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Immunotherapy based on Immune Checkpoint blockade (ICB) has become a significant therapeutic option for advanced Non Small Cell Lung Cancer (NSCLC) patients. However, there is an urgent need to find novel biomarkers that to reliably stratify good responders to immunotherapy. Currently, the available biomarkers are not specific enough. Exosomes are small membrane vesicles with sizes of 30-100 nm secreted by most cell types including cancer cells. Exosomes operate as an intercellular communication system by sending proteins, mRNA and miRNAs among other relevant RNA molecules. Exosomes enriched with miRNAS are involved in proliferation, differentiation, maduration and immune cell activation. Moreover, in cancer cells miRNA and other small RNA molecule expressions are dysregulated. Exosomes produced from cancer patient's plasma have been shown to be accurate diagnostic tools for the disease. In this study we profiled miRNAs and other small RNA cargo by exosome of plasma samples from 77 Non Small Cell Lung Cancer (NSCLC) metastatic patients before and after the first cycle of immunotherapy to evaluate the potentiality of predicting response to immunotherapy. We perform exosomes together with RNA isolation and small RNAseq sequencing from plasma samples before and after the first ICB cycle. Two independent softwares were used to identify small RNAs (RNAtoolbox and mirMaster). Prior ICB treatment we did not find differentially expressed miRNAs or other small RNA between good and bad responders. Interestingly we identified 12 exosomal miRNA differentially expressed between good and bad responders after the first cycle of ICB. Intriguingly, levels of miR-134-5p, miR-142-3p, miR-143-3p among others previously associated with NSCLC were found to be considerably higher in the good responder group than the bad responder group. Regarding other smallRNA molecules we observed a great variety and variability of piRNA, rRNA, scaRNA, lncRNA, snoRNA, snRNA, miscRNA and circRNA. To address the function of miRNA and other differentially expressed RNA molecules, we consulted KEGG, GO and Reactome for gene regulatory networks. Interestingly, KEGG results show pathways in cancer as top hit and Reactome highlight Immune System and cancer hits. In conclusion, we observed that patients that have favorable response to ICB have distinctive plasma exosomal miRNA patterns that could be used as possible biomarkers for predicting the effectiveness of immunotherapy in advanced NSCLC patients Citation Format: Maria Garrido-Barros, Javier Oliver, Juan Luis Onieva, Beatriz Martinez-Galvez, Jaime Duddelman, Antonio Rueda, Elisabeth Perez, Emilio Alba, Inmaculada Ramos, Juan Zafra, Manuel Cobo, Isabel Barragán. Dynamic characterization of small RNAs in non small cell lung cancer exosomes under immune-checkpoint inhibitor treatments. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3746.

The irruption of the immunotherapy for the treatment of non-small cell lung cancer (NSCLC) based on Immune Checkpoint Inhibitors (ICI) PD-1 and PD-L1 inhibitors is considered a treatment revolution. However, only a small percentage of patients benefit with ICI treatment over the long term, and precise biomarkers that can recognize these individuals before or early during treatment have so far eluded. PD-L1 expression and tumor mutational burden (TMB) are the most well studied biomarkers for predicting response to PD- (L)1 blockade-based ICI prior to treatment. TMB is still being clinically assessed whereas PD-L1 has several drawbacks for prediction of persistent benefit. Our aim is to evaluate the basal state and dynamic changes of cell-free DNA (cfDNA) concentration to predict and monitor response in NSCLC patients starting ICI. A total of 248 cfDNA concentration measurements were performed from 87 NSCLC patients. The quantification was done before the start of the treatment, at the second ICI cycle, after 6 and 12 months in treatment, and at progression if it was within the first 12 months. Quality and quantity of the cfDNA was assessed using Qubit High Sensitivity and Bioanalyzer 2100. We first explored the association with response of the basal cell-free DNA concentration using Mann-Whitney-Wilcoxon test. The longitudinal analysis between different time points was tested with Wilcoxon signed-rank tests. Response was ascertained using RECIST parameters at 3, 6, and 12 months. We evaluated the utility of the cfDNA concentration as a prognostic factor using Mantel-Cox test. The response association results indicate that early and long-term response is associated with lower levels of basal cfDNA (p Citation Format: Isabel Barragan, Elisabeth Perez-Ruiz, Juan Luis Onieva, Maria Garrido-Ramos, Beatriz Martinez-Galvez, Jaime Dubbelman, Emilio Alba, Juan Zafra, Manuel Cobo, Javier Oliver, Antonio Rueda-Dominguez. Cell-free DNA concentration as a prognostic biomarker in patients with non-small cell lung cancer under immunotherapy treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1044.

