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.
Because of small populations, it is difficult to assess differences in the immunocomposition and their associated survival depending on smoking behaviour and tumor subtype for non‐small cell lung cancer patients. Here, we used an in silico approach of all available material to identify these differences and the associations with survival. Immune cell fractions showed different, sometimes opposing associations depending on the tumor subtype and smoking status.
Circulating tumour cells (CTCs) can be used to monitor cancer longitudinally, but their use in non-small cell lung cancer (NSCLC) is limited due to low numbers in the peripheral blood. Through ...diagnostic leukapheresis (DLA) CTCs can be obtained from larger blood volumes.
Patients with all stages of NSCLC were selected. One total body blood volume was screened by DLA before and after treatment. Peripheral blood was drawn pre- and post DLA for CTC enumeration by CellSearch. CTCs were detected in the DLA product (volume equalling 2 × 10
leucocytes) and after leucocyte depletion (RosetteSep, 9 mL DLA product). Single-cell, whole-genome sequencing was performed on isolated CTCs.
Fifty-six patients were included. Before treatment, CTCs were more often detected in DLA (32/55, 58%) than in the peripheral blood (pre-DLA: 18/55, 33%; post DLA: 13/55, 23%, both at p < 0.01). CTCs per 7.5 mL DLA product were median 9.2 times (interquartile range = 5.6-24.0) higher than CTCs in 7.5 mL blood. RosetteSEP did not significantly improve CTC detection (pretreatment: 34/55, 62%, post treatment: 16/34, 47%) and CTCs per mL even decreased compared to DLA (p = 0.04).. Patients with advanced-stage disease with DLA-CTC after treatment showed fewer tumour responses and shorter progression-free survival (PFS) than those without DLA-CTC (median PFS, 2.0 vs 12.0 months, p < 0.01). DLA-CTC persistence after treatment was independent of clinical factors associated with shorter PFS (hazard ratio (HR) = 5.8, 95% confidence interval (CI), 1.4-35.5, p = 0.02). All evaluable CTCs showed aneuploidy.
DLA detected nine times more CTCs than in the peripheral blood. The sustained presence of CTCs in DLA after treatment was associated with therapy failure and shortened PFS.
The study was approved by the Medical Ethical Committee (NL55754.042.15) and was registered in the Dutch trial register (NL5423).
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.
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.
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.
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.
The aim of this study was to determine the efficacy of early tocilizumab treatment for hospitalized patients with COVID-19 disease. Open-label randomized phase II clinical trial investigating ...tocilizumab in patients with proven COVID-19 admitted to the general ward and in need of supplemental oxygen. The primary endpoint of the study was 30-day mortality with a prespecified 2-sided significance level of alpha = 0.10. A post-hoc analysis was performed for a combined endpoint of mechanical ventilation or death at 30 days. Secondary objectives included comparing the duration of hospital stay, ICU admittance and duration of ICU stay and the duration of mechanical ventilation. A total of 354 patients (67% men; median age 66 years) were enrolled of whom 88% received dexamethasone. Thirty-day mortality was 19% (95% CI 14%-26%) in the standard arm versus 12% (95% CI: 8%-18%) in the tocilizumab arm, hazard ratio (HR) = 0.62 (90% CI 0.39-0.98; p = 0.086). 17% of patients were admitted to the ICU in each arm (p = 0.89). The median stay in the ICU was 14 days (IQR 9-28) in the standard arm versus 9 days (IQR 5-14) in the tocilizumab arm (p = 0.014). Mechanical ventilation or death at thirty days was 31% (95% CI 24%-38%) in the standard arm versus 21% (95% CI 16%-28%) in the tocilizumab arm, HR = 0.65 (95% CI 0.42-0.98; p = 0.042). This randomized phase II study supports efficacy for tocilizumab when given early in the disease course in hospitalized patients who need oxygen support, especially when concomitantly treated with dexamethasone.
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 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.
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 × 10
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,
< 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,
< 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.
•Lung cancer subtypes are differentiated by conserved methylated loci.•Immune modulating genes with ks-score ≥ 70 % are under methylation control.•Lung cancer exhibits selective suppression of ...antigen presentation and processing.
Non-small-cell lung cancer exhibits a range of transcriptional and epigenetic patterns that not only define distinct phenotypes, but may also govern immune related genes, which have a major impact on survival.
We used open-source RNA expression and DNA methylation data of the Cancer Genome Atlas with matched non-cancerous tissue to evaluate whether these pretreatment molecular patterns also influenced genes related to the immune system and overall survival.
The distinction between lung adenocarcinoma and squamous cell carcinoma are determined by 1083 conserved methylation loci and RNA expression of 203 genes which differ for >80 % of patients between the two subtypes. Using the RNA expression profiles of 6 genes, more than 95 % of patients could be correctly classified as having either adeno or squamous cell lung cancer. Comparing tumor tissue with matched normal tissue, no differences in RNA expression were found for costimulatory and co-inhibitory genes, nor genes involved in cytokine release. However, genes involved in antigen presentation had a lower expression and a wider distribution in tumor tissue.
Only a small number of genes, influenced by DNA methylation, determine the lung cancer subtype. The antigen presentation of cancer cells is dysfunctional, while other T cell immune functions appear to remain intact.
As therapies have become more and more dependent on tumor as well as patient characteristics, obtaining tumor material has become of great importance. Liquid biopsies hold much potential as shown by ...a large amount of evidence across several studies. Clinical applications for circulating tumor cells (CTCs) are unfortunately still lacking. In part this is due to a lack of studies comparing liquid biopsies to conventional diagnostics and response measurements as well as studies showing that liquid biopsies can be used to switch therapies leading to improved outcomes. However, liquid biopsies using ctDNA for specific markers such as EGFR, ALK, ROS1 or RET have clinical applications because specific drugs are available.
Background
The clinical relevance of epidermal growth factor receptor (
EGFR
) copy number gain in patients with
EGFR
mutated advanced non-small cell lung cancer on first-line tyrosine kinase ...inhibitor treatment has not been fully elucidated.
Objective
We aimed to estimate
EGFR
copy number gain using amplicon-based next generation sequencing data and explored its prognostic value.
Patients and Methods
Next generation sequencing data were obtained for 1566 patients with non-small cell lung cancer.
EGFR
copy number gain was defined based on an increase in
EGFR
read counts relative to internal reference amplicons and normal controls in combination with a modified
z
-score ≥ 3.5. Clinical follow-up data were available for 60 patients treated with first-line EGFR-tyrosine kinase inhibitors.
Results
Specificity and sensitivity of next generation sequencing-based
EGFR
copy number estimations were above 90%.
EGFR
copy number gain was observed in 27.9% of
EGFR
mutant cases and in 7.4% of
EGFR
wild-type cases.
EGFR
gain was not associated with progression-free survival but showed a significant effect on overall survival with an adjusted hazard ratio of 3.14 (95% confidence interval 1.46–6.78,
p
= 0.003). Besides
EGFR
copy number gain, osimertinib in second or subsequent lines of treatment and the presence of T790M at relapse revealed significant effects in a multivariate analysis with adjusted hazard ratio of 0.43 (95% confidence interval 0.20–0.91,
p
= 0.028) and 0.24 (95% confidence interval 0.1–0.59,
p
= 0.001), respectively.
Conclusions
Pre-treatment
EGFR
copy number gain determined by amplicon-based next generation sequencing data predicts worse overall survival in
EGFR
-mutated patients treated with first-line EGFR-tyrosine kinase inhibitors. T790M at relapse and subsequent treatment with osimertinib predict longer overall survival.
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