The role of sex and gender in the diagnosis and treatment of lung cancer: the 6th ICAPEM Annual Symposium

The incidence and mortality of lung cancer in women are rising, with both increasing by 124% between 2003 and 2019. The main risk factor for lung cancer is tobacco use, but indoor radon gas exposure is one of the leading causes in nonsmokers. The most recent evidence demonstrates that multiple factors can make women more susceptible to harm from these risk factors or carcinogens. For this consensus statement, the Association for Lung Cancer Research in Women (ICAPEM) invited a group of lung cancer experts to perform a detailed gender-based analysis of lung cancer. Clinically, female patients have different lung cancer profiles, and most actionable driver alterations are more prevalent in women, particularly in never-smokers. Additionally, the impact of certain therapies seems to be different. In the future, it will be necessary to carry out specific studies to improve the understanding of the role of certain biomarkers and gender in the prognosis and evolution of lung cancer (AU)

The role of sex and gender in the diagnosis and treatment of lung cancer: the 6th ICAPEM Annual Symposium.

The incidence and mortality of lung cancer in women are rising, with both increasing by 124% between 2003 and 2019. The main risk factor for lung cancer is tobacco use, but indoor radon gas exposure is one of the leading causes in nonsmokers. The most recent evidence demonstrates that multiple factors can make women more susceptible to harm from these risk factors or carcinogens. For this consensus statement, the Association for Lung Cancer Research in Women (ICAPEM) invited a group of lung cancer experts to perform a detailed gender-based analysis of lung cancer. Clinically, female patients have different lung cancer profiles, and most actionable driver alterations are more prevalent in women, particularly in never-smokers. Additionally, the impact of certain therapies seems to be different. In the future, it will be necessary to carry out specific studies to improve the understanding of the role of certain biomarkers and gender in the prognosis and evolution of lung cancer.

Determinants of activity and efficacy of anti-PD1/PD-L1 therapy in patients with advanced solid tumors recruited in a clinical trials unit: a longitudinal prospective biomarker-based study.

Immune-checkpoint inhibitors (ICI) have revolutionized the therapeutic landscape of cancer. However, optimal patient selection is still an unmet need. One-hundred-forty-six patients with metastatic cancer candidates to ICI at the Hospital Clinic of Barcelona Clinical Trials Unit were prospectively recruited in this observational study. Blood samples were collected at different timepoints, baseline LIPI score calculated and pre-ICI archived tissues retrieved to evaluate PD-L1, tumor-infiltrating lymphocytes (TILs) and PD1 mRNA levels. Tumor assessments were centrally reviewed by RECIST 1.1 criteria. Associations with overall response rates (ORR), durable clinical benefit (DCB), progression-free survival (PFS) and overall survival (OS) were performed with univariable/multivariable logistic and Cox regressions, where appropriate. At a median follow-up of 26.9 months, median PFS and OS were 2.7 and 12.9 months. Response rates were 17.8% with duration of response (DOR) of 4.4 months. LIPI score was independently associated with PFS (p = 0.025) and OS (p < 0.001). Immunotherapy-naïve status was independently associated with better PFS (p = 0.005). Time-to-best response (TTBR) and ORR (p < 0.001 both) were associated with better OS at univariate analysis. PFS and DOR were moderately correlated with OS (p < 0.001 both). A PD-L1 10% cut-off detected worse/best responders in terms of ORR (univariate p = 0.011, multivariate p = 0.028) and DCB (univariate p = 0.043). PD1 mRNA levels were strikingly associated to complete responses (p = 0.021). To resume, in our prospective observational pan-cancer study, baseline LIPI score, immunotherapy-naïve status, cancer type and RT before starting ICI were the most relevant clinical factors independently correlated with immunotherapy outcomes. Longer TTBR seemed to associate with better survival, while PD1 mRNA and PD-L1 protein levels might be tumor-agnostic predictive factors of response to ICI and should be furtherly explored.

Tumor microenvironment gene expression profiles associated to complete pathological response and disease progression in resectable NSCLC patients treated with neoadjuvant chemoimmunotherapy.