Background: Pembrolizumab improves overall survival (OS) when added to platinum-based doublet chemotherapy (PDC) in patients with aNSCLC. However, progression-free survival (PFS) and OS remain disappointing for most patients and can vary depending on programmed death-ligand 1 (PD-L1) status. This highlights the need for additional therapeutic options. Canakinumab is a monoclonal anti-interleukin-1β antibody that inhibits pro-tumor inflammation and potentially enhances anti-tumor immune responses, with the potential to synergize with programmed death 1 inhibitors plus chemotherapy. Methods: CANOPY-1 is a randomized, double-blind phase 3 study investigating the addition of canakinumab or placebo (PBO) to 1L pembrolizumab + PDC. Patients with previously untreated stage IIIB/C or IV NSCLC, of any histology and no known EGFR or ALK alterations, were randomized 1:1 and stratified based on PD-L1 status, geographic region, and histology to canakinumab 200 mg or PBO every 3 weeks, plus pembrolizumab and histology-guided PDC for 4 cycles, followed by maintenance canakinumab or PBO, with pembrolizumab ± pemetrexed. The primary endpoints were investigator-assessed PFS (Response Evaluation Criteria In Solid Tumors 1.1) and OS. Exploratory biomarker analyses were also investigated and will be presented at the time of the congress. The cut-off dates for these analyses were May 18, 2020 (PFS) and August 9, 2021 (OS). Results: A total of 643 patients were randomized to canakinumab (n=320) or PBO (n=323) in combination with pembrolizumab + PDC. Baseline characteristics were well balanced across treatment arms. Median PFS was 6.8 months for both treatment arms (HR 0.85, 95% CI, 0.67-1.09; one-sided P=0.102). Median OS was 20.8 and 20.2 months for the canakinumab and PBO arms, respectively (HR 0.87, 95% CI, 0.70-1.10; one-sided P=0.123). Grade 3/4 adverse events (AEs) were reported for 205 (64.1%) patients in the canakinumab arm and 191 (59.3%) patients in the PBO arm, and fatal AEs were reported for 37 (11.6%) and 47 (14.6%) patients, respectively. AEs (any grade) leading to discontinuation of any study drug were reported for 72 (22.5%) patients in the canakinumab arm and 61 (18.9%) patients in the PBO arm. Conclusions: The addition of canakinumab to pembrolizumab plus PDC did not statistically improve PFS nor OS for 1L treatment of patients with aNSCLC. No unexpected safety findings were observed with the addition of canakinumab to pembrolizumab plus PDC. Citation Format: Daniel S. Tan, Enriqueta Felip, Gilberto Castro, Benjamin J. Solomon, Alastair Greystoke, Byoungchul Cho, Manuel Cobo, Tae Min Kim, Sandip Ganguly, Enric Carcereny, Luis Paz-Ares, Jaafar Bennouna, Marina Garassino, Michael Schenker, Sang-We Kim, Bijoyesh Mookerje, Vanessa Q. Passos, Stephanie Deudon, Bharani Dharan, Yuanbo Song, Rafael Caparica, Bruce E. Johnson. Canakinumab in combination with first-line (1L) pembrolizumab plus chemotherapy for advanced non-small cell lung cancer (aNSCLC): Results from the CANOPY-1 phase 3 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT037.

