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Background Previous studies have demonstrated that IgG1 mAbs as cetuximab, stimulate Antibody-Dependent Cell Cytotoxicity (ADCC). Among immune checkpoint inhibitors, avelumab is the only fully human IgG1 anti-PD-L1 mAb with ADCC properties. Anti-PD-L1 and anti-EGFR mediated NK cytotoxicity is evaluated. Methods LDH release was analyzed to study NK-mediated cytotoxicity by LDH Cytotoxicity Assay Kit and results was correlated to the level of PD-L1, EGFR and MHC-I cell surface expression analyzed by flow cytometry. NK-mediated cytotoxicity of the combination of anti-PD-L1 and anti-EGFR mAbs was studied in a panel of NSCLC cells lines encompassing different tumor types, using as effector NK cells isolated from healthy donors or NSCLC patients. Results PD-L1/EGFR/MHC-I expression levels correlated with enhanced ADCC lysis by the combination of avelumab and cetuximab as demonstrated by LDH assay, CD16 and CD107a mRNA. No significant difference in avelumab plus cetuximab-mediated ADCC between NK cells from healthy donors or from NSCLC patients was observed with a trend in favor of cancer patients, indicating that NK from cancer patients maintain lytic activity. ADCC capability of NK cells isolated from patients enrolled in the phase II study CAVE (Cetuximab-AVElumab)-lung, a single arm phase II clinical study of the combination of avelumab plus cetuximab in the second line treatment of metastatic non small cell lung cancer (NSCLC) patients (EUDRACT 2017- 004195-58) study resulted significantly enhanced after the experimental treatment compared to untreated baseline and healthy donors samples. Conclusions The combination of anti-EGFR and anti PD-L1 IgG1 antibodies is synergistic in terms of ADCC, where each antibody complements each other by promoting a more permissive immune reaction against the tumor, active also in otherwise immune-resistant cancers. Legal entity responsible for the study Universita degli studi della Campania Luigi Vanvitelli. Funding Merck KGaA. Disclosure F. Ciardiello: Advisory / Consultancy, Research grant / Funding (institution): Roche; Advisory / Consultancy, Research grant / Funding (institution): Bayer; Advisory / Consultancy, Research grant / Funding (institution): Amgen; Advisory / Consultancy: Merck; Advisory / Consultancy: Pfizer; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Servier; Advisory / Consultancy: BMS; Advisory / Consultancy: Cellgene; Advisory / Consultancy: Lilly; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Ipsen. F. Morgillo: Advisory / Consultancy: MSD; Advisory / Consultancy: Lilly; Research grant / Funding (institution): AstraZeneca. All other authors have declared no conflicts of interest.

Background Immune checkpoint inhibitors (ICIs) in patients with pretreated advanced NSCLC (aNSCLC) showed an overall survival (OS) benefit over standard chemotherapy in phase III randomized clinical trials (RCTs). Nevertheless, a significant portion of patients do not benefit from ICIs. The identification of biomarkers to select patients most likely to respond to ICIs is greatly needed in clinical practice. The role of baseline clinical and blood biomarkers as prognostic of response to ICIs was investigated in patients with aNSCLC and a prognostic ImmunoScore is defined. Methods We retrospectively reviewed clinical data of aNSCLC patients consecutively treated with single agents anti PD-1 or anti PD-L1 as 2nd (81.8%) or ≥ 3rd (18.2%) line at University Hospitals of Valencia and Naples. ECOG PS, sites of metastases, neutrophil to lymphocyte ratio (NLR), LDH and albumin levels were recorded at baseline. The impact of these variables on PFS and OS was assessed through survival analyses (Kaplan Meier method), univariate (log rank test) and multivariate analyses (Cox proportional hazard model). Results The analysis included 132 pts. Median PFS and OS were 3 (95% CI 2.74-3.26) and 9 (95% CI 5.90-12.02) months respectively. The univariate analysis for PFS showed that baseline NLR>4 (p = 0.001), albumin 1 (HR 2.96, p 400 U/l (p = 0.089). The multivariate analysis for PFS confirmed as statistically significant independent negative prognostic factors PS > 1 (p = 0.001) and liver metastases (p = 0.011). Finally, according to PS, liver metastases, NLR and albumin three different prognostic groups at high (3-4 RF), intermediate (1-2 RF) and low (0 RF) risk for OS were defined. Median OS was respectively 5 (95% CI 3.45-6.55), 7 (95% CI 4.98-9.02) and 23 (95% CI 16.53-29.47) months (p Conclusion Baseline evaluation of clinical and blood biochemical parameters can be a tool to predict outcome in patients treated with ICIs for aNSCLC. Moreover, combining them in ImmunoScore may help to identify pts candidates to second or subsequent Iines of therapy who most likely will benefit from ICIs. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Background cfDNA analysis could represent an alternative to invasive biopsy in patients with NSCLC to better understand disease profile. We report a real-world experience of genomic assessment of an Italian patients’ cohort. Methods We evaluated 58 non-oncogene-addicted aNSCLC patients treated at our Institution from January 2018 to May 2019. All patients were characterized using cfDNA NGS Guardant360® platform (Guardant Health, Redwood City, CA). Association between a positive result (defined as cfDNA detected) and clinical features was assessed using logistic regression test. Using online library dataset (mycancergenome.org) and literature data, we divided patients in genomic clusters and performed a descriptive analysis. Results The median age of patients was 67 (range 38-85), 44 men and 14 women, 8.6% never smokers, 41.4% former and 50% current smokers. 