PURPOSE: Neoadjuvant chemotherapy plus nivolumab has been shown to be effective in resectable non–small-cell lung cancer (NSCLC) in the NADIM trial (ClinicalTrials.gov identifier: NCT03081689 ). The 3-year overall survival (OS) and circulating tumor DNA (ctDNA) analysis have not been reported. METHODS: This was an open-label, multicenter, single-arm, phase II trial in which patients with stage IIIA NSCLC, who were deemed to be surgically resectable, were treated with neoadjuvant paclitaxel (200 mg/m² once a day) and carboplatin (area under curve 6) plus nivolumab (360 mg) once on day 1 of each 21-day cycle, for three cycles, followed by adjuvant nivolumab monotherapy for 1 year (240 mg once every 2 weeks for 4 months, followed by 480 mg once every 4 weeks for 8 months). The 3-year OS and ctDNA analysis were secondary objectives of the trial. RESULTS: OS at 36 months was 81.9% (95% CI, 66.8 to 90.6) in the intention-to-treat population, rising to 91.0% (95% CI, 74.2 to 97.0) in the per-protocol population. Neither tumor mutation burden nor programmed cell death ligand-1 staining was predictive of survival. Conversely, low pretreatment levels of ctDNA were significantly associated with improved progression-free survival and OS (hazard ratio [HR], 0.20; 95% CI, 0.06 to 0.63, and HR, 0.07; 95% CI, 0.01 to 0.39, respectively). Clinical responses according to RECIST v1.1 criteria did not predict survival outcomes. However, undetectable ctDNA levels after neoadjuvant treatment were significantly associated with progression-free survival and OS (HR, 0.26; 95% CI, 0.07 to 0.93, and HR, 0.04; 95% CI, 0.00 to 0.55, respectively). The C-index to predict OS for ctDNA levels after neoadjuvant treatment (0.82) was superior to that of RECIST criteria (0.72). CONCLUSION: The efficacy of neoadjuvant chemotherapy plus nivolumab in resectable NSCLC is supported by 3-year OS. ctDNA levels were significantly associated with OS and outperformed radiologic assessments in the prediction of survival.

Platinum-based chemotherapy remains the standard of care for most lung cancer cases. However chemoresistance is often developed during the treatment, limiting clinical utility of this drug. Recently, the ability of tumor cells to adapt their metabolism has been associated to resistance to therapies. In this study, we first described the metabolic reprogramming of Non-Small Cell Lung Cancer (NSCLC) in response to cisplatin treatment. Cisplatin-resistant versions of the A549, H1299, and H460 cell lines were generated by continuous drug exposure. The long-term metabolic changes, as well as, the early response to cisplatin treatment were analyzed in both, parental and cisplatin-resistant cell lines. In addition, four Patient-derived xenograft models treated with cisplatin along with paired pre- and post-treatment biopsies from patients were studied. Furthermore, metabolic targeting of these changes in cell lines was performed downregulating PGC-1α expression through siRNA or using OXPHOS inhibitors (metformin and rotenone). Two out of three cisplatin-resistant cell lines showed a stable increase in mitochondrial function, PGC1-α and mitochondrial mass with reduced glycolisis, that did not affect the cell cycle. This phenomenon was confirmed in vivo. Post-treatment NSCLC tumors showed an increase in mitochondrial mass, PGC-1α, and a decrease in the GAPDH/MT-CO1 ratio. In addition, we demonstrated how a ROS-mediated metabolism reprogramming, involving PGC-1α and increased mitochondrial mass, is induced during short-time cisplatin exposure. Moreover, we tested how cells with increased PGC-1a induced by ZLN005 treatment, showed reduced cisplatin-driven apoptosis. Remarkably, the long-term metabolic changes, as well as the metabolic reprogramming during short-time cisplatin exposure can be exploited as an Achilles' heel of NSCLC cells, as demonstrated by the increased sensitivity to PGC-1α interference or OXPHOS inhibition using metformin or rotenone. These results describe a new cisplatin resistance mechanism in NSCLC based on a metabolic reprogramming that is therapeutically exploitable through PGC-1α downregulation or OXPHOS inhibitors.

Lung cancer is a major public health problem due to its high incidence and mortality rate. The altered metabolism in lung cancer is key for the diagnosis and has implications on both, the prognosis and the response to treatments. Although Cancer-associated fibroblasts (CAFs) are one of the major components of the tumor microenvironment, little is known about their role in lung cancer metabolism. We studied tumor biopsies from a cohort of 12 stage IIIA lung adenocarcinoma patients and saw a positive correlation between the grade of fibrosis and the glycolysis phenotype (Low PGC-1α and High GAPDH/MT-CO1 ratio mRNA levels). These results were confirmed and extended to other metabolism-related genes through the in silico data analysis from 73 stage IIIA lung adenocarcinoma patients available in TCGA. Interestingly, these relationships are not observed with the CAFs marker α-SMA in both cohorts. To characterize the mechanism, in vitro co-culture studies were carried out using two NSCLC cell lines (A549 and H1299 cells) and two different fibroblast cell lines. Our results confirm that a metabolic reprogramming involving ROS and TGF-β signaling occurs in lung cancer cells and fibroblasts independently of α-SMA induction. Under co-culture conditions, Cancer-Associated fibroblasts increase their glycolytic ability. On the other hand, tumor cells increase their mitochondrial function. Moreover, the differential capability among tumor cells to induce this metabolic shift and also the role of the basal fibroblasts Oxphos Phosphorylation (OXPHOS) function modifying this phenomenon could have implications on both, the diagnosis and prognosis of patients. Further knowledge in the mechanism involved may allow the development of new therapies.