Flow cytometry is a technique for analyzing cells that are suspended in a buffered salt-based solution and flow past one or more lasers. Visible light scatter and one or more fluorescence characteristics are assessed for each particle. The most common use of flow cytometry is immunophenotyping. In traditional flow cytometry analysis, a region around a population of cells is manually drawn (gating) in two-dimensional scatter plots. This enables the measuring of specific groups of cells. However, manual gating could increase the overall error rate of the study and makes the analysis hardly reproducible since it is done in less controlled settings. Furthermore, it represents a bottleneck in the analysis of large amounts of data. Some processes, such as gating, have recently been automated using R packages. In addition, dimensionality reduction approaches have been developed in the flow cytometry environment to take advantage of information from several markers at once. Despite this, none of them are integrated in a harmonized way, and none of them allow back-gating to emphasize the study population and eliminate false positives. To tackle this problem, our team has developed flowTOTAL (github.com/ImmunoOncology/flowTOTAL), a user-friendly command line workflow to analyze flow cytometry data. The major attractive feature is the facility to perform with one command not only a traditional analysis, but also an unsupervised analysis. As input, the user has to indicate the folder with the .FCS files, the metadata associated with each file, and the marker to be used during back-gating. The pipeline is divided into three main sections: preprocessing, traditional analysis, and unsupervised analysis. During preprocessing each. FCS will be subjected to correcting for fluorescence spillover (compensation), detection of anomalies by checking flow rate and signal acquisition as well as removing doublets based on forward scatter (QC). For the traditional analysis, auto-gating will be performed for the identification of the target population using back-gating. For each set of given markers, the number of events obtained and the scatter plot will be generated. Finally, in the unsupervised analysis, the population of interest will be specified and the pipeline will proceed with normalization, dimensionality reduction using PCA or UMAP and finally a clustering approach for subpopulation identification. In addition, differential abundance analysis can be performed with metadata information. flowTOTAL is presented as a standardization for the analysis of flow cytometry data, comprising all the necessary steps for comprehensive analysis and allowing mass analysis. Furthermore, it goes beyond the simple quantification of particles, since the implementation of more complex methodologies allows for the discovery of subpopulations that are not present in the traditional analysis but have a significant biological role. Citation Format: Juan Luis Onieva, Patricia Cháves, Javier Oliver, María Garrido-Barros, Juan Zafra, Belén Sojo, Alfonso Sánchez, Martina Álvarez, Pedro Jiménez, Emilio Alba, Miguel Berciano, Antonio Rueda, Manuel Cobo-Dols, Elisabeth Pérez, Isabel Barragán. flowTOTAL: A comprehensive bioinformatics workflow for flow cytometry automatic analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1910.

There is growing evidence supporting that long-term survival of neoadjuvant chemo-immunotherapy for locally advanced NSCLC patients can be achieved. However, some patients invariably progress within the short-term. Identification of patients at high risk of progression is needed to achieve a better control of the disease. Concentrations of baseline ctDNA have been shown to be of prognostic significance. Patients and methods 42 pre-treatment plasma samples from the NADIM clinical trial (NCT03081689), in which resectable stage IIIA NSCLC patients were treated with neoadjuvant chemo-immunotherapy with Nivolumab, were analyzed by NGS, using the Oncomine Pan-Cancer Cell-Free Assay™ (Thermo Fisher Scientific®). Variant calling, annotation and filtering were performed on the Ion Reporter (v5.14) platform using the OncomineTagSeq Pan-Cancer Liquid Biopsy workflow (v2.3). The final variant matrix was obtained from vcf files as generated from Ion Reporter (v5.14) platform and applying an internal pipeline (R-code is available upon request). Progression disease was evaluated by RECIST criteria V1.1. Results A total of 116 variants were detected in 88.10% (N=37) of the plasma samples collected before neoadjuvant treatment. The average number of variants detected per sample was 3.13. The most frequently mutated genes were TP53, which accounts for 59.52% of the detected variants, followed by PIK3CA (30.95%), MAP2K1 (30.95%), APC (23.81%), MTOR (9.52%) and KIT (9.52%). Patients in whom a GNA11 mutation was detected in the plasma sample by NGS showed worsen progression free survival (PFS) (HR: 14. 