BACKGROUND: Neoadjuvant chemoimmunotherapy for non-small cell lung cancer (NSCLC) has improved pathological responses and survival rates compared with chemotherapy alone, leading to Food and Drug Administration (FDA) approval of nivolumab plus chemotherapy for resectable stage IB-IIIA NSCLC (AJCC 7th edition) without ALK or EGFR alterations. Unfortunately, a considerable percentage of tumors do not completely respond to therapy, which has been associated with early disease progression. So far, it is impossible to predict these events due to lack of knowledge. In this study, we characterized the gene expression profile of tumor samples to identify new biomarkers and mechanisms behind tumor responses to neoadjuvant chemoimmunotherapy and disease recurrence after surgery. METHODS: Tumor bulk RNA sequencing was performed in 16 pretreatment and 36 post-treatment tissue samples from 41 patients with resectable stage IIIA NSCLC treated with neoadjuvant chemoimmunotherapy from NADIM trial. A panel targeting 395 genes related to immunological processes was used. Tumors were classified as complete pathological response (CPR) and non-CPR, based on the total absence of viable tumor cells in tumor bed and lymph nodes tested at surgery. Differential-expressed genes between groups and pathway enrichment analysis were assessed using DESeq2 and gene set enrichment analysis. CIBERSORTx was used to estimate the proportions of immune cell subtypes. RESULTS: CPR tumors had a stronger pre-established immune infiltrate at baseline than non-CPR, characterized by higher levels of IFNG, GZMB, NKG7, and M1 macrophages, all with a significant area under the receiver operating characteristic curve (ROC) >0.9 for CPR prediction. A greater effect of neoadjuvant therapy was also seen in CPR tumors with a reduction of tumor markers and IFNγ signaling after treatment. Additionally, the higher expression of several genes, including AKT1, BST2, OAS3, or CD8B; or higher dendritic cells and neutrophils proportions in post-treatment non-CPR samples, were associated with relapse after surgery. Also, high pretreatment PD-L1 and tumor mutational burden levels influenced the post-treatment immune landscape with the downregulation of proliferation markers and type I interferon signaling molecules in surgery samples. CONCLUSIONS: Our results reinforce the differences between CPR and non-CPR responses, describing possible response and relapse immune mechanisms, opening the possibility of therapy personalization of immunotherapy-based regimens in the neoadjuvant setting of NSCLC.

Characterization of the MicroRNA Cargo of Extracellular Vesicles Isolated from a Pulmonary Tumor-Draining Vein Identifies miR-203a-3p as a Relapse Biomarker for Resected Non-Small Cell Lung Cancer.

In resected non-small cell lung cancer (NSCLC), post-surgical recurrence occurs in around 40% of patients, highlighting the necessity to identify relapse biomarkers. An analysis of the extracellular vesicle (EV) cargo from a pulmonary tumor-draining vein (TDV) can grant biomarker identification. We studied the pulmonary TDV EV-miRNAome to identify relapse biomarkers in a two-phase study (screening and validation). In the screening phase, a 17-miRNA relapse signature was identified in 18 selected patients by small RNAseq. The most expressed miRNA from the signature (EV-miR-203a-3p) was chosen for further validation. Pulmonary TDV EV-miR-203a-3p was studied by qRT-PCR in a validation cohort of 70 patients, where it was found to be upregulated in relapsed patients (p = 0.0194) and in patients with cancer spread to nearby lymph nodes (N+ patients) (p = 0.0396). The ROC curve analysis showed that TDV EV-miR-203a-3p was able to predict relapses with a sensitivity of 88% (AUC: 0.67; p = 0.022). Moreover, patients with high TDV EV-miR-203a-3p had a shorter time to relapse than patients with low levels (43.6 vs. 97.6 months; p = 0.00703). The multivariate analysis showed that EV-miR-203a-3p was an independent, predictive and prognostic post-surgical relapse biomarker. In conclusion, pulmonary TDV EV-miR-203a-3p is a promising new relapse biomarker for resected NSCLC patients.

Overall Survival and Biomarker Analysis of Neoadjuvant Nivolumab Plus Chemotherapy in Operable Stage IIIA Non-Small-Cell Lung Cancer (NADIM phase II trial).

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.

