Despite the development of predictive biomarkers to shape treatment paradigms and outcomes, de novo EGFR TKI resistance advanced non-small cell lung cancer (NSCLC) remains an issue of concern. We explored clinical factors in 332 advanced NSCLC who received EGFR TKI and molecular characteristics through 65 whole exome sequencing of various EGFR TKI responses including; de novo (progression within 3 months), intermediate response (IRs) and long-term response (LTRs) (durability > 2 years). Uncommon EGFR mutation subtypes were significantly variable enriched in de novo resistance. The remaining sensitizing EGFR mutation subtypes (exon 19 del and L858R) accounted for 75% of de novo resistance. Genomic landscape analysis was conducted, focusing in 10 frequent oncogenic signaling pathways with functional contributions; cell cycle, Hippo, Myc, Notch, Nrf2, PI-3-Kinase/Akt, RTK-RAS, TGF-β, p53 and β-catenin/Wnt signaling. Cell cycle pathway was the only significant alteration pathway among groups with the FDR p-value of 6 × 10–4. We found only significant q-values of < 0.05 in 7 gene alterations; CDK6, CCNE1, CDK4, CCND3, MET, FGFR4 and HRAS which enrich in de novo resistance [range 36–73%] compared to IRs/LTRs [range 4–22%]. Amplification of CDK4/6 was significant in de novo resistance, contrary to IRs and LTRs (91%, 27.9% and 0%, respectively). The presence of co-occurrence CDK4/6 amplification correlated with poor disease outcome with HR of progression-free survival of 3.63 [95% CI 1.80–7.31, p-value < 0.001]. The presence of CDK4/6 amplification in pretreatment specimen serves as a predictive biomarker for de novo resistance in sensitizing EGFR mutation.

Background: Previous studies from our group reported paracrine signal-induced peripheral mononuclear cell (PBMC) gene upregulation in various cancer types through epigenetic regulation. We speculated protein expression on circulating T- lymphocytes which might represent T-lymphocyte trafficking before infiltrating into tumor microenvironment. The possibility to use protein expression on circulating T-lymphocytes as a biomarker to discriminate early stage lung cancer has been explored.Methods: PBMC gene expression was explored, using 4 independent gene expression microarray datasets (GSE12771, GSE13255, GSE20189 and GSE3934). We selected 3 candidate proteins, C5AR1, NLRP3 and CLEC4A, based on their significant protein expression in tumor-infiltrating lymphocytes but not in normal lymphoid tissue. A validation study using automated flow cytometry was conducted in 121 participants including 44 treatment-naïve early stage non-small cell lung cancer patients (NSCLC), 19 non-malignant pulmonary diseases and 62 healthy individuals. Results: The ratio of C5AR1, NLRP3 and CLEC4A specific antibody staining to CD3 positive was significantly higher in early stage NSCLC compared to healthy control. Median ratio of C5AR1, NLRP3 and CLEC4A expression in early stage NSCLC were 0.65 [range 0.27-0.96; 95% CI: 0.55-0.70], 0.83 [range 0.27-0.99; 95% CI: 0.73-0.86] and 0.75 [range 0.21-0.98; 95% CI: 0.65-0.81], respectively. While, median ratio of C5AR1, NLRP3 and CLEC4A expression in healthy control were 0.21 [range 0.05-0.81; 95% CI: 0.23-0.42, p-value <0.001], 0.32 [range 0.04-0.94; 95% CI: 0.28-0.46, p-value <0.001] and 0.22 [range 0.04-0.97; 95% CI: 0.27-0.48, p-value < 0.001], respectively. This led to 87.5-100% sensitivity, 50.9-64.7% specificity and 71.4-75% accuracy. However, those PBMC protein expressions could not discriminate early stage NSCLC from malignant-mimic inflammation and infection.Conclusions: Our proof-of-principle findings strengthen the hypothesis that malignancies generate distinctive protein expression fingerprints on circulating T-lymphocytes. Trial registration: This trial was registered with clinicaltrials.in.th, Number TCTR20190508003.

Background: Changes in gene expression profiling of peripheral blood mononuclear cells (PBMC) appear to represent the host’s response to the cancer cells via paracrine signaling. We speculated that protein expression on circulating T-lymphocytes represent T-lymphocyte trafficking before infiltration into the tumor microenvironment. The possibility of using protein expression on circulating T-lymphocytes as a biomarker to discriminate early-stage non-small cell lung cancer (NSCLC) was explored in this study.Methods: Four independent PBMC gene expression microarray datasets (GSE12771, GSE13255, GSE20189 and GSE3934) were analyzed. We selected C5AR1, CLEC4A and NLRP3 based on their significant protein expression in tumor-infiltrating lymphocytes, but not in normal lymphoid tissue. A validation study using automated flow cytometry was conducted in 141 study participants including 76 treatment-naive early-stage non-small cell lung cancer patients (NSCLC), 12 individuals with non-malignant pulmonary diseases, and 53 healthy individuals. Results: Median ratios of C5AR1, CLEC4A and NLRP3 specific antibody staining to CD3 positive cells in early-stage NSCLC patients compared to healthy controls were 0.014 [0-0.37] vs. 0.01 [0-0.07, p=0.13], 0.03 [0-0.87] vs. 0.02 [0-0.13, p=0.10] and 0.19 [0-0.60] vs. 0.09 [0.02-0.31, p<0.0001], respectively. Median fluorescence intensity (MFI) of CD3+C5AR1+, CD3+CLEC4A+ and CD3+NLRP3+ expression in early-stage NSCLC patients compared to healthy volunteers was 185 [64.2-4801] vs. 107.5 [27-229, p<0.0001], 91.2 [42.4-2355] vs. 71.25 [46.2-103, p=0.0005], and 1585 [478-5224] vs. 758.5 [318-1976, p<0.0001], respectively. NLRP3:CD3 ratio, CD3+C5AR1+, CD3+CLEC4A+ and CD3+NLRP3+ MFI were significantly higher in early-stage NSCLC than healthy volunteers with an area under the ROC curve of 0.69-0.76. The CD3+NLRP3+ MFI provided the most distinguishable expression at 71.5% sensitivity and 70% specificity. Furthermore, CD3+NLRP3+ MFI potentially discriminated between early-stage NSCLC from malignant-mimic inflammation and infection pulmonary disease.Conclusions: Our proof-of-principle findings strengthen the hypothesis that malignancies generate distinctive protein expression fingerprints on circulating T-lymphocytes.