95%; CI: 2.6-71, P-value with Fold Discovery Rate correction: 0.019). Finally, ctDNA levels at Mutant Allele Frequency (MAF) below 1% at baseline were associated with improved PFS (P=0.025). At 30 months, PFS was 80.30% for these patients compared with 58.33% in patients with ctDNA levels ≥ 1%. Conclusions Molecular profiling of liquid biopsies collected before neoadjuvant chemo-immunotherapy using NGS can identify patients at high risk of progression who might require more aggressive adjuvant treatment in order to achieve a better control of the disease. Citation Format: Mariano Provencio, Roberto Serna-Blasco, Ernest Nadal, Amelia Insa, M. Rosario Garcia-Campelo, Diego Pereiro Corbacho, Manuel Domine, Margarita Majem, Delvys Rodriguez-Abreu, Alex Martinez-Marti, Javier de Castro, Manuel Cobo, Guillermo Lopez-Vivanco, Edel del Barco, Reyes Bernabe, Nuria Viñolas, Isidoro Barneto, Santiago Viteri, Eva Pereira, Ana Royuela, Marta Casarrubios, Clara Salas, Edwin R Parra, Ignacio Wistuba, Virginia Calvo, Raquel Laza-Briviesca, Bartomeu Massuti, Alberto Cruz-Vermudez, Atocha Romero. High levels of baseline ctDNA constitute a poor prognostic factor in progression-free survival in patients receiving neo-adjuvant chemo-immunotherapy: Results from NADIM clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 560.

Background: We have recently demonstrated that antibodies generated by vaccination (anti-EGFVacAbs) potentiate the effects of EGFR TKIs in epidermal growth factor receptor mutant (EGFR-mut) non-small cell lung cancer (NSCLC) cells growing in vitro and prevent the emergence of resistance to EGFRTKIs (1). Based on these preclinical results we have started the Epical Phase Ib clinical trial(NCT03623750), testing anti-EGF vaccination in combination with EGFR TKI in advanced EGFR-mut NSCLC patients. Methods: The EPICAL trial is a Multicenter, Open-Label, Exploratory Phase Ib Clinical Study in which 23 patients have been enrolled. Anti-EGF VacAbs is a vaccine for intramuscular administration that is designed to be used in conjunction with standard of care treatment which, in this study, is afatinib. Blood samples have been collected at baseline and every three months and sent to a central laboratory. The presence of EGFR mutations has been determined on purified DNA by 5′-nuclease real-time PCR (Taqman®) assay in presence of PNA. All the serum samples have been analyzed by ELISA to determine the levels of EGF, TGFα, HGF and GAS6, together with anti-EGF antibody titers. Sera of patients were added to EGFR-mutant cell lines growing in vitro and Western blot was used to analyze EGFR, AKT andERK activation. Results: Median EGF levels in the sera of enrolled patients was 179.5 pg/mL at baseline and no anti-EGFantibodies were detected. Three months after first immunization, median serum EGF levels decreased to30.8 pg/mL (p=0.0008) while the anti-EGF vaccination titers increased to 1:6000 (p=0.0012). Anti-EGF titers remained high and EGF levels low in the patient's sera for the duration of the trial. Regarding serum TGFα and HGF levels baseline were 19.4 pg/mL and 861.2 pg/mL, respectively, and after three months dropped to 7.9 pg/mL (p=0.0037) and 341.3 pg/mL (p=0.0259). In EGFR-mutant cell lines in culture, the activation of the EGFR, AKT or ERK was abrogated by the sera of immunized patients. Conclusions: Anti-EGF vaccination induces high anti-EGF antibody titers and effectively reducing the levels of EGF and other growth factors in the blood of advanced NSCLC patients. 1J Thorac Oncol. 2018 Sep;13(9):1324-1337. Citation Format: Silvia Garcia-Roman, Jordi Codony, Miguel Angel Molina-Vila, Nuria Jordana-Ariza, Beatriz Garcia, Monica Garzon, Clara Mayo de las Casas, Maria Jose Catalan, Andres Aguilar, Santiago Viteri, Andres F. Cardona, Delvys Rodríguez, Manuel Cobo, Noemi Reguart, Erik d'Hondt, Rafael Rosell. Evolution of antibody titers, growth factor levels and in vitro activity in the sera of patients enrolled in the EPICAL trial of afatinib combined with anti-EGF vaccination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 951.