Approximately 30% of tumor biopsies from patients with advanced-stage lung adenocarcinomas yield insufficient tissue for successful molecular subtyping. We have analyzed the clinical utility of next-generation sequencing (NGS) of cell-free circulating tumor DNA (ctDNA) in patients with inadequate tumor samples for tissue genotyping. We conducted the study in a multi-institutional prospective cohort of clinically unselected patients with advanced-stage lung adenocarcinomas with insufficient tissue for EGFR, ALK or ROS1 genotyping across 12 Spanish institutions (n = 93). ctDNA NGS was carried out by Guardant Health (Guardant360, Redwood City, CA), using a hybrid-capture-based 73-gene panel. Variants were deemed actionable if they were part of the OncoKB precision oncology knowledge database and classified in four levels of actionability based on their clinical or preclinical evidence for drug response. Eighty-three out of 93 patients (89%) had detectable levels of ctDNA. Potentially actionable level 1-4 genomic alterations were detected in 53 cases (57%), of which 13 (14%) had level 1-2A alterations (Food and Drug Administration-approved and standard-care biomarkers according to lung cancer guidelines). Frequencies of each genomic alteration in ctDNA were consistent with those observed in unselected pulmonary adenocarcinomas. The majority of the patients (62%), particularly those with actionable alterations (87%), had more than one pathogenic variant in ctDNA. The median turnaround time to genomic results was 13 days. Twelve patients (13%) received genotype-matched therapies based on ctDNA results, deriving the expected clinical benefit. Patients with co-occurring pathogenic alterations had a significantly shorter median overall survival as compared with patients without co-occurring pathogenic alteration (multivariate hazard ratio = 5.35, P = 0.01). Digital NGS of ctDNA in lung cancers with insufficient tumor samples for tissue sequencing detects actionable variants that frequently co-occur with other potentially clinically relevant genomic alterations, allowing timely initiation of genotype-matched therapies.

Background: Immunotherapy is being tested in early‐stage non‐small cell lung cancer (NSCLC), and achieving higher rates of complete pathological responses (CPR) as compared to standard of care. Early identification of CPR patients has vital clinical implications. In this study, we focused on basal peripheral immune cells and their treatment‐related changes to find biomarkers associated to CPR. Methods: Blood from 29 stage IIIA NSCLC patients participating in the NADIM trial (NCT03081689) was collected at diagnosis and post neoadjuvant treatment. More than 400 parameters of peripheral blood mononuclear cells (PBMCs) phenotype and plasma soluble factors were analyzed. Results: Neoadjuvant chemoimmunotherapy altered more than 150 immune parameters. At diagnosis, 11 biomarkers associated to CPR were described, with an area under the ROC curve >0.70 and p ‐value <.05. CPR patients had significantly higher levels of CD4 ⁺ PD‐1 ⁺ cells, NKG2D, and CD56 expression on T CD56 cells, intensity of CD25 expression on CD4 ⁺ CD25hi ⁺ cells and CD69 expression on intermediate monocytes; but lower levels of CD3 ⁺ CD56 ^– CTLA‐4 ⁺ cells, CD14 ⁺⁺ CD16 ⁺ CTLA‐4 ⁺ cells, CTLA‐4 expression on T CD56 cells and lower levels of b‐NGF, NT‐3, and VEGF‐D in plasma compared to non‐CPR. Post treatment, CPR patients had significantly higher levels of CD19 expression on B cells, BCMA, 4‐1BB, MCSF, and PARC and lower levels of MPIF‐1 and Flt‐3L in plasma compared to non‐CPR. Conclusions: Patients achieving CPR seem to have a distinctive peripheral blood immune status at diagnosis, even showing different immune response to treatment. These results reinforce the different biology behind CPR and non‐CPR responses.

Purpose: Follicular lymphoma (FL) is the most frequent indolent non-Hodgkin lymphoma. Around 20% of patients suffer early disease progression within 24 months (POD24) of diagnosis. This study examined the significance of circulating tumor DNA (ctDNA) in predicting response to therapy and POD24 in patients with FL. Experimental Design: We collected 100 plasma samples, before and during the treatment, from 36 patients with FL prospectively enrolled in 8 Spanish hospitals. They were treated with a chemotherapy-rituximab regimen and followed up for a median of 3.43 years. We performed targeted deep sequencing in cell-free DNA (cfDNA) and tumor genomic DNA from 31 diagnostic biopsy samples. Results: Of the alterations detected in the diagnostic tissue samples, 73% (300/411) were also identified in basal cfDNA. The mean numbers of alterations per basal cfDNA sample in patients who suffered progression of disease within 24 months (POD24-pos) or did not achieve complete response (non-CR) were significantly higher than in POD24-neg or CR patients (unpaired samples t test, P = 0.0001 and 0.001, respectively). Pretreatment ctDNA levels, as haploid genome equivalents per milliliter of plasma, were higher in patients without CR (P = 0.02) and in POD24-pos patients compared with POD24-neg patients (P < 0.001). Dynamic analysis showed that ctDNA levels decreased dramatically after treatment, although the reduction was more significant in patients with CR and POD24-neg patients. Conclusions: Basal ctDNA levels are associated with the risk of early progression and response to treatment in FL. cfDNA monitoring and genotyping during treatment and follow-up predict response to treatment and early progression.