A Pilot Study to Evaluate Early Predictive Value of Thorax Perfusion-CT in Advanced NSCLC.

BACKGROUND: The role of perfusion computed tomography (pCT) in detecting changes in tumor vascularization as part of a response to antiangiogenic therapy in non-small cell lung cancer (NSCLC) remains unclear. METHODS: In this prospective pilot study (IMPACT trial, NCT02316327), we aimed to determine the ability of pCT to detect early changes in blood flow (BF), blood volume (BV), and permeability (PMB), and to explore whether these changes could predict the response at day +42 in patients with advanced, treatment-naive, non-squamous NSCLC treated with cisplatin and gemcitabine plus bevacizumab. RESULTS: All of the perfusion parameters showed a consistent decrease during the course of treatment. The BV difference between baseline and early assessment was significant (p = 0.013), whereas all perfusion parameters showed significant differences between baseline and day +42 (p = 0.003, p = 0.049, and p = 0.002, respectively). Among the 16 patients evaluable for efficacy, a significant decline in BV at day +7 from baseline was observed in tumors with no response (p = 0.0418). CONCLUSIONS: Our results confirm that pCT can capture early changes in tumor vasculature. A substantial early decline of BV from baseline might identify tumors less likely responsive to antiangiogenic-drugs.

Pretreatment Tissue TCR Repertoire Evenness Is Associated with Complete Pathologic Response in Patients with NSCLC Receiving Neoadjuvant Chemoimmunotherapy.

PURPOSE: Characterization of the T-cell receptor (TCR) repertoire may be a promising source for predictive biomarkers of pathologic response to immunotherapy in locally advanced non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: In this study, next-generation TCR sequencing was performed in peripheral blood and tissue samples of 40 patients with NSCLC, before and after neoadjuvant chemoimmunotherapy (NADIM clinical trial, NCT03081689), considering their complete pathologic response (CPR) or non-CPR. Beyond TCR metrics, tissue clones were ranked by their frequency and spatiotemporal evolution of top 1% clones was determined. RESULTS: We have found a positive association between an uneven TCR repertoire in tissue samples at diagnosis and CPR at surgery. Moreover, TCR most frequently ranked clones (top 1%) present in diagnostic biopsies occupied greater frequency in the total clonal space of CPR patients, achieving an AUC ROC to identify CPR patients of 0.967 (95% confidence interval, 0.897-1.000; P = 0.001), and improving the results of PD-L1 tumor proportion score (TPS; AUC = 0.767; P = 0.026) or tumor mutational burden (TMB; AUC = 0.550; P = 0.687). Furthermore, tumors with high pretreatment top 1% clonal space showed similar immune cell populations but a higher immune reactive gene expression profile. Finally, the selective expansion of pretreatment tissue top 1% clones in peripheral blood of CPR patients suggests also a peripheral immunosurveillance, which could explain the high survival rate of these patients. CONCLUSIONS: We have identified two parameters derived from TCR repertoire analysis that could outperform PD-L1 TPS and TMB as predictive biomarkers of CPR after neoadjuvant chemoimmunotherapy, and unraveled possible mechanisms of CPR involving enhanced tumor immunogenicity and peripheral immunosurveillance.

Clinical and molecular parameters associated to pneumonitis development in non-small-cell lung cancer patients receiving chemoimmunotherapy from NADIM trial.