Melanoma comprises a variety of malignant cell types from the skin, the mucous membranes, and the pigmented cells of the eye. Its incidence and mortality rates continue to rise worldwide, mainly driven by increased ultraviolet light exposure. Although screening improvements have led to a better diagnosis in the early stages, the outcome for patients with Metastatic Melanoma (MM) remains poor and the survival rate is considerably low. Recently, Immune Checkpoint Inhibitors (ICB) have revolutionized the treatment of MM, although only 30% of patients enjoy clinical benefit and a plethora of different molecular mechanisms is postulated to cause resistance. Several biomarkers are currently on the spotlight as putative predictors of response; however, they are not sufficiently informative and robust. Circular RNAs (circRNAs) are a class of single-stranded stable RNAs produced by 5′-to-3′ transcription of coding gene exons or long non-coding RNAs that act as translation templates, RNA-binding protein regulators, or miRNA-binding sponges. Here, we present an exploratory study to evaluate the potential use of circRNA as ICB response predictors. We recruited 23 MM patients treated with Nivolumab and categorized them as good responders (10 patients) and bad responders (11 patients). All RNA-seq with ribosomal depletion was performed on RNA isolated from pre-treatment FFPE tumor biopsies. Two bioinformatic tools that use de novo assembly for the identification of circRNAs (Starchip and Ciri) were employed independently to characterize the MM circRNAs profile and to identify a specific pattern for responders. Overall, a total of 731 circRNAs were detected. Interestingly, the number of circRNAs were comparatively higher in bad responders (11.917 reads, 321 circRNAs) versus good responders (7368 reads, 176 circRNAs), with 234 common circRNAs. Additionally, we also explored the biological function of the identified circRNAs in MM, by interrogating KEGG, GO and Reactome databases to delineate the gene regulatory networks specifically associated with the response-related phenotypes. The circRNAs exclusively expressed in patients presenting resistance to Nivolumab were associated with Transcriptional Misregulation in Cancer and Viral Infections processes. In contrast, circRNAs expressed uniquely in good responders were enriched in Generic Transcription pathways. Importantly, amongst the identified circRNAs, CDR1-AS was differentially downregulated in good responders (536 vs 1580 reads, p-value 0.002). Remarkably, CDR1-AS acts as miRNA-binding sponge of miRNA-7 in the context of invasion and migration in melanoma. In conclusion, our exploratory study paves the way to the utilization of novel biomarkers associated to immunotherapy failure in MM patients. Moreover, our results suggest that CDR1-AS is a novel putative predictor of ICB response. Citation Format: Juan Luis Onieva, Javier Oliver, Aurora Laborda-Illanes, Maria Rosario Chica-Parrado, Alicia Garrido-Aranda, Cynthia Robles-Podadera, Daniel Prieto, Elena Gallego, Alfonso Sanchez, Iñaki Comino, Vanessa De Luque, Martina Alvarez, Maria Jose Lozano, Pedro Jimenez, Miguel Angel Berciano-Guerrero, Emilio Alba, Manuel Cobo, Isabel Barragan. Evaluation of circular RNA profiling in metastatic melanoma patients treated with immune checkpoint blockade [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 281.

