Programmed cell death protein-1/programmed cell death ligand-1 (PD-1/PD-L1) pathway blockade is a promising new cancer therapy. Although PD-1/PD-L1 treatment has yielded clinical benefits in several types of cancer, further studies are required to clarify predictive biomarkers for drug efficacy and to understand the fundamental mechanism of PD-1/PD-L1 interaction between host and tumor cells. Here, we show that exosomes derived from lung cancer cells express PD-L1 and play a role in immune escape by reducing T-cell activity and promoting tumor growth. The abundance of PD-L1 on exosomes represented the quantity of PD-L1 expression on cell surfaces. Exosomes containing PD-L1 inhibited interferon-gamma (IFN-γ) secretion by Jurkat T cells. IFN-γ secretion was restored by PD-L1 knockout or masking on the exosomes. Both forced expression of PD-L1 on cells without PD-L1 and treatment with exosomes containing PD-L1 enhanced tumor growth in vivo. PD-L1 was present on exosomes isolated from the plasma of patients with non-small cell lung cancer, and its abundance in exosomes was correlated with PD-L1 positivity in tumor tissues. Exosomes can impair immune functions by reducing cytokine production and inducing apoptosis in CD8⁺T cells. Our findings indicate that tumor-derived exosomes expressing PD-L1 may be an important mediator of tumor immune escape.

Epithelial‐mesenchymal transition (EMT) is associated with reduced sensitivity to many chemotherapeutic drugs, including EGFR tyrosine kinase inhibitors. Here, we investigated if this reduced sensitivity also contributes to resistance to crizotinib, an ALK inhibitor of lung cancer that exhibits the EML4‐ALK translocation. We established a crizotinib‐resistant subline (H2228/CR), which was derived from the parental H2228 cell line by long‐term exposure to increasing concentrations of crizotinib. Characteristics associated with EMT, including morphology, EMT marker proteins, and cellular mobility, were analyzed. Compared with H2228 cells, the growth of H2228/CR cells was independent of EML4‐ALK, and H2228/CR cells showed cross‐resistance to TAE‐684 (a second‐generation ALK inhibitor). Phenotypic changes to the spindle‐cell shape were noted in H2228/CR cells, which were accompanied by a decrease in E‐cadherin and increase in vimentin and AXL. In addition, H2228/CR cells showed increased secretion and expression of TGF‐β1. Invasion and migration capabilities were dramatically increased in H2228/CR cells. Applying TGF‐β1 treatment to parental H2228 cells for 72 h induced reversible EMT, leading to crizotinib resistance, but this was reversed by the removal of TGF‐β1. Suppression of vimentin in H2228/CR cells by siRNA treatment restored sensitivity to crizotinib. Furthermore, these resistant cells remained highly sensitive to the Hsp90 inhibitors, similar to the parental H2228 cells. In conclusion, we suggest EMT is possibly involved in acquired resistance to crizotinib, and that HSP90 inhibitors could be a promising option for the treatment of EMT.

Background: Epidermal growth factor receptor (EGFR)-activating mutations are major determinants in predicting the tumor response to EGFR tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC). Noninvasive test for the detection of EGFR mutations is required, especially in NSCLC patients from whom tissue is not available. In this study, we assessed the feasibility of detection of EGFR mutations in free DNA circulating in plasma. Methods: Plasma samples of 60 patients with partial response to gefitinib were analyzed to detect EGFR-activating mutations in exons 19 and 21. Forty (66.7%) of patients had tumor EGFR mutation results. EGFR mutations in plasma were detected using the peptide nucleic acid (PNA)-mediated polymerase chain reaction (PCR) clamping method. All clinical data and plasma samples were obtained from 11 centers of the Korean Molecular Lung Cancer Group (KMLCG). Results: Of the 60 patients, 39 were female and the median age was 62.5 years. Forty-three patients never smoked, 53 had adenocarcinomas, and seven had other histologic types. EGFR-activating mutation was detected in plasma of 10 cases (exon 19 deletion in seven and exon 21 L858R point mutation in three). It could not be found in plasma after treatment for 2 months. When only patients with confirmed EGFR mutation in tumor were analyzed, 17% (6 of 35) of them showed positive plasma EGFR mutation and the mutation type was completely matched with that in tumor. There was no statistically significant difference in clinical parameters between patients with EGFR mutations in plasma and those without EGFR mutations. Conclusions: The detection rate of EGFR mutations from plasma was not so high despite highly sensitive EGFR mutation test suggesting that more advances in detection methods and further exploration of characteristics of circulating free DNA are required.

