Objectives: In non‐small cell lung cancer (NSCLC), the immune system and possibly its composition affect survival. In this in silico study, the immune infiltrate composition in NSCLC patients was evaluated.Methods: Gene expression data of tumors from early NSCLC patients were obtained from Gene Expression Omnibus (GEO). With CIBERSORT, 22 immune cell fractions were estimated.Results: The immune infiltrate of 1430 pretreatment NSCLC patients contained mostly plasma cells, macrophages and CD8 T cells. Higher fractions of resting mast and CD4 T‐helper cells were associated with longer overall survival (OS) (HR = 0.95, P < 0.01; HR = 0.98, = 0.04, respectively) and higher fractions of M2 macrophages and active dendritic cells with shorter survival (HR = 1.02, P = 0.03; HR = 1.03, P = 0.05, respectively). Adenocarcinoma patients with survival data (n = 587) showed higher fractions of resting mast and resting CD4 T cells, and lower M0 macrophages than squamous cell carcinoma (n = 254), which were associated with OS (HR = 0.95, P = 0.04; HR = 0.97, P = 0.01; HR = 1.03, P = 0.01, respectively). Fractions of memory B cells, naïve CD4 T cells and neutrophils had different associations with survival depending on the subtype. Smokers had had higher fractions of regulatory T cell, follicular helper T cell, neutrophil and M2 macrophage, which were associated with shorter survival (HR = 1.3, P < 0.01; HR = 1.13, P = 0.02; HR = 1.09, P = 0.03; HR = 1.04, P = 0.02, respectively).Conclusion: Pretreatment differences in immune cell composition in NSCLC are associated with survival and depend on smoking status and histological subtype. Smokers' immune composition is associated with lower survival.

Background Non-small cell lung cancer (NSCLC) patients treated with checkpoint inhibitors show long lasting responses, but it is hard to predict which patients will profit from this treatment with the currently used marker, programmed death ligand 1 (PD-L1). We hypothesized that circulating tumor cells (CTC) or tumor derived extracellular vesicles (tdEV) are markers of treatment efficacy. Methods Patients with advanced NSCLC treated with checkpoint inhibitors were included. Blood was drawn at baseline (T0) and at 4 weeks of treatment (T1) for analysis of CTC and tdEV using CellSearch®. Tumor response was classified as partial or complete response based on the response evaluation criteria in solid tumors (RECISTv1.1) measured 4–6 weeks after start of treatment. Durable response was defined as stable disease, partial or complete response without disease progression at 6 months. Analyses were adjusted for covariables including PD-L1 expression. Results We included 104 patients (30 with a tumor response, 74 non-responders, 2 responses not evaluable due to early death); 63 patients provided T1 samples. All patients were treated with PD-L1 inhibitors. The majority of patients received second (85%) or third line (treatment with nivolumab monotherapy (89%). CTC were present in 33/104 patients at T0 (32%) and 17/63 at T1 (27%), 9/63 patients had CTC (14%) at both time points. The presence of CTC, both at T0 (OR = 0.28, p = 0.02,) and T1 (OR = 0.07, p < 0.01), was an independent predictive factor for a lack of durable response and was associated with worse progression free and overall survival. More tdEV were associated with shorter survival but not with response rate. Conclusion CTC occur in one third of advanced NSCLC patients and their presence is a predictive factor for a worse durable response rate to checkpoint inhibitors. tdEV are associated with shorter survival but not with response.

PURPOSE: Immune checkpoint inhibitors (ICIs) are increasingly being used in non–small-cell lung cancer (NSCLC), yet biomarkers predicting their benefit are lacking. We evaluated if on-treatment changes of circulating tumor DNA (ctDNA) from ICI start (t₀) to after two cycles (t₁) assessed with a commercial panel could identify patients with NSCLC who would benefit from ICI. PATIENTS AND METHODS: The molecular ctDNA response was evaluated as a predictor of radiographic tumor response and long-term survival benefit of ICI. To maximize the yield of ctDNA detection, de novo mutation calling was performed. Furthermore, the impact of clonal hematopoiesis (CH)–related variants as a source of biologic noise was investigated. RESULTS: After correction for CH-related variants, which were detected in 75 patients (44.9%), ctDNA was detected in 152 of 167 (91.0%) patients. We observed only a fair agreement of the molecular and radiographic response, which was even more impaired by the inclusion of CH-related variants. After exclusion of those, a ≥ 50% molecular response improved progression-free survival (10 v 2 months; hazard ratio [HR], 0.55; 95% CI, 0.39 to 0.77; P = .0011) and overall survival (18.4 v 5.9 months; HR, 0.44; 95% CI, 0.31 to 0.62; P < .0001) compared with patients not achieving this end point. After adjusting for clinical variables, ctDNA response and STK11/ KEAP1 mutations (HR, 2.08; 95% CI, 1.4 to 3.0; P < .001) remained independent predictors for overall survival, irrespective of programmed death ligand-1 expression. A landmark survival analysis at 2 months (n = 129) provided similar results. CONCLUSION: On-treatment changes of ctDNA in plasma reveal predictive information for long-term clinical benefit in ICI-treated patients with NSCLC. A broader NSCLC patient coverage through de novo mutation calling and the use of a variant call set excluding CH-related variants improved the classification of molecular responders, but had no significant impact on survival.

Circulating cell-free DNA (ccfDNA) may contain DNA originating from the tumor in plasma of cancer patients (ctDNA) and enables noninvasive cancer diagnosis, treatment predictive testing, and response monitoring. A recent multicenter evaluation of workflows by the CANCER-ID consortium using artificial spiked-in plasma showed significant differences and consequently the importance of carefully selecting ccfDNA extraction methods. Here, the quantity and integrity of extracted ccfDNA from the plasma of cancer patients were assessed. Twenty-one cancer patient-derived cell-free plasma samples were selected to compare the Qiagen CNA, Maxwell RSC ccfDNA plasma, and Zymo manual quick ccfDNA kit. High-volume citrate plasma samples collected by diagnostic leukapheresis from six cancer patients were used to compare the Qiagen CNA (2 mL) and QIAamp MinElute ccfDNA kit (8 mL). This study revealed similar integrity and similar levels of amplified short-sized fragments and tumor-specific mutants comparing the CNA and RSC kits. However, the CNA kit consistently showed the highest yield of ccfDNA and short-sized fragments, while the RSC and ME kits showed higher variant allelic frequencies (VAFs). Our study pinpoints the importance of standardizing preanalytical conditions as well as consensus on defining the input of ccfDNA to accurately detect ctDNA and be able to compare results in a clinical routine practice, within and between clinical studies.

Molecular profiling from liquid biopsy, in particular cell-free DNA (cfDNA), represents an attractive alternative to tissue biopsies for the detection of actionable targets and tumor monitoring. In addition to PCR-based assays, Next Generation Sequencing (NGS)-based cfDNA assays are now commercially available and are being increasingly adopted in clinical practice. However, the validity of these products as well as the clinical utility of cfDNA in the management of patients with cancer has yet to be proven. Within framework of the Innovative Medicines Initiative (IMI) program CANCER-ID we evaluated the use of commercially available reference materials designed for ctDNA testing and cfDNA derived from Diagnostic Leukaphereses (DLA) for inter- and intra-assay as well as intra- and inter-laboratory comparisons. In three experimental setups, a broad range of assays including ddPCR, MassARRAY and various NGS-based assays were tested. We demonstrate that both reference materials with predetermined VAFs and DLA samples are extremely useful for the performance assessment of mutation analysis platforms. Moreover, our data indicate a substantial variability of NGS assays with respect to sensitivity and specificity highlighting the importance of extensive validation of the test performance before offering these tests in clinical routine practice.

Circulating tumor cells (CTCs) detected by CellSearch are prognostic in non-small-cell lung cancer (NSCLC), but rarely found. CTCs can be extracted from the blood together with mononuclear cell populations by diagnostic leukapheresis (DLA), therefore concentrating them. However, CellSearch can only process limited DLA volumes (≈2 mL). Therefore, we established a protocol to enumerate CTCs in DLA products with Isolation by SizE of Tumor cells (ISET), and compared CTC counts between CellSearch^® and ISET. DLA was performed in NSCLC patients who started a new therapy. With an adapted protocol, ISET could process 10 mL of DLA. CellSearch detected CTCs in a volume equaling 2 × 10⁸ leukocytes (mean 2 mL). CTC counts per mL were compared. Furthermore, the live cell protocol of ISET was tested in eight patients. ISET successfully processed all DLA products—16 with the fixed cell protocol and 8 with the live cell protocol. In total, 10–20 mL of DLA was processed. ISET detected CTCs in 88% (14/16), compared to 69% (11/16, p < 0.05) with CellSearch. ISET also detected higher number of CTCs (ISET median CTC/mL = 4, interquartile range [IQR] = 2–6, CellSearch median CTC/mL = 0.9, IQR = 0–1.8, p < 0.01). Cells positive for the epithelial cell adhesion molecule (EpCAM+) per mL were detected in similar counts by both methods. Eight patients were processed with the live cell protocol. All had EpCAM+, CD45−, CD235- cells isolated by fluorescence-activated cell sorting (FACS). Overall, ISET processed larger volumes and detected higher CTC counts compared to CellSearch. EpCAM+ CTCs were detected in comparable rates.