Background: We have previously described that EGF blocks the activity of EGFR tyrosine kinase inhibitors (EGFR-TKIs) in EGFR mutant NSCLC cells, an effect that is reversed by anti-EGF antibodies generated by vaccination (anti-EGF VacAbs) (1). In this study we aimed to determine the effect of EGF and anti-EGF Vac Abs in the activity of different kinase inhibitors in tumor cell lines with different genetic alterations. Methods: Anti-EGF VacAbs were obtained by immunizing rabbits with recombinant EGF. The cell lines used in this study were H2228 and H3122 (NSCLC, EML4-ALK positive), H23 (NSCLC, KRAS-G12C), DLD1 (colorectal carcinoma, KRAS-G13D), PC9 and PC9-GR4 (NSCLC, EGFR-mut). Tumor cell lines were treated with EGF, anti-EGF VacAbs and combinations with the kinase inhibitors alectinib, crizotinib, brigatinib (ALK inhibitors), trametinib (MEK inhibitor), dacomitinib and osimertinib (EGFR inhibitors). Cell viability was analyzed by MTT, changes of total and phosphorylated proteins were determined by Western blot and emergence of resistance by direct microscopic examination in low density cultures. Results: EGF significantly decreased the antitumor activity of alectinib, crizotinib and brigatinib in ALK translocated cells (H2228 and H3122), trametinib in KRAS mutant cells (H23 and DLD1) and osimertinib and dacomitinib in EGFR mutant cells (PC9). In combination with these TKIs, the anti-EGF VacAbs reversed the effects of EGF and significantly potentiated the antitumor activity of all the kinase inhibitors, blocking the activation of EGFR, Akt and Erk. Finally, the addition of the anti-EGF VacAbs to the culture medium delayed the appearance of resistant clones to kinase inhibitors. Conclusions: Anti-EGF VacAbs potentiate the antitumor effects of ALK, MEK and EGFR kinase inhibitors in tumor cell lines and delay the emergence of resistance in vitro. A clinical trial is currently testing anti-EGF vaccination in combination with afatinib in EGFR-mut advanced NSCLC patients.(1)Anti-Epidermal Growth Factor Vaccine Antibodies Enhance the Efficacy of Tyrosine Kinase Inhibitors and Delay the Emergence of Resistance in EGFR Mutant Lung Cancer Cells” Codony-Servat J, García-Roman S, Molina-Vila MÁ, et al. J Thorac Oncol. 2018. Citation Format: Jordi Codony-Servat, Silvia Garcia-Roman, Miguel Ángel Molina-Vila, Jordi Bertran-Alamillo, Ana Giménez-Capitán, Santiago Viteri, Andrés F. Cardona, Delvys Rodríguez, Manuel Cobo, Noemi Reguart, Niki Karachaliou, Erik d'Hondt, Rafael Rosell. Anti-EGF antibodies generated by vaccination significantly improve the activity of kinase inhibitors in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1450.

Background High TMB is predictive of PFS benefit with anti-PD-(L)1 ± anti-CTLA-4 therapy in mNSCLC. Preliminary results from MYSTIC (NCT02453282), an open-label, Phase III trial of first-line durvalumab (D; anti-PD-L1), ± tremelimumab (T; anti-CTLA-4), vs platinum-based chemotherapy (CT) in mNSCLC, indicate that blood TMB (bTMB, ≥16 mut/Mb; GuardantOMNI [Guardant Health]) from circulating tumor DNA (ctDNA) correlates positively with tissue (t) TMB (≥10 mut/Mb) (Spearman’s correlation coefficient = 0.6) and is predictive of survival benefit with D±T vs CT. Efficacy outcomes were assessed using additional exploratory cut-offs for bTMB and ≥10 mut/Mb for tTMB. Methods Pts with EGFR and ALK wild-type, immunotherapy/CT-naïve mNSCLC were randomized (1:1:1) to D (20 mg/kg i.v. q4w); D (20 mg/kg i.v. q4w) + T (1 mg/kg i.v. q4w up to 4 doses); or CT. bTMB was evaluated with the GuardantOMNI platform. tTMB was evaluated with the FoundationOne tissue NGS platform. bTMB cut-off was defined as ≥20 mut/Mb (bTMB≥20). Data cut-off: Oct 4, 2018 (OS); Jun 1, 2017 (PFS). Results 1118 pts were randomized. Baseline sample datasets were: bTMB 809; tTMB 460 pts, with baseline characteristics balanced