of serum VEGF and MMP-7 together reached a good accuracy with a fair AUC, and should be suggested when a noninvasive evaluation of a PE is required.

Background:
BRCA1-associated protein 1 (BAP1) gene is located at chromosome region 3p21.1, a genomic region that is deleted in several human malignancies, including approximately 30-60% of mesotheliomas(1). In this study, we retrospectively investigated BAP1 status in 41 unrelated patients with mesothelioma who had a history of environmental fibrous mineral exposure (erionite or asbestos). We have also reviewed histological tpe and clinical characteristics of the analyzed patients.

Methods:
A total of 41 malignant mesothelioma cases were reviewed histopathologically. Representative areas were selected and 4-mm-diameter tissue microarrays were composed from paraffin blocks. Immunohistochemistry (IHC) was performed on paraffin tissue sections prepared from microarrays with a monoclonal antibody against BAP1. Cases with loss of nuclear staining were considered as loss of BAP1 expression.

Results:
Satisfactory results were obtained in 37 patients (25 females, 12 males; mean age 56 yrs). Thirty-one cases were pleural, 5 cases were peritoneal and 1 case was paratesticular mesotheliomas. Histologically, 31 cases were epithelioid, and 6 cases were biphasic type. Overall all loss of BAP1 expression was 31/37 [83, 8 % ( 87, 1% pleural, 80% peritoneal, 0 paratesticular)]. Histologically, all biphasic types and 25/31 (80, 6%) epithelioid types showed BAP1 expression loss.

Conclusion:
Loss of BAP1 expression seems to be a frequent event in Turkish malignant mesotheliomas. However, in our small cohort, no significant correlation was found between tumor type and localization. We need to demonstrate both somatic and germ-like mutations in familial cases especially from erionite villages. References:1. Carbone M. BAP1 and Cancer. Nat Rev Cancer; 13: 153–159,1.2013.

Background:
Malignant pleural mesothelioma (MPM) is a low-incidence, aggressive, asbestos-related tumor, whose treatment options are currently limited. MPM is a heterogeneous tumor with three main histological subtypes: epithelioid (E), sarcomatoid (S) and biphasic (B). S- and B- MPMs are rarer and have a poorer prognosis than the E-subtype. In the present study we compared the expression profile of 117 genes with a crucial role in cancer between the E- and S/B- subtypes, in order to identify histology-specific molecular markers.

Methods:
Gene expression analysis was performed by Nanostring system directly on RNA from 38 formalin-fixed and paraffin-embedded tissues of MPM patients (25 E-subtype, 13 S/B-subtypes). After data normalization, differences of gene expression levels between the two groups were evaluated by a non-parametric Mann-Whitney U-test (p-value < 0.05).

Results:
39 genes were differentially expressed. In particular, 21 genes were statistically up-regulated and 18 down-regulated in E- compared to S/B-subtypes (Table 1). Figure 1



Conclusion:
The identification of gene expression profiles specific for each histological subtype could improve the clinical approach to MPM. In this study we found genes differentially expressed between E- and S/B-subtypes. In detail, up-regulated genes in E-MPM encode for proteins involved in epithelial cell differentiation and regulation of apoptosis, whereas down-regulated genes belong to pathways related to extracellular matrix, cell adhesion and angiogenesis. Moreover, some of the deregulated genes have been already described to influence the sensitivity to chemotherapy, such as ASS1, to play an important role in the mesenchymal transition, like MMP9, and others, among which ESR2, have been proposed as potential therapeutic targets. Our results reveal genes activated or inactivated in a histotype-dependent manner as new potential biomarkers for MPM, however, further studies are needed to better understand their clinical value.

Background:
Phenotypic and genomic heterogeneity may contribute to the pathogenesis of Malignant Mesothelioma (MM). We have implemented a novel clinical study in which multiple samples of tumour and normal pleura are obtained from individual patients undergoing surgery for the management of MM.

Methods:
Patients undergoing routine video-assisted thoracoscopic surgery (VATS) undergo a baseline FDG-PET scan. Extra samples are taken from both PET positive and PET negative areas of pleura, along with normal pleural samples. Corresponding fresh and formalin fixed tissue samples are obtained. Patients are subsequently treated as per standard of care practice, along with detailed clinical follow up and serial PET imaging. Standard H&E will be performed on formalin fixed samples to look for morphological heterogeneity in tumour cells and stroma and the findings will be correlated with PET imaging.

Results:
Six (6) patients (4 epithelioid, 1 sarcomatoid and 1 biphasic subtype) with median age of 70 (62-81) have been recruited into the project so far. All patients underwent an FGD-PET scan prior to VATS procedure. Multiple samples from PET avid and PET non-avid areas were collected and stored from 5 out of 6 patients. Preliminary results show that high quality samples were obtained. The H&E analysis shows that tumours from PET avid area are morphologically different to PET not avid areas (Figure 1). Further IHC analysis of various MM specific markers is underway. Figure 1



Conclusion:
This preliminary data demonstrate the feasibility of our clinical approach. We therefore propose to create a unique research platform in which MM samples from multiple sites from each patient will be integrated with clinical, imaging and therapeutic response. Further proof-of-principle studies will explore: (i) regional heterogeneity and the response to therapies; (ii) variability in the detection of predictive biomarkers within the same patient; (iii) genomic heterogeneity within MM, and its relationship to mutational changes in normal pleura.

Background:
Malignant pleural mesothelioma (MPM) is a rare and aggressive, treatment-resistant cancer. Pemetrexed, an inhibitor of thymidylate synthase (TS), is used worldwide for MPM as a first-line chemotherapy regimen. However, there is little consensus for a second-line chemotherapy. S-1, a highly effective dihydropyrimidine dehydrogenase (DPD)-inhibitory fluoropyrimidine, mainly acts via a TS inhibitory mechanism similar to pemetrexed. Orotate phosphoribosyltransferase (OPRT) is a key enzyme related to the first step activation of 5-fluorouracil (5-FU) for inhibiting RNA synthesis. We investigated 5-FU related-metabolism proteins, especially focusing on OPRT expression, in MPM.