BACKGROUND: Pneumonitis (Pn) is one of the main immune-related adverse effects, having a special importance in lung cancer, since they share affected tissue. Despite its clinical relevance, Pn development remains an unpredictable treatment adverse effect, whose mechanisms are mainly unknown, being even more obscure when it is associated to chemoimmunotherapy. METHODS: In order to identify parameters associated to treatment related Pn, we analyzed clinical variables and molecular parameters from 46 patients with potentially resectable stage IIIA non-small-cell lung cancer treated with neoadjuvant chemoimmunotherapy included in the NADIM clinical trial (NCT03081689). Pn was defined as clinical or radiographic evidence of lung inflammation without alternative diagnoses, from treatment initiation to 180 days. RESULTS: Among 46 patients, 12 developed Pn (26.1%). Sex, age, smoking status, packs-year, histological subtype, clinical or pathological response, progression-free survival, overall survival and number of nivolumab cycles, were not associated to Pn development. Regarding molecular parameters at diagnosis, Pn development was not associated to programmed death ligand 1, TPS, T cell receptor repertoire parameters, or tumor mutational burden. However, patients who developed Pn had statistically significant lower blood median levels of platelet to monocyte ratio (p=0.012) and teratocarcinoma-derived growth factor 1 (p=0.013; area under the curve (AUC) 0.801), but higher median percentages of natural killers (NKs) (p=0.019; AUC 0.786), monocytes (p=0.017; AUC 0.791), MSP (p=0.006; AUC 0.838), PARN (p=0.017; AUC 0.790), and E-Cadherin (p=0.022; AUC 0.788). In addition, the immune scenario of Pn after neoadjuvant treatment involves: high levels of neutrophils and NK cells, but low levels of B and T cells in peripheral blood; increased clonality of intratumoral T cells; and elevated plasma levels of several growth factors (EGF, HGF, VEGF, ANG-1, PDGF, NGF, and NT4) and inflammatory cytokines (MIF, CCL16, neutrophil gelatinase-associated lipocalin, BMP-4, and u-PAR). CONCLUSIONS: Although statistically underpowered, our results shed light on the possible mechanisms behind Pn development, involving innate and adaptative immunity, and open the possibility to predict patients at high risk. If confirmed, this may allow the personalization of both, the surveillance strategy and the therapeutic approaches to manage Pn in patients receiving chemoimmunotherapy.

LungBEAM: A prospective multicenter study to monitor stage IV NSCLC patients with EGFR mutations using BEAMing technology.

OBJECTIVES: The aim of LungBEAM was to determine the value of a novel epidermal growth factor receptor (EGFR) mutation test in blood based on BEAMing technology to predict disease progression in advanced non-small cell lung cancer (NSCLC) patients treated with first- or second-generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs). Another goal was to monitor the dynamics of EGFR mutations, as well as to track EGFR exon 20 p.T790M (p.T790M) resistance during treatment, as critical indicators of therapeutic efficacy and patient survival. METHODS: Stage IV NSCLC patients with locally confirmed EGFR-TKI sensitizing mutations (ex19del and/or L858R) in biopsy tissue who were candidates to receive first- or second-generation EGFR-TKI as first-line therapy were included. Plasma samples were obtained at baseline and every 4 weeks during treatment until a progression-free survival (PFS) event or until study completion (72-week follow-up). The mutant allele fraction (MAF) was determined for each identified mutation using BEAMing. RESULTS: A total of 68 of the 110 (61.8%) patients experienced a PFS event. Twenty-six patients (23.6%) presented with an emergent p.T790M mutation in plasma at some point during follow-up, preceding radiologic progression with a median of 76 (interquartile ratio: 54-111) days. Disease progression correlated with the appearance of p.T790M in plasma with a hazard ratio (HR) of 1.94 (95% confidence interval [CI], 1.48-2.54; p < 0.001). The HR for progression in patients showing increasing plasma sensitizing mutation levels (positive MAF slope) versus patients showing either decreasing or unchanged plasma mutation levels (negative or null MAF slopes) was 3.85 (95% CI, 2.01-7.36; p < 0.001). CONCLUSION: Detection and quantification of EGFR mutations in circulating tumor DNA using the highly sensitive BEAMing method should greatly assist in optimizing treatment decisions for advanced NSCLC patients.

Blood biomarkers associated to complete pathological response on NSCLC patients treated with neoadjuvant chemoimmunotherapy included in NADIM clinical trial.

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.

Monitoring HOTTIP levels on extracellular vesicles for predicting recurrence in surgical non-small cell lung cancer patients.