between treatment arms. bTMB≥20 was associated with improved OS (D+T vs CT: HR 0.49 [95% CI 0.32, 0.74]; D vs CT: HR 0.72 [95% CI 0.50, 1.05]) and PFS (D+T vs CT: HR 0.53 [95% CI 0.34, 0.81]; D vs CT: HR 0.77; [95% CI 0.52, 1.13]); 24-mo survival: D+T 48.1% (95% CI 35.5, 59.7), D 33.8% (95% CI 23.4, 44.5) and CT 19.4% (95% CI 11.0, 29.5). Data for tTMB are shown (table). Additional cut-offs will be presented. Conclusion MYSTIC provides the most comprehensive data set to date supporting TMB as a predictive biomarker of OS benefit with immunotherapy. In exploratory analyses, bTMB≥20 was associated with OS and PFS benefit with D±T vs CT, with the greatest magnitude of benefit observed for pts receiving D+T. bTMB ≥20 mut/MbbTMB Citation Format: Solange Peters, Byoung Chul Cho, Niels Reinmuth, Ki Hyeong Lee, Alexander Luft, Myung-Ju Ahn, Paul Baas, Manuel Cobo Dols, Alexey Smolin, David Vicente, Vladimir Moiseyenko, Scott J. Antonia, Kazuhiko Nakagawa, Sarah B. Goldberg, Edward Kim, Rajiv Raja, Philip Brohawn, Delyth Clemett, Piruntha Thiyagarajah, Urban Scheuring, Feng Liu, Naiyer Rizvi. Tumor mutational burden (TMB) as a biomarker of survival in metastatic non-small cell lung cancer (mNSCLC): Blood and tissue TMB analysis from MYSTIC, a Phase III study of first-line durvalumab ± tremelimumab vs chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT074.

Introduction and objective. To evaluate and compare quantitatively aberrant methylation in the promoter area of EGFR gene in patients (p) with in non small cell lung cancer (NSCLC) lung tumor tissue (TT), lung normal tissue (NT), blood (B), bronchial aspirate (BA) and sputum (S). Correlate these methylation patterns with clinical variables. Methods. DNA was extracted from samples of B, S and BA from patients with NSCLC prior to surgery and resected TT and NT. Methylation patterns were analyzed by the bisulfite conversion and subsequent pyrosequencing (QIagen PyroMark System). We analyzed three CpG islands in the promoter region of EGFR gene. Results. 43p were included, median age 66 years (range 46-79), 35 men and 8 women. Histology: 26p adenocarcinoma, 14 squamous and 3 others. 2p had EGFR mutation. Smoking status: 4p never smokers, 21p former smokers, 18p smokers. Significant differences in methylation percentage (%Mth) were observed between TT and S in the following islands: CpG2 (11.8vs7.1, p = 0.008), CpG3 (10vs7.4, p = 0.037) and in overall promoter area (10.6vs7. 6, p = 0.034). The %Mth was higher in women vs men in TT CpG1 island (16.2vs24.9, p = 0.042) and TT CpG2 island (15.8vs26.5, p = 0.013). The %Mth was higher in squamous histology compared to adenocarcinoma in NT CpG2 island (22.6vs14.1, p = 0.017) and S CpG1 island (13.7vs8.2, p = 0.047). However, the %Mth was higher in the overall promoter area of adenocarcinoma respect to squamous histology (19.7vs12.8 TT, p = 0.042). The %Mth was higher in overall promoter area of NT respect Stage I vs II vs III (22.3 vs 13 vs 17.5, p = 0.03). The %Mth was higher in former smokers compared to current smokers and non smokers in NT CpG3 (22.8 vs 13 vs 14.1, p = 0.032). The %Mth in NT CpG3 was higher in never smokers and former smokers > 5 years, respect to current smokers and former smokers 5 years patients. * This study was funded by the Carlos III Health Institute (PS09/00308), Spain Note: This abstract was not presented at the conference. Citation Format: Rocio Lavado-Valenzuela, Enrique Bermejo, Roberto Mongil, Francisco Páez, Carlos Pagés, Miguel Angel Berciano, Ricardo Arrabal, Dolores Bautista, Jose Luis De la Cruz, Cynthia Robles, Manuel Benavides, Manuel Cobo. Quantitative determination of EGFR gene aberrant methylation in NSCLC: a comparative analysis in tumor tissue, normal lung tissue, peripheral blood, sputum and bronchial aspirate. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C20.