Methods:
Fifteen MPM patients who were diagnosed between July 2004 and December 2013 were enrolled. We examined the protein levels of 5-FU metabolism-related enzymes (TS, DPD, OPRT, and thymidine phosphorylase [TP]) in 14 cases.

Results:
High TS, DPD, OPRT, and TP expressions were seen in 28.6%, 71.4%, 85.7%, and 35.7% of patients, respectively. We found that OPRT expression was extremely high in MPM tissue. We experienced one remarkable case of highly effective S-1 combined therapy for pemetrexed refractory MPM. This case also showed high OPRT protein expression.

Conclusion:
The present study suggests that OPRT expression is high in MPM tumors. Although pemetrexed is mainly used for MPM chemotherapy as a TS inhibitor, S-1 has potential as an anticancer drug not only as a TS inhibitor but also inhibiting RNA synthesis through the OPRT pathway. This is the first report investigating OPRT protein expressions in MPM.

Background:
Photodynamic therapy (PDT) and external beam radiotherapy (RT) have been used as adjuvant therapies directed at increasing local control in patients undergoing surgical resection for malignant pleural mesothelioma (MPM). In patients with MPM treated with a surgically-based multimodality treatment program that involves intraoperative PDT and postoperative RT for MPM, we have previously demonstrated that expression/activation of epidermal growth factor receptor (EGFR)/STAT3 signaling correlates with increased pleural recurrence rates and decreased overall survival. We assessed if activation of STAT3 through EGFR pathway activation mediates resistance of lung cancer cells to either PDT or ionizing RT.

Methods:
Tumor samples from patients with MPM undergoing lung-sparing surgery/intraoperative PDT and postoperative RT were analyzed for expression of growth factor and inflammatory signaling pathways using both IHC and Nanostring techniques. For in vivo assays, Balbc mice with syngeneic Ab12 MPM tumors were treated with partial surgical resection followed by PDT. In vitro studies involved human MPM cell lines derived from subjects enrolled on tissue acquisition with a pTRIPZ expression vector designed to allow doxycycline-induced STAT3 or EGFR shRNA expression.

Results:
Ninety-three consecutive patients were assessed. Patients undergoing surgery/PDT and RT with median time to local recurrence of <12 months demonstrated elevated EGFR/STAT3 expression levels and increased plasma IL-6 after tumor resection as compared to patients with a median time to local recurrence of > 12 months. In vivo studies in Balbc mice undergoing incomplete surgical resection of Ab12 MPM flank tumors demonstrated activation of STAT3 and EGFR signaling as well as increased plasma IL-6. Moreover, this activation was associated with decreased efficacy of postoperative PDT as compared to mice bearing equivalent sized tumors undergoing PDT without surgery and the effects of surgery on PDT were abolished by pretreating animals with the Cox-2 inhibitor celecoxib. Finally, using a novel 3D in vitro MPM cell culture system, we found that activation of EGFR/STAT3/Cox-2 signaling pathways significantly decreased PDT and RT mediated cellular cytotoxicity. Conversely, inhibition of these pathways enhanced PDT and RT efficacy.

Conclusion:
Both EGFR and STAT3 are activated in the wound healing/inflammatory response to surgical injury, and EGFR/STAT3 activation leads to increased cox-2 expression. Taken with the above results, this suggests that surgically mediated activation of these pathways, possibly through STAT3-mediated growth factor/inflammatory signaling pathways, impairs the potential efficacy of intraoperative/post-operative PDT and RT and that inhibition of this response to surgery might enhance clinical outcomes in patients with MPM.

Background:
Malignant pleural mesothelioma (MPM) is a rare and aggressive malignancy with dismal prognosis. Cisplatin and pemetrexed combination has been established as standard 1st line chemotherapy for advanced MPM. There is no approved 2nd line therapy for MPM and early phase clinical trials have shown that programmed death-1 (PD-1) inhibitors are safe and effective. However, the majority of patients with advanced MPM are excluded from clinical trials due to their poor performance status (PS). We report a single institution’s experience with nivolumab, a humanized IgG4 monoclonal anti PD-1 antibody, in patients with poor PS outside of clinical trials.

Methods:
Patients with advanced MPM were treated at Baylor Clinic with nivolumab (3mg/kg every 2 weeks) through a Nivolumab Expanded Access Program (EAP). Response rate (RR) and disease control rate (DCR) were evaluated at 8 weeks (RECIST 1.1 criteria). All patients were assessed for treatment related adverse events (CTCAE 4.0). PD-L 1 expression on tissue samples were quantified by commercially available PD-L1 IHC 28-8 pharmDx assay.

Results:
Between 12/2015 and 6/2016, six patients were enrolled in a Nivolumab EAP. Five patients (83%) were males and the median age was 65 years (range 38-78). Four patients had received at least one line of platinum-based chemotherapy and two refused 1st line chemotherapy due to poor PS. Three patients had PS of 2 and three patients had PS of 3. Tumor histology included: epithelioid (n=3), biphasic (n=2), and sarcomatoid (n=1). Median treatment duration was 16 weeks (range 8-28 weeks). At 8 weeks, four patients had partial response (PR), one patient had stable disease (SD), and one patient had progressive disease(PD). Then RR was 67% and DCR was 83%. The four PR patients had PD-L1 expression of 0%, 35%, 40% and 70%, one SD patient had PD-L1 expression of 10%, and one PD patient had PD-L1 expression of 5%. All six patients had subjective clinical response with improved PS. Two patients had G2 fatigue, one patient had G1 depression. Five patients are still on treatment.