In resected non-small cell lung cancer (NSCLC), postsurgical recurrence is the major factor affecting long-term survival. The identification of biomarkers in extracellular vesicles (EV) obtained from serial blood samples after surgery could enhance early detection of relapse and improve NSCLC outcome. Since EV cargo contains long non-coding RNAs (lncRNAs), we aimed to analyze whether the oncogenic lncRNA HOTTIP, which higher expression in tumor tissue was related to worse outcome in NSCLC, could be detected in EV from NSCLC patients and serve as recurrence biomarker. After purification of EVs by ultracentrifugation in 52 serial samples from 18 NSCLC patients, RNA was isolated and HOTTIP was quantified by Real time PCR. We observed that patients that relapsed after surgery displayed increased postsurgical EV HOTTIP levels in comparison with presurgical levels. In the relapsed patients with several samples available between surgery and relapse, we observed an increment in the EV HOTTIP levels when approaching to relapse, which indicated its potential utility for monitoring disease recurrence. When we focused in EV HOTTIP levels in the first post-surgical sample, we observed that the detection of an increment of the expression levels in comparison to presurgical sample, predicted recurrence with high sensitivity (85.7%) and specificity (90.9%) and that patients had shorter time to relapse and shorter overall survival. In conclusion, our pilot study showed that EV HOTTIP is a potential biomarker for monitoring disease recurrence after surgery in NSCLC.

Role of the epithelial-mesenchymal transition-related circular RNA, circ-10720, in non-small-cell lung cancer.

BACKGROUND: Circular RNAs (circRNAs) are non-coding RNAs with a circular structure that have recently emerged as important regulators of tumorogenesis. Recently, several circRNAS, including circ-10720 have been related to epithelial-mesenchymal transition (EMT) process. In the present study, we have analyzed the role of circ-10720 in non-small-cell lung cancer (NSCLC) and studied its prognostic relevance in resected stage I-IIIa NSCLC patients. METHODS: Circ-10720 expression was analyzed using a custom TaqMan assay in four NSCLC cell lines (HCC44, A549, H23 and H1299) and in the normal immortalized lung cell line BEAS2B. Silencing of circ-10720 was performed using two custom siRNAs which were transfected using lipofectamine 2000. Protein levels were evaluated by Western blot and immunofluorescence. Wound healing and invasion assays were performed to evaluate the impact the circRNA on cell motility. Apoptosis was analyzed by evaluation of Caspase 3-7 activity and proliferation by MTS assay. Moreover, the expression levels of the circRNA were studied in 119 resected NSCLC patients. The expression in tumor tissue was correlated with the main clinicopathological characteristics and with time to relapse (TTR). RESULTS: Circ-10720 was overexpressed in HCC44 and A549 and underexpressed in H23 and H1299 NSCLC cell lines in comparison to BEAS2B normal immortalized lung cell line. CircRNA knockdown in the two circ-10720 overexpressing cell lines was associated with a decrease of Vimentin (VIM) and an increase of E-cadherin (CDH1) protein levels, loss of mesenchymal phenotype, and a significant reduction of migration and invasion capacity. After silencing circ-10720, the apoptosis rate increased and the proliferation was significantly reduced. Furthermore, circ-10720 was upregulated in tumor vs. normal tissue from 119 resected NSCLC patients. In the group of patients not receiving adjuvant treatment, those with high levels of circ-10720 had a shorter TTR than those with low levels and emerged as an independent prognostic value in the multivariate analysis. In tumor tissue, circ-10720 levels positively correlated with the EMT gene Twist1 levels. CONCLUSIONS: Circ-10720 regulates EMT, apoptosis and proliferation and acts as a biomarker of relapse in NSCLC.

Pleural effusion is a negative prognostic factor for immunotherapy in patients with non-small cell lung cancer (NSCLC): The pluie study.