Introduction and objective. Quantifying and comparing the degree of methylation in the promoter region of APC gene and coding region of FHIT gene in patients (p) with non small cell lung cancer (NSCLC) in different samples. Correlate these methylation patterns with clinical variables. Methods. DNA was extracted from peripheral blood simples (B), sputum (S) and bronchial aspirate (BA), obtained from patients with NSCLC prior to the completion of surgery, as well as resected lung tumor tissue (TT) and normal lung tissue (TN). Methylation patterns were analyzed by bisulfito conversion and subsequent pyrosequencing (QIagen PyroMark System).We analyzed 5 CpG islands in the promoter region of the APC gene and 5 CpG islands in the coding region of the FHIT gene. Results: We analyzed 20p, with a median age of 64 years (range 48-70), 16 men and 4 women. Smoking status: 2p never smokers, 11p former smokers, 7p current smokers. Histology: 10p adenocarcinoma, 9p squamous, 1p large cell. Stage: I 8p, II 8p, III 4p. No statistically significant differences were observed between the samples studied to any of the islands analyzed. The degree of methylation in TT CpG1 was higher in smokers and former smokers 5 years, mean 4 (0-10) vs 0, p=0.022. There was no other difference when analyzing the degree of methylation as a function of the variables age, sex, smoking status, cumulative tobacco consumption, histological type and clinical stage. Respect FHIT gene, no statistically significant differences were found between the tissues studied respect to any of the CpG islands analyzed and likewise, no differences were observed when analyzed for degree of methylation depending on the clinical variables studied. Conclusions. The area of the APC gen promoter and FHIT coding region analyzed showed a low degree of methylation, with no significant difference observed between the samples studied. The degree of methylation in the TT CpG1 island of the APC gene was higher in current smokers and former smokers *This study was funded by the Carlos III Health Institute (PS09/00308), Spain. Note: This abstract was not presented at the conference. Citation Format: Rocio Lavado-Valenzuela, Sr., Carlos Pagés, Sr., Roberto Mongil, Sr., Francisco Páez, Sr., Enrique Bermejo, Sr., Miguel Angel Berciano, Sr., Dolores Bautista, Sr., Cynthia Robles, Jr., Ricardo Arrabal, Sr., Jose Luis De la Cruz, Sr., Manuel Benavides, Sr., Manuel Cobo, Sr.. Quantitative determination of methylation patterns in FHIT and APC genes in NSCLC: comparative analysis in tumor and normal lung tissue, peripheral blood, sputum and bronchial aspirate. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A36.

Background: The CpG island methylator phenotype is associated with distinct clinicophatological characteristics as Estrogen Receptor (ESR1) positive and amplification of HER2 in breast cancer.Objetive: To evaluate epigenetics differences in tumor-related genes to ERS1 and HER2/neu status in primary breast cancer.Material and Methods: We quantified methylation levels of promoter of 5 genes (ERS1, RAR- β, 14-3-3 sigma, APC, E-Cadherin) which are to confer growth adventage to cells, in 107 women with breast cancer and 108 control subjects. Real Time QMS-PCR SYBR green (methylation-specific PCR) was used to analyze the hypermethylation. Tumours were classified as phenotype basal, luminal A, Luminal B and phenotype HER2+.Results: Ours analyses revealed low or absent methylation ESR1and 14-3-3σ in healthy controls and significant differences between breast cancer patients (pts) and healthy controls in relative serum levels of methylated gene promoters ESR1 (p=0.0112) and 14-3-3s (p=0.0047). Presence of methylated ESR1 in serum of breast cancer patients was associated with ER-negative phenotype (p=0.0179). Of the available cases, 60 pts (56%) were Luminal A, 10 pts (9.3%) Luminal B, 13 pts (12%) Basal like and 9 pts (8.4%) HER2+. We observed that methylated ERS1 was preferably associated with phenotype Basal Like and worse interval progression free and survival global though p>0.05 and the amplification HER2+ was correlation with significant more frequent methylation gene (p Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1151.