Conclusion:
Nivolumab is a safe and effective treatment option for advanced MPM patients with poor PS. Although tumor PD-L1 overexpression is related to treatment response, subjective clinical response is observed in advanced MPM patients regardless of tumor PD-L1 status. Our experience of nivolumab in advanced MPM patients with poor PS outside of clinical trials provides additional data to ongoing clinical trials.

Background:
The treatment of MPM is not well defined. Although it has been shown that extrapleural pneumonectomy (EPP) followed by hemithoracic RT may improve survival in early-stage disease, this therapeutic approach is declining. This might be due to the recent publication of a randomized trial (SAKK 17/04) which assessed the effect of high-dose hemithoracic RT after EPP. The study reported poor outcomes and the results did not support the use of hemithoracic RT. The major weakness of the SAKK study is the heterogeneity of the radiation techniques and schedules, and that only 12 patients received IMRT. On this background we conducted the present study to assess the outcome of MPM patients treated with EPP and adjuvant IMRT.

Methods:
This is a retrospective multicenter study, including 5 academic centers in Italy. Seventy patients treated with EPP and adjuvant IMRT were enrolled. The majority (95%) of patients had an epithelioid histology. Sixty patients were affected by stage III-IVA disease. Fifty-four (77%) patients received neoadjuvant/adjuvant chemotherapy. The IMRT dose ranged between 50-60 Gy in 25-27 fractions. Radiation was interrupted in two patients due to systemic progression of disease.

Results:
Median follow-up was 14 months (range, 0-83 months). Rates of local control, loco-regional control, distant-metastases free survival (DMFS) and overall survival (OS) at 2 years were 73%, 63%, 39%, and 63%, respectively. Patients with stage I-II had a better 2-year OS and DMFS than those with advanced disease: 60% vs. 40% (p=0.09), and 78% vs. 32% (p=0.03), respectively. Three fatal pneumonitis were reported. Other major toxicities were: Grade 2-3 pneumonitis in 2 cases, 1 bronchial fistula, and 1 Grade 3 esophagitis.

Conclusion:
This multicenter study showed that EPP followed by hemithoracic IMRT is associated with high rate of local and loco-regional control, and showed that long-term survival can be achieved, even if some patients may experienced life-threatening lung toxicity. Distant metastasis represent the predominant pattern of failure.

Background:
In a single-arm Phase II trial on 78 patients with advanced mesothelioma, promising objective response rate (ORR, 50%) and overall survival (OS, median: 17.0 months) after treatment with cisplatin and low-dose gemcitabine in long infusion (C-GILI) were reported (Kovac et al, Anticancer Drugs 23:230-38, 2012). Here we present a randomized Phase II clinical trial, comparing cisplatin/pemetrexed (CP) and C-GILI.

Methods:
Eligible patients had histologically confirmed malignant mesothelioma, were chemonaive, had performance status (PS) 0-2, adequate organ function to receive cisplatin-based chemotherapy and signed informed consent. Patients were randomized between group A (pemetrexed 500 mg/m2 and cisplatin 75 mg/m2, both on day 1 every 3 weeks) and group B (gemcitabine 250 mg/m2 in 6-hours infusion, d1 and d 8, and cisplatin 75 mg/m2 on d 2, every 3 weeks). The primary endpoint was progression-free survival (PFS); secondary endpoints were ORR, toxicity, quality of life and OS. After progression, cross-over to the alternative regimen was recommended.

Results:
Ninety-six patients entered the trial. Median age was 63 years, 75% were male and 68% had documented exposure to asbestos. Patients were randomized between Group A (CP, 51 pts) and Group B (C-GILI, 45 pts). With ORR over 45%, both regimens were effective. The main Grade 3-4 toxicity was neutropenia: 13.7% for arm A and 33.3% for arm B. Details on demographics, histologic features and effects of treatment are presented in the Table. Median PFS and OS for all patients are 9.4 and 18.6 months, respectively.

Conclusion:
Both arms of primary treatment were effective and well tolerated. Overall survival is among the longest reported so far. This trial confirms the value of C-GILI for treatment of mesothelioma. This treatment may find its indication as second-line treatment, as well as in the first line for deprivileged patients for whom the costs of pemetrexed may be prohibitive.

		Group A CP, 51 pts	Group B C-GILI, 45 pts
DEMOGRAPHICS	Male/Female	35/16	37/8
	Median age	63	64
	PS 0-1/PS 2	40/11	36/9
	epitheliod/biphasic/ sarcomatoid/unspec	42/2/4/3	32/8/4/2
PRIMARY TREATMENT	ORR, %	48.9	50.0
	PFS (months, median)	10.6	8.6
	% of pts with any grade ≥ 3 toxicity	51.1	55.6
SECOND-LINE TREATMENT AND SURVIVAL	% of pts crossing-over to alternative combination	77.8	61.1
	OS (months, median)	20.6	18.6

Background:
To analyze rates of definitive radiation therapy (RT) utilization for malignant pleural mesothelioma (MPM) and evaluate the association between RT and overall survival (OS).

Methods:
The National Cancer Data Base (NCDB) was queried to identify patients with non-metastatic MPM diagnosed between 2004 and 2013. Definitive RT was defined as receipt of 40-65 Gy of external beam radiation therapy to the chest wall, lungs, or pleura. Multivariate logistic regression was performed to identify predictors of RT receipt. OS was estimated using the Kaplan-Meier method. Cox proportional hazards models were used to identify predictors of mortality. Propensity score matching was performed to verify the effect of definitive RT on OS.