BACKGROUND: Pleural effusion (PE) is a common metastatic site of NSCLC, associated with poor outcomes. As very few data are available about immune checkpoint inhibitors (ICI) and PE, we aimed to assess the clinical outcome of PE in NSCLC treated with ICI. METHOD: Multicenter international retrospective study of patients with metastatic NSCLC treated with ICI, between 2012 and 2019. Stratification according to the presence of PE at ICI baseline or appearance under ICI treatment (PE group) versus no history of PE (non-PE group). Primary endpoints were overall survival (OS) and early death rate (EDR, OS ≤ 3 months). RESULTS: A total of 538 patients were included: 196 in the PE group and 342 in the non-PE group. In the PE group, median age was 64, 31.6 % were female, 77.6 % had non-squamous histology, PD-L1 was ≥50 % in 38.6 % of cases (95 missing). PE was more likely associated with >2 metastatic sites (70.4 % vs. 50 %) and worse performance status (PS ≥ 2, 30.8 % vs 23.1 %). Globally, the overall median OS was 9.7 months [95 %CI: 8.1-11.8]; 6.3 [95 % CI: 4.0-8.6] in PE vs. 11.4 [95 %CI: 9.7-13.8] in the non-PE respectively, P = 0.002. Overall the EDR was 31.4 %; higher in the PE group (38.3 % vs. 27.5 %; OR 1.63, 95 %CI: 1.13-2.37, P = 0.01). In the PE PD-L1≥50 % group, EDR was 33.3 %. In multivariate analysis, after adjustment on PS, liver/intracranial/bone metastasis, ICI line and dNLR, PE remained an independent prognostic factor for OS [HR: 1.38, 95 %CI: 1.09-1.74, P = 0.007]. In the PE group, PE appeared under ICI for 31 patients (16.4 %). We observed lower EDR in this group compared to patients whom PE was already present (29.0 % vs 40.5 %, P = 0.2). CONCLUSION: PE is associated with worse immunotherapy outcomes in NSCLC treated with ICI, including in patients with ≥50 % PD-L1 tumors. Thus, in these patients, combination strategies should be explored.

Molecular characterization of advanced non-small cell lung cancer patients by cfDNA analysis: experience from routine laboratory practice.

BACKGROUND: Analysis of circulating free DNA (cfDNA) by the real-time PCR cobas® EGFR Mutation Test v2 (cobas® EGFR Test) is a diagnostic approach used in clinical practice for the characterization of advanced non-small cell lung cancer (NSCLC) patients. The test additionally outputs a semiquantitative index (SQI) which reflects the proportion of mutated versus wild-type copies of the EGFR gene in cfDNA with potential use as a biomarker. CfDNA concentration and cfDNA fragmentation pattern have also shown potential utility as biomarkers for cancer patients. We evaluated the implementation of EGFR testing and cfDNA related parameters in NSCLC patients in routine clinical setting as biomarkers for disease stage and diagnosis. METHODS: A prospective cohort of 173 locally advanced or metastatic NSCLC TKI-naïve patients analyzed by the cobas® EGFR Test were included in the study. Reproducibility of the test was assessed in 56 patients. The concentration of cfDNA and fragment size pattern was measured using fluorometry and microchip electrophoresis respectively. RESULTS: The test showed high diagnostic accuracy when compared to the gold standard of biopsy tumor tissue testing. The SQI value showed a moderate reproducibility (r2=0.70) and did not correlate with cfDNA concentration (r2=0.17, P=0.28) or disease stage (stage III patients SQI =9.1±3.1 and stage IV patients SQI =11.5±4.8, P=0.41). We found differences in SQI values according to the type of EGFR mutation (Ex19Del mutations, SQI =13.6; p.L858R, SQI =8.88; P=0.001). Stage IV patients had higher concentrations of cfDNA (P<0.0001) and higher fractions of cfDNA 100-250 base pairs (bp) fragments (P=0.01) compared to stage III patients. From the ROC curve analysis, cfDNA concentration showed higher AUC compared to cfDNA 100-250 bp fragments (0.86 vs. 0.71). We obtained a cut-off value for cfDNA concentration of 20.3 ng/mL with 72.3% sensitivity and 95% specificity for predicting disease stage in TKI-naïve advanced NSCLC patients. CONCLUSIONS: The study indicates that cfDNA analysis in plasma for EGFR testing by RT-PCR is an accurate and fast method to initially stratify NSCLC patients in a real-world clinical setting. However, the SQI has limited clinical value. The cfDNA concentration and fragmentation pattern have clear potential clinical utility for tumor staging in NSCLC patients.

EBUS-TBNA Cytological Samples for Comprehensive Molecular Testing in Non-Small Cell Lung Cancer.