14 patients (24.2%) had squamous cell carcinoma and 44 (75.8%) had non-squamous NSCLC, 50 of 58 at stage IV at the time of testing. Most patients were PS 0-1 per ECOG scale and has been treated with at least one systemic regimen before performing NGS. 47 of 58 patients (81%) has been considered positive at the analysis. No targetable genomic alterations per local authority has been found. More than 40 different mutated genes were detected, the most common alterations involved TP53 (60% of patients), KRAS (34%), PDGFRα (17%), EGFR (15% - not-TKI-sensitive), PI3KCA (15%), CDKN2A (15%). Chemotherapy (but not immunotherapy) and extra-thoracic radiotherapy before testing increased the probability to result positive at NGS of 4.5 and 2 folds respectively. Six mutational clusters have been identified. Most patients had genomic alterations in Replication Stress - DNA damage/repair, cell cycle - (72%) and Tyrosine Kinase Receptor/Growth Factor pathway (62%), less common mutations affected PI3K/AKT/mTOR and Hormone signaling (26% and 9% respectively). Conclusions Our experience demonstrated the feasibility of NGS-based NSCLC patients genomic profiling. The widespread of NGS platforms (and the subsequent reduction of costs) should encourage clinicians to use these methods with all patients to guarantee access to developing therapeutics and clinical trials. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure F. Ciardiello: Advisory / Consultancy, Research grant / Funding (institution): Roche; Advisory / Consultancy, Research grant / Funding (institution): Amgen; Advisory / Consultancy, Research grant / Funding (institution): Merck; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Ipsen; Advisory / Consultancy, Research grant / Funding (institution): Bayer; Advisory / Consultancy: BMS; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Servier; Advisory / Consultancy: Celgene; Advisory / Consultancy: Lilly; Advisory / Consultancy: Pfizer. F. Morgillo: Advisory / Consultancy: MSD; Advisory / Consultancy: Lilly; Research grant / Funding (institution): AstraZeneca. All other authors have declared no conflicts of interest.

Background In the era of precision medicine, cancer treatment strategies are aimed to be patient-tailored. Here, we report a translational study conducted on a NSCLC patient where the potentiality of ex vivo spheroidal cultures and peripheral blood biomarkers were investigated to analyze correspondence with treatment response. Methods We developed a protocol to generate ex vivo spheroidal cultures from patient’s surgical sample that has been used to assess drug sensitivity through cell proliferation MTT assay. Primary tumor tissue, patient-derived spheroids and patient’s circulating tumor DNA (ctDNA) were characterized through immunohistochemistry (IHC), immunofluorescence and next generation sequencing (NSG) analysis. Tumor infiltrating lymphocytes (TILs) from surgical specimen were analyzed by FACS and the analysis of TCR repertoire was conducted using Spectratyping technique. Moreover, peripheral blood samples collected at different time points underwent qPCR analysis to assess cytokines expression and flow cytometry to study peripheral blood mononuclear cells (PBMCs). All these data were correlated with clinical and radiological evaluations. Results Immunohistochemistry, immunofluorescence, NGS analysis and TCR repertoire assay showed elevated concordance among primary tumor tissue, spheroids and ctDNA. Cisplatin-based chemotherapy and anti-PD-1 treatment sensitivity assessed in spheroidal cultures allowed us to anticipate patient response to chemo- and immunotherapy. Furthermore, circulating cytokines expression levels and lymphocytes subpopulations profiling correlated with clinical and radiological response to first line anti-PD-1 therapy. Conclusions This approach demonstrated the feasibility of using spheroidal cultures, cytokines expression levels and PBMCs profiling to follow patient’s disease history and response to treatment in parallel with clinical and radiological evaluation. Legal entity responsible for the study Universita degli Studi della Campania Luigi Vanvitelli. Funding Has not received any funding. Disclosure F. Ciardiello: Advisory / Consultancy, Research grant / Funding (institution): Roche; Advisory / Consultancy, Research grant / Funding (institution): Bayer; Advisory / Consultancy, Research grant / Funding (institution): Amgen; Advisory / Consultancy: Merck; Advisory / Consultancy: Pfizer; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Servier; Advisory / Consultancy: BMS; Advisory / Consultancy: Cellgene; Advisory / Consultancy: Lilly; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Ipsen. F. Morgillo: Advisory / Consultancy: MSD; Advisory / Consultancy: Lilly; Research grant / Funding (institution): AstraZeneca. All other authors have declared no conflicts of interest.