Results:
Among 14,090 MPM patients, 3.6% received RT. Younger age, lower co-morbidity score, private insurance, surgical resection, and receipt of chemotherapy were associated with increased RT utilization. Patients who received RT had higher crude 2 and 5-year OS rates (33.9% and 12.6%, respectively) compared to patients who did not (19.5% and 5.3%, respectively; p<0.001). On multivariable analysis and propensity matched analysis, definitive RT was associated with improved survival (adjusted hazard ratio [adj HR] 0.78, 95% CI 0.70-0.87) and (adj HR 0.77, 95% CI 0.67-0.89), respectively. Compared to no therapy, surgery and RT (adj HR 0.41, 95% CI 0.31-0.54) and trimodality therapy (adj HR 0.47, 95% CI 0.40-0.55) were associated with the best survival.

Comparison of Overall Survival According to Definitive RT

	2-yr rate	95% CI	5-yr rate	95% CI	p	Adjusted HR	95% CI
No RT	19.5%	18.8-20.3	5.3%	4.9-5.8	<0.001	1.00	Ref
RT	33.9%	29.4-38.4	12.6%	9.4-16.3	<0.001	0.78	0.70-0.87

Conclusion:
The rate of definitive RT utilization for non-metastatic MPM has remained low over the past decade. Patients who received RT had improved OS, suggesting a role for increased utilization, particularly with the advancement in RT techniques. Combined modality therapy was associated with a greater improvement in survival than any single modality treatment.

Background:
Positron Emission Tomography/Computed Tomography (PET/CT) is well recognized as an important modality to detect malignant neoplasm, which plays a crucial role in the assessment of patients with malignant pleural mesothelioma (MPM). T1a category of IMIG classification refers to an early tumor that involves the parietal pleura only. And, there are a certain number of patients with more earlier clinical stage than T1a, i.e., radiological and thoracoscopical T0 stage. Those patients with T0 stage have neither visible pleural tumor nor pathologic PET/CT findings, and they are sometimes misdiagnosed as nonspecific pleuritis after a complete investigation including thoracoscopical biopsies. Those patients will turn out to be malignant during follow-up period. The purpose of this study was to investigate when tumor growth came to be detectable with PET/CT in patients with very early clinical stage of MPM whose PET/CT had been negative.

Methods:
Seven histologically-proven MPM patients with T0/T1a clinical stage were followed up with PET/CT (Epithelioid/unknown;6/1). A surgical thoracoscopy(VATS) or medical thoracoscopy with narrow band and autofluorescence imaging in addition to white light had been performed to obtain adequate materials of pleura and to diagnose T1-stage macroscopically. No patients had received talc pleurodesis. The initial thoracic CT scans showed pleural effusion without thickening of pleura, and all of the initial PET/CT evaluation was negative. The radiological examinations, including PET/CT and contrast and/or plain CT for loco-regional disease were reviewed to see tumor growth that came to be able to detect with PET scan. Interpretation of positive PET scan for malignancy is SUV>2.5.

Results:
The median interval between the initial PET/CT and the follow-up PET scan to come to identify malignant nodules for the first time was 32 months (5 - 46 months). Two patients with epithelioid and unknown MPM showed a positive finding of PET scan at the site of pleural intervention (thoracoscopy/thoracentesis), of which interval were 5 and 8 months, respectively. PET-positive pleural nodules, 5.1 ～ 8.2 mm in diameter, were demonstrated in 4 patients. And, a diffuse pleural thickening with positive PET scan, 6.5 mm in thickness, was shown in 1 patient with epithelioid MPM.

Conclusion:
PET/CT is a valuable modality for detecting the progression of T0/T1a tumors > 5 mm in diameter. Interval between PET-negative T0/T1a tumor and PET-positive tumor growth is more than 2 years (median; 32 months), and tumor seeding at the site of previous pleural intervention is an early manifestation of MPM.

Background:
Whilst the impact of clinico-pathological factors on the prognosis of malignant pleural mesothelioma (MPM) is well understood, socio-economic and geographic factors have received less attention. Although the majority of Australians reside in major cities, a dispersed population lives in regional and remote areas, where access to clinical services may be limited. We investigated the association between geographic and socio-economic factors and treatment provision and survival in a large series of patients from New South Wales.

Methods:
All patients awarded compensation by the NSW Dust Diseases Board (2002-2009) following diagnosis with MPM were assessed. Geographic remoteness, distance from oncological multidisciplinary teams (MDT) and socioeconomic status according to the index of relative socio-economic advantage and disadvantage (IRSAD), were assessed with known prognostic factors using Kaplan Meir and Cox-regression analysis. Chi-square testing compared categorical variables to analyse the impact of these factors upon clinical features and treatment received. Cancer Registry incidence data was assessed to allow comparison of the compensated DDB cohort to all NSW MPM cases.

Results:
We assessed 910 patients: Geographic remoteness (major city 67%; regional/remote 33%), distance to MDT (<10km 65%, <50km 92%). Geographic distribution was comparable to cancer registry data. Median overall survival was 10.0 months. On multivariate analysis, non-epithelioid histological subtype (HR.2.19); male gender (HR=1.37); age >70 (HR=1.39) and IRSAD status by decreasing quintile (HR=1.07) were independent prognostic factors, with a pronounced survival difference between highest and lowest IRSAD quintiles (8.4 vs 12.8 months). A trend to improved survival when residing in major cities (10.6 vs 8.8 months; p=0.162) and within 50km of MDT (10.3 vs 7.8 months;p=0.539) was noted. Patients geographic location and distance to MDT affected the use of palliative radiotherapy (p<0.05) however did not impact chemotherapy, adjuvant radiotherapy or extrapleural pneumonectomy provision. Socioeconomically disadvantaged patients were less likely to receive chemotherapy (40.3% vs 47.7%; p=0.032), with pronounced disparity between the most socioeconomically advantaged and disadvantaged quintiles (54.2% vs 37.6%;p=0.001).

Conclusion:
Despite ‘universal’ health care and the support of a compensation scheme, socioeconomic disadvantage was an independent prognostic factor for MPM in NSW Australia. A significant reduction in chemotherapy utilisation was noted, particularly in highly socioeconomically deprived areas. Furthermore, a trend to improved survival was noted in patients residing in major cities within closer proximity to oncology units, though treatment provision did not differ. Prospective research analysing specific factors including comorbidity, income, and individual preference will be required to better understand these findings in both compensated and non-compensated individuals.

Background:
Malignant pleural mesothelioma (MM) remains an almost universally fatal disease with limited treatment options. Immunotherapy represents a rapidly emerging therapeutic strategy for multiple malignancies including MM. However durable therapeutic responses occur in a minority (~30%) of patients. Better understanding of the quantities and immunophenotype of circulating and intrapleural immune cells is essential to design and apply personalized immunotherapeutic strategies.

Methods:
We used a comprehensive flow cytometry panel (PLoS One. 2015 Mar 23;10 (3):e0121546) to prospectively characterize circulating and pleural fluid immune cells in patients with MPM (n=12) and normal volunteers (circulating cells only, n=50). Matched blood and pleural samples were available from 11 patients, including samples from 9 patients enrolled into a Phase I study investigating the intrapleural administration of the modified vaccine strain measles virus (MV-NIS), MC1023. Pre- and post-treatments samples were available from 7 patients. The immune cell counts and cell fractions were compared using a false discovery rate (FDR) of 10% and the non-parametric Mann-Whitney test and the Wilcoxon matched-pairs signed rank test (blood versus pleural fluid and different time points). (p ≤ 0.05)

Results:
Cell counts and immune phenotype of circulating immune cells differ between of patients with MPM differ from normal volunteers (31 of 86 cell types). Patients with MPM had fewer B-lymphocytes, more pro-inflammatory monocytes (CD14+CD16+) and exhausted CD4 and CD8 T-lymphocytes (CD4 and CD8 PD1+TIM3+ double positive cells). As expected we observed characteristic differences between blood and pleural fluid in MPM patients. For example there are a higher numbers of antigen experienced, PD-1 expressing CD4 and CD8-memory cells within the pleural fluid compared to the peripheral blood. The intrapleural administration of a modified vaccine strain measles (MV-NIS) triggered changes in pleural and circulating immune cells. While these changes varied among patients, we observed increased CD80 and CD86 expression on CD14+ monocytes in the pleural fluid down.

Conclusion:
Comprehensive flow-cytometric immunophenotying of blood and pleural fluid is feasible in patients with MPM. This approach may help to identify patients who may respond favorably to specific immunotherapeutic interventions such as immune checkpoint inhibitors or facilitate longitudinal immune monitoring during clinical trials. Additional data is needed and we are continuing to prospectively analyze samples from patients with MPM.

Background:
Nucleoside transporter proteins mediates the transport of nucleosides and nucleoside analog drugs across the plasma membrane. The human equilibrative nucleoside transporter 1 (hENT1) is a nucleoside transporter protein that mediates cellular entry of gemcitabine, cytarabine, and fludarabine. The hENT1 expression has been demonstrated to be related with prognosis and activity of gemcitabine-based therapy in breast, ampullary, lung, and pancreatic cancer. In the same way, RRM1 encodes the regulatory subunit of ribonucleotide reductase and is a molecular target of gemcitabine. Previous studies showed increased RRM1 expression on continuous exposure of cell lines to gemcitabine and suggested improved survival for patients with low RRM1 expression.

Methods:
We measured hENT1 and RRM1 messenger RNA (mRNA) levels by quantitative real-time reverse transcription-polymerase chain reaction in tumor samples from 29 patients with advanced pleural mesothelioma (APM) treated in second line with gemcitabine based chemotherapy correlating these data with clinical parameters and disease outcomes (overall response rate-ORR, progression free survival-PFS and overall survival-OS).

Results:
The median age was 60-yo (r, 33-70 years), 15 patients were males (51%), 34% of cases undergone debulking surgery and the median follow-up was 13.4 months (95%CI 6.4-34). All patients were treated with Pem based chemotherapy in first line achieving a PFS of 8.1 months (95%CI 3.7-12.6), while PFS with second-line Gem based chemotherapy was 6.3 months (95%CI 3.6-8.8). 62% and 34.5% of patients had low levels of mRNA for hENT1 and RRM1, respectively. On univariate survival analysis, the hENT1 expression was associated with OS (p=0.001) and PFS (p=0.022). Specifically, those patients with overexpression of hENT1 showed a shorter OS p=0.021) and a shorter PFS (p=0.033). In contrast, mRNA expression of RRM1 did not influence the OS (p = 0.44) but it modifies positively the PFS (p=0.034). Multivariate analysis found that combined hENT1 (p=0.001) and RMM1 (p=0.012) predict survival in patients with APM treated with Gem based regimens.

Conclusion:
hENT1 mRNA expression carries prognostic information in patients with APM and combined with RRM1 holds promise as a predictive biomarkers in gemcitabine treated patients.

Background:
Prognostic models play an important role in the design and analysis of mesothelioma treatment trials. The European Organisation for Research and Treatment of Cancer (EORTC) developed a well-known tool in 1998 to predict overall survival (OS) in patients with malignant mesothelioma. In this study, we built and assessed the performance of a new mesothelioma prognostic model OS using data from multiple CALGB clinical trials data.

Methods:
This study included 595 mesothelioma patients from fifteen completed CALGB treatment trials accrued between June 1984 and August 2009. We split the cohort of patients into two parts - 67% of patients as training and 33% as testing. We developed a Cox model using the training set with PS, age, WBC count, and platelet count as prognostic variables. To compare the EORTC and our new models, the concordance of predicted survival times and risk scores were estimated by concordance C (c-index) (Harrell et al. 1996) and AUC score at 6-months (Patrick et al. 2000). 95% confidence intervals were calculated for the c-index. Based on the prediction model fit from training set, we partitioned testing set patients into high-risk and low-risk groups using the median for their risk score values for the new model. For the EORTC model, the cut off of 1.27 from the original paper was used to assign the high-risk and low-risk groups. A Log-rank test was used to compare the survival curves of these two groups. We also compared our results with a model using PS alone.

Results:
For OS, the EORTC model c-index was 0.55 (0.52, 0.58) and P = 0.0007 comparing high- and low- risk patients for testing set. The new model c-index was 0.60 (0.56, 0.64), with P < 0.000001 for testing set. Using the new model, the median OS in the high-risk and low-risk groups in the testing set were 5.16 (4.70, 6.37) and 10.41 (7.95, 14.32) months, respectively. PS alone produced c-index of 0.55 (0.53, 0.57) and P = 0.0002 for testing set. The AUC scores at 6-months for testing set generated by EORTC and PS alone models are 0.62 and 0.66. The new model generated AUC scores at 6-months of 0.70.

Conclusion:
Our new model performs better than the EORTC model or PS alone for survival prognostication in patients with mesothelioma.

Background:
Biphasic mesothelioma has a poor prognosis. There is no clear evidence on the role of multimodality treatment in patients with biphasic mesothelioma. The aim of this study was to analyse the impact of pathological features on survival, to determine which patients may benefit from multimodality treatment.

Methods:
Between January 2005 and December 2015, 214 patients with biopsy-proven biphasic mesothelioma were retrospectively identified to fulfil our inclusion criteria. The primary outcome was survival measured from time of diagnosis. Two slides were reviewed for each patient by a specialist thoracic pathologist. Slides were stained with Hematoxylin and Eosin (H+E) and the immunohistochemically-stained slides were digitally scanned and analysed using a Hamamatsu Nanozoomer scanner (Hamamatsu ‘NDP.View2’). The proportion of epithelioid and sarcomatoid components on each slide was mapped and its area in mm[2] recorded as a percentage of the total tumour area studied. Necrosis and lymphovascular invasion were analyzed. Patients with no slides available were excluded from the analysis (n=96). All eligible patients (n=118) were followed up until May 2016.

Results:
One hundred and eighteen patients were included in the analysis, 106 (89.9%) were male with a median age of 73 (range 53 - 91). Twenty-eight patients (23.7%) underwent Pleurectomy Decortication (PD) and 90 patients received medical treatment alone, either with chemotherapy or best supportive care. The median overall survival (OS) was 11.2 months (range 0.3 – 36.2). At 1 year and 2 years, 49.1% and 6.4% of patients were alive respectively. Univariable analysis revealed both age and PD to be associated with improved survival (p=0.004 and p=0.004). Patients treated with PD had OS of 12.8 months (range 5.6 - 36), compared to 9.2 months (range 0.3 – 31.8) in patients receiving medical treatment alone. No lymphovascular invasion was identified in any specimen. Necrosis was not correlated with survival (p=0.76). The proportion of epithelioid (p=0.45) or sarcomatoid (p=0.60) component within the specimen did not correlate significantly with overall survival. This remained true when patients undergoing surgical PD (epitheloid p=0.42 and sarcomatoid p=0.60) and medical treatment (epitheloid p=0.43 and sarcomatoid p=0.11) were analysed as separate subgroups.

Conclusion:
The prognosis for patients with biphasic mesothelioma remains poor, even after multimodality treatment including pleurectomy decortication (PD). However, necrosis and the proportion of sarcomatoid histology is not helpful in selecting patients with more favourable prognosis, who may benefit from a multimodality approach.

Background:
Mesothelioma data has been collected by the National Lung Cancer Audit (NLCA) since it was introduced in 2004 to improve standards of care for patients in the UK and ultimately improve outcomes. The first mesothelioma-specific report combining data submitted to the audit from 2008-2012 was reported in 2014, capturing approximately 85 per cent of total incident mesothelioma cases. This same year, the NLCA switched from using a bespoke dataset to use the generic Cancer Outcomes and Services Dataset (COSD), linked to other National Cancer Registration and Analysis Service (NCRAS) registry datasets, as its primary data source. This dataset change has allowed data for all mesothelioma cases diagnosed during 2014, in England, to be analysed for the first time and reported here.

Methods:
Using 2014 COSD data submitted to the NLCA for all hospital trusts in England, we have analysed demographic, diagnostic and active treatment data items and in particular, have calculated the proportion of cases receiving histological subtype confirmation, palliative chemotherapy and per cent surviving to one year after diagnosis, both nationally and by strategic cancer network (SCN).

Results:
There were 2179 cases of pleural mesothelioma diagnosed in England in 2014. Histological confirmation of diagnosis was very high, but the proportion of mesothelioma cases without histological sub-classification (M9050/3) was 47%. This unspecified mesothelioma rate varied from 32.6 up to 74.4% by cancer network across England. Overall, palliative chemotherapy was given to 51% of patients with performance status (PS) 0-1, however at network level, this varied from 42.2% up to 77.4%. For all cases of mesothelioma, the 1 year overall survival was 43% with variation by network from 37.5 to 55.6% with adjusted odds ratios (OR) ranging from OR 0.8 up to 1.56.

Conclusion:
There has been improvement in the proportion of mesothelioma patients in England receiving histological subtyping compared to previous years and in the proportion of patients with good PS being treated with palliative chemotherapy. However, there is still marked variation across the country and addressing this may improve national outcomes further. Cancer networks and individual hospitals should examine their results and implement mechanisms to ensure best practice is being followed.

Background:
Prognosis of Malignant Pleural Mesothelioma (MPM) is poor and median survival is about 12 months. If any associations can be established between biomarkers and MPM, there will be benefit to clinical practice. In this study, the aim is to examine expression levels of the genes selected from relevant literature and utilizing in silico methods in the determination of prognosis of MPM.

Methods:
The study group consisted of 54 MPM patients treated by chemotherapy. The expression of 6 genes; sestrin 1 (SESN1), laminin subunit alpha 4 (LAMA4), midkine (MKN), fibulin-3 (EFFL-3), syndecan-1 (SOC-1) and hyaluronan-2 (HYA-2) were examined by qPCR in the tumor tissues. SESN1 and LAMA4 were identified using an in house R based script “Unsupervised Survival Analysis Tool." MKN, EFFL-3, SOC1 and HYA-2 were determined according to existing literature. We used two housekeeping genes; glucose-6-phosphate dehydrogenase (G6PD), TATA-box binding protein (TBP) as controls. qPCR Relative quantification of gene expression was based on the geometric mean G6PD, TBP. The relation between gene expression and prognosis was determined by categorizing samples into two groups using all possible cut-off values, analyzed by the Log Rank test (Log-rank test with multiple cut-offs). Cut-offs generating a p value less than 0.05, between %10-90 percentiles were considered significant.

Results:
Mean age of study group was 62.5± 9.7 years (r:36-82). Of the patients, 43 (79.6%) had epithelioid cell type mesothelioma. The median survival (MS) for all patients was 10 (±1.18 SE) months (CI 95%;7.69 to 12.30). Twenty-five patients (46.3%) survived less than 12 months, 29 (53.7%) more than 12 months, and 4 (7.4%) were still surviving at the end of the study. The clinical factors that was associated with survival were histopathology (p=0.004) and stage (p=0.036). MKN, EFFL-3, SOC1, HYA-2, SESN-1 were found to be associated with survival time by univariate analyses. After the correction with histopathology and stage, MKN (p=0.041), SOC1 (p=0.015), HYA-2 (p=0.003), SESN-1 (p=0.028) were found to be related with survival time. Additionally, in only epithelioid type MPM patients, HYA-2 expression was found to be related with survival time according to multivariate analyses (p=0.016).

Conclusion:
High MKN expression is potential biomarker of poor prognosis and high SOC1, HYA-2, SESN-1 expressions are potential biomarkers of good prognosis in MPM patients, and should be further investigated. High HYA-2 expression also can be utilized as good prognosis biomarker for epithelioid type MPM. *This study was partly supported by General Directorate of Health Researches, Republic of Turkey, Ministry of Health.

Background:
Malignant Pleural Mesothelioma (MPM) is an uncommon thoracic malignancy which remains a challenge in management. In recent years the use of surgery has been widely debated especially the use of extrapleural pneumonectomy. (EPP) Following EPP radiotherapy has been widely used to reduce local control with varied results. In patients that are not surgical candidates definitive intensity modulated radiotheapy (IMRT) based treatment has become an option in addition to systemic therapy (Zaruder et al.). We sought to report our results in a unique middle-eastern population with low-level asbestos exposure for both: IMRT – post – EPP and with IMRT used as definitive therapy for patients who were unresectable.

Methods:
Complete medical records of MPM patients (n=28) treated with IMRT at the Davidoff Center were reviewed with Helsinki committee approval. Patients were divided into two groups: post- EPP(n=17) and without surgery(n=11). Patients following EPP were treated with IMRT to 54Gy to the entire hemithorax. Patients without surgery were treated with pleural IMRT (P-IMRT) to the entire hemithorax to 54Gy. cisplatinum\pemetrexed chemotherapy was used in 18\28 patients. Patients were grouped by asbestos exposure (9/28-32%) and Mediterranean/Arabic (58%) vs. caucasian ethnicity(42%). Patients were followed for outcomes and toxicity until death.

Results:
28 patients, predominately male, (82%) were treated at a single center in Israel between 8/2007 and 3/2016. For patients post-EPP 56% received sequential chemotherapy with IMRT. 94% had epitheliod histology. 11/17 (65%) had disease progression with a median time TTP of 12 months(range 1-72mos) . 23% of remain alive without evidence of disease. Only 2/17 (11%) experienced local failure. The Median OS for this group is 23.6 months(1- 100 months) . For the 11 patients treated with definitive P-IMRT, 90% received platinum based chemotherapy. 81% were epitheliod histology. 54% have experienced progression with median TTP of 12 months. Median overall survival for the cohort is 13.5 months (range 8-49months) . Of note no episodes of grade 3 or greater radiation pneumonitis were seen in the entire cohort.

Conclusion:
This is the first Israeli report of outcomes following definitive therapy for mesothelioma. IMRT was delivered without toxicity. The local control following EPP was excellent with encouraging OS. P-IMRT can be delivered to unresectable patients with encouraging overall survival and time to progression. Further work must be done to sequence systemic therapy with IMRT.

Background:
Dysregulation of FAS/FASL apoptosis-related pathway may lead to cancer cells immune escape and influence platinum-based chemotherapy outcome, which is currently the mainstay treatment for malignant pleural mesothelioma (MPM). FAS-670 A>G (rs1800682) and FASL-844 C>T (rs763110) single nucleotide polymorphisms (SNPs) are two functional promoter polymorphisms of FAS and FASL genes, respectively which may alter their transcriptional levels. They have been previously correlated with clinical outcome of non-small cell lung cancer (NSCLC), breast and bladder cancers. Therefore, we aim to investigate the influence of these polymorphisms on clinical outcome of MPM patients.

Methods:
In this cohort study [NCT02269878], 68 epithelioid MPM Egyptian patients treated with first-line platinum-based chemotherapy were recruited from Department of Clinical Oncology and Nuclear Medicine, Ain Shams University and El-Nasr hospital for health insurance in the period between April 2014 and May 2015. The genotype analysis was performed using TaqMan[®] SNP Genotyping assay. We evaluated the association between the selected polymorphisms and response rate, progression free survival (PFS) and overall survival (OS) at 18 months.

Results:
The mean age of patients was 55.5 ± 9.5 years and 45.6 % of them received Platinum in combination with pemetrexed, while 54.4% received platinum in combination with gemcitabine. FASL-844 CC genotype was more common than expected in early stage tumors (p = 0.042). There was no association between the investigated polymorphisms and response rate or 18-months OS. However, the median PFS for carriers of FASL-844 CC genotype was 14 months (95 % CI: 12.8 - 15.2 months) and 9 months (95 % CI: 7.2 - 10.8 months) for carriers of FASL-844 CT/TT genotypes (Log-Rank: 6.2; p = 0.013).Also, the number of platinum-based cycles and tumor stage were found to be significant variables by univariate analysis (p =<0.001, p = 0.006, respectively). Multivariate cox proportional hazards regression showed that the carriers of FASL-844 CT/TT genotypes were still more susceptible to disease progression than carriers of FASL-844 CC genotype (adjusted HR = 4.40 , 95 % CI: 1.62 - 11.89, p = 0.004).

Conclusion:
Our results suggest that FASL-844 C/T polymorphism could predict PFS in MPM patients receiving Platinum-based chemotherapy; therefore, this should be further evaluated as potential marker for the prediction of clinical outcome in patients with MPM.

Background:
Malignant pleural mesothelioma (MPM) is a rare cancer with relatively poor outcome. Only stage (TNM) and histotype can be considered prognostic factors, but TNM still results inaccurate and difficult to be classified. Several studies investigated the use of tumor volume (TV) for response assessment, but its role as predictor of survival is unclear. A cut-off of 600 cm[3 ]seemed to divide patients (pts) with different prognosis. Our objective is to assess the association between baseline TV, stage/TNM and overall survival (OS).

Methods:
We retrospectively selected 49 MPM pts treated from August 2002 to January 2012. All pts had a digitally available baseline chest computed tomography (CT), performed before any treatment and up to 3 months after histological diagnosis. CT staging was carried out by two thoracic radiologists according to TNM staging system (7[th] Edition). Pleural disease volume mesaurements were obtained by a computer system. Major prognostic variables (age, sex, histology, TV, stage/TNM, treatment) were collected. Pts were divided in 2 groups according to baseline TV (large volume >600cm[3]; small volume <=600cm[3]). Association of volume groups, stage, T, N, M separately and OS was tested using Cox models adjusted by age, sex, histology and surgery.

Results:
Thirty-three pts were men, 16 women; median age was 62 years (range 25-78). Forty pts had epithelioid MPM, 7 mixed histology, 2 unknown histology. Four pts were diagnosed in early stage (I-II) and 45 in advanced stage (III-IV). The mean baseline TV was 494.15 cm[3 ](range 17.91- 2,329.03). Pts with small volume had a slight but not statistically significant tendency to survive longer than pts with large volume (3-year OS=32% vs 21%, respectively). The HR was 1.5 (95% CI=0.6-3.7) for large volume pts, 4.3 (p=0.08;95%CI=0.8-22.1) and 7.5 (p=0.02;95%CI=1.4-39.9) for stage III and IV, 7.0 (p=0.001;95%CI=2.3-21) and 5.4 (p=0.005;95%CI=1.7-17.4) for T3 and T4, respectively. Regarding N and M, not statistically significant results were observed.

Conclusion:
Coherently with the available literature, we report an association between baseline TV and prognosis; however it seems weak and barely near to statistical significancy. On the contrary, stage, in particular T3, showed a stronger association with prognosis. Considering the small sample and the wide 95% CI, our results should be interpreted with caution; nevertheless they open a critical question on the TV prognostic role and suggest a greater relevance of adjacent organs infiltration in predicting prognosis. Further collaborative studies are needed.

Background:
Malignant pleural mesothelioma (MPM) is a dreadful disease, and the treatment strategy of it has not been established yet. Our experience of trimodality therapy with extrapleural pneumonectomy (EPP), radiation therapy, and chemotherapy for epithelioid MPM is reported.

Methods:
26 consecutive EPP for epithelioid MPM which were performed from June 2006 to December 2015 in our hospital were reviewed. We have instituted a trimodality therapy protocol consisting of EPP, adjuvant 45-50.4 Gy hemithoracic radiation therapy, and adjuvant CDDP plus PEM chemotherapy. 21 patients have been treated with this protocol. However, 5 patients were given induction CDDP plus PEM chemotherapy, and referred to us. They were scheduled to undergo EPP and adjuvant hemithoracic radiation therapy. Overall survival was calculated using Kaplan-Meier method. This was one institutional retrospective study.

Results:
Median age at EPP was 61 years old. Female was 7, and male was 19. Right side was 15, and left side was 11. Median EPP time was 7 hours 22 minutes. No blood transfusion during EPP was 12 cases (46%). 30 day mortality was zero. Atrial fibrillation was the most common morbidity, and developed in 9 patients (35%). IMIG pathological stage was stage IV in 1, stage III in 18, stage II in 3, and stage Ib in 4. Adjuvant 45-50.4 Gy radiation therapy was completed for 21 patients (81%). 5 patients (19%) could not undergo chemotherapy. 18 patients (69%) underwent trimodality therapy. Postoperative median follow-up period was 5 years and 2 months. Five year survival, two year survival, and median survival of all 26 patients were 40%, 55%, and 30.4 months.Figure 1



Conclusion:
The five year survival and median survival of trimodality therapy with EPP, 45-50.4 Gy hemithoracic radiation therapy, and CDDP plus PEM chemotherapy for epithelioid MPM is 40% and 30.4 months. This treatment strategy is feasible, and the prognosis has been very improved.