Clinical guidelines promote the identification of several targetable biomarkers to drive treatment decisions in advanced non-small cell lung cancer (NSCLC), but half of all patients do not have a viable biopsy. Specimens from endobronchial-ultrasound transbronchial needle aspiration (EBUS-TBNA) are an alternative source of material for the initial diagnosis of NSCLC, however their usefulness for a complete molecular characterization remains controversial. EBUS-TBNA samples were prospectively tested for several biomarkers by next-generation sequencing (NGS), nCounter, and immunohistochemistry (PD-L1). The primary objectives were to assess the sensitivity of EBUS-TBNA samples for a comprehensive molecular characterization and to compare its performance to the reference standard of biopsy samples. Seventy-two EBUS-TBNA procedures were performed, and 42 NSCLC patients were diagnosed. Among all cytological samples, 92.9% were successfully genotyped by NGS, 95.2% by nCounter, and 100% by immunohistochemistry. There were 29 paired biopsy samples; 79.3% samples had enough tumor material for genomic genotyping, and 96.6% for PD-L1 immunohistochemistry. A good concordance was found between both sources of material: 88.9% for PD-L1, 100% for NGS and nCounter. EBUS-TBNA is a feasible alternative source of material for NSCLC genotyping and allows the identification of patient candidates for personalized therapies with high concordance when compared with biopsy.

Multiplex RNA-based detection of clinically relevant MET alterations in advanced non-small cell lung cancer.

MET inhibitors have shown activity in non-small-cell lung cancer patients (NSCLC) with MET amplification and exon 14 skipping (METΔex14). However, patient stratification is imperfect, and thus, response rates have varied widely. Here, we studied MET alterations in 474 advanced NSCLC patients by nCounter, an RNA-based technique, together with next-generation sequencing (NGS), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT-PCR), exploring correlation with clinical benefit. Of the 474 samples analyzed, 422 (89%) yielded valid results by nCounter, which identified 13 patients (3%) with METΔex14 and 15 patients (3.5%) with very-high MET mRNA expression. These two subgroups were mutually exclusive, displayed distinct phenotypes and did not generally coexist with other drivers. For METΔex14, 3/8 (37.5%) samples positive by nCounter tested negative by NGS. Regarding patients with very-high MET mRNA, 92% had MET amplification by FISH and/or NGS. However, FISH failed to identify three patients (30%) with very-high MET RNA expression, among which one received MET tyrosine kinase inhibitor treatment deriving clinical benefit. Our results indicate that quantitative mRNA-based techniques can improve the selection of patients for MET-targeted therapies.

Prospective Evaluation of Single Nucleotide Variants by Two Different Technologies in Paraffin Samples of Advanced Non-Small Cell Lung Cancer Patients.

Targeted therapies are a new paradigm in lung cancer management. Next-generation sequencing (NGS) techniques have allowed for simultaneous testing of several genes in a rapid and efficient manner; however, there are other molecular diagnostic tools such as the nCounter® Vantage 3D single nucleotide variants (SNVs) solid tumour panel which also offer important benefits regarding sample input and time-to-response, making them very attractive for daily clinical use. This study aimed to test the performance of the Vantage panel in the routine workup of advanced non-squamous non-small cell lung cancer (NSCLC) patients and to validate and compare its outputs with the Oncomine Solid Tumor (OST) panel DNA kit, the standard technique in our institution. Two parallel multiplexed approaches were performed based on DNA NGS and direct digital detection of DNA with nCounter® technology to evaluate SNVs. A total of 42 advanced non-squamous NSCLC patients were prospectively included in the study. Overall, 95% of samples were successfully characterized by both technologies. The Vantage panel accounted for a sensitivity of 95% and a specificity of 82%. In terms of predictive values, the probability of truly presenting the SNV variant when it is detected by the nCounter panel was 82%, whereas the probability of not presenting the SNV variant when it is not detected by the platform was 95%. Finally, Cohen's Kappa coefficient was 0.76, indicating a substantial correlation grade between OST and Vantage panels. Our results make nCounter an analytically sensitive, practical and cost-effective tool.

Long non-coding RNA NANCI/NKX2-1 duplex impacts prognosis in stage I non-small-cell lung cancer / La combinación de NANCI/NKX2-1 de ARN no codificante de cadena larga afecta al pronóstico del cáncer de pulmón de células no pequeñas en estadio I

BACKGROUND: NANCI, an intergenic long non-coding RNA (lncRNA) is essential for buffering NKX2-1 expression during embryonic development and in adult tissue. We analyzed NANCI and NKX2-1 in human lung embryonic samples and adult lung tissues and evaluated their potential as prognostic markers in stage I non-small cell lung cancer (NSCLC). METHODS AND RESULTS: NANCI and NKX2-1 expression was assessed by TaqMan assays in 18 human embryonic samples from 8 to 13 weeks, 59 non-tumoral (NT) lung tissue samples, and 98 stage I NSCLC tumor samples. NANCI and NKX2-1 expression in embryonic and NSCLC samples were downregulated in comparison to adult NT tissue. Patients with low expression of NANCI had shorter disease-free survival (DFS) and overall survival (OS) than those with high levels (47.6 vs 69.3 months, P = 0.032 and 57.7 vs 77.6 months, P = 0.021, respectively). When the expression levels of NANCI and NKX2-1 were evaluated in combination, four groups were identified (high NANCI/high NKX2-1, low NANCI/high NKX2-1, high NANCI/low NKX2-1 and low NANCI/low NKX2-1) with differential impact on DFS (P = 0.042) and OS (P = 0.024). Interestingly, the high NANCI/high NKX2-1 duplex group had longer DFS and OS than the other three groups (71.25 vs 46.3 months, P = 0.009 and 81.3 vs 56.1 months, P = 0.004, respectively). In the multivariate analysis, the high NANCI/high NKX2-1 duplex was identified as an independent prognostic factor for longer DFS (HR 0.346, 95% CI, 0.169-0.709; P = 0.004) and OS (HR 0.309, 95% CI, 0.121-0.786; P = 0.014). CONCLUSIONS: NANCI and the NANCI-NKX2-1 duplex impacts prognosis in stage I NSCLC patients

CONTEXTO GLOBAL: NANCI, un ARN intergénico largo no codificante (lncRNA) es esencial para regular la expresión de NKX2-1 durante el desarrollo embrionario y en el tejido adulto. Analizamos la expresión de NANCI y NKX2-1 en muestras embrionarias de pulmón humano y tejidos pulmonares adultos, y evaluamos su potencial como marcadores pronósticos en el cáncer de pulmón de células no pequeñas (CPCNP) en estadio I. MÉTODOS Y RESULTADOS: La expresión de NANCI y NKX2-1 se evaluó mediante ensayos TaqMan® en 18 muestras embrionarias humanas de 8 a 13 semanas, 59 muestras de tejido pulmonar no tumoral (NT) y 98 muestras de tumor de CPCNP en estadio i. La expresión de NANCI y NKX2-1 en muestras embrionarias y NSCLC se encontraba reducida en comparación con el tejido NT adulto. Los pacientes con baja expresión de NANCI tuvieron una supervivencia libre de enfermedad (SLE) y una supervivencia general (SG) más cortas que aquellos con niveles altos (47,6 frente a 69,3 meses; p = 0,032 y 57,7 frente a 77,6 meses; p = 0,021, respectivamente). Cuando se evaluaron los niveles de expresión de NANCI y NKX2-1 combinados se identificaron 4 grupos (NANCI alto/NKX2-1 alto, NANCI bajo/NKX2-1 alto, NANCI alto/NKX2-1 bajo y NANCI bajo/NKX2-1 bajo) con impacto variable en la SLE (p = 0,042) y la SG (p = 0,024). Curiosamente, la combinación de NANCI alto/NKX2-1 alto presentó unas SLE y SG más largas que los otros 3 grupos (71,25 frente a 46,3 meses; p = 0,009 y 81,3 frente a 56,1 meses; p = 0,004, respectivamente). En el análisis multivariante, la combinación de NANCI alto/NKX2-1 alto se identificó como un factor de pronóstico independiente para una SLE más larga (HR: 0,346; IC 95%: 0,169-0,709; p = 0,004), al igual que la SG (HR: 0,309; IC 95%: 0,121-0,786; p = 0,014). CONCLUSIONES: NANCI y la combinación de NANCI-NKX2-1 afecta al pronóstico de los pacientes con CPCNP en estadio i