EGFR genotyping in pulmonary adenocarcinoma patients who develop pleural effusions is mostly performed using cytology or cell block slides with low sensitivity. Liquid biopsy using the supernatant of pleural effusions may be more effective because they contain many components released by cancer cells. Extracellular vesicles (EVs) are known to carry oncogenic double-stranded DNA that is considered a notable biomarker. Here, we investigate the efficiency of liquid biopsy using cell-free DNA (cfDNA) and extracellular vesicle-derived DNA (EV-derived DNA) from the supernatant of pleural effusions for EGFR genotyping in patients with pulmonary adenocarcinoma. Fifty pleural effusion samples from patients with pulmonary adenocarcinoma were evaluated. The supernatant, after removing the cell pellet by centrifugation, was used for liquid biopsy, and EVs were isolated from the pleural effusion by ultracentrifugation. EV-derived DNA and cfDNA were extracted separately, and EGFR genotyping was performed by the PNA clamping method. Among 32 patients who were EGFR-tyrosine kinase inhibitor (TKI) naïve with a known tissue EGFR genotype, liquid biopsy using EV-derived DNA from the pleural effusion supernatant showed 100% matching results with tissue EGFR genotyping in 19 EGFR mutant cases and detected three additional EGFR mutations in patients with wild-type (WT) tissue. Liquid biopsy using cfDNA from pleural effusion supernatants missed two cases of tissue-based EGFR mutations and found two additional EGFR mutation cases. In 18 patients who acquired resistance to EGFR-TKI, EGFR genotyping using EV-derived DNA from the pleural effusion supernatant detected the T790 M mutation in 13 of 18 (72.2%) patients, and this mutation was detected in 11 (61.1%) patients using cfDNA. By contrast, only three patients were found to present the T790 M mutation when using cell block or cytology slides. Liquid biopsy using the supernatant of pleural effusions showed significantly improved results for EGFR genotyping compared to those using conventional cell block or cytology samples. Liquid biopsy using EV-derived DNA is promising for EGFR genotyping, including T790 M detection in pulmonary adenocarcinoma patients who develop pleural effusions.

Journal of Medical Internet Research - International Scientific Journal for Medical Research, Information and Communication on the Internet #Preprint #PeerReviewMe: Warning: This is a unreviewed preprint. Readers are warned that the document has not been peer-reviewed by expert/patient reviewers or an academic editor, may contain misleading claims, and is likely to undergo changes before final publication, if accepted, or may have been rejected/withdrawn. Readers with interest and expertise are encouraged to sign up as peer-reviewer, if the paper is within an open peer-review period. Please cite this preprint only for review purposes or for grant applications and CVs (if you are the author). Background: Lung cancer patients experience various symptoms during treatment. Although pulmonary rehabilitation is an effective way to improve these symptoms, limited medical environment makes it difficult to provide seamless adequate rehabilitation for lung cancer patients. Objective: This study aims to investigate the effects of pulmonary rehabilitation for patients with non-small cell lung cancer by means of a personalized pulmonary rehabilitation program that uses real-time mobile patient health data. Methods: A prospective clinical trial was conducted with 64 non-small cell lung cancer patients aged between 20 and 80 years, diagnosed with lung cancer at Asan Medical Center. A 12-week personalized pulmonary rehabilitation program was administered to investigate the efficacy of the rehabilitation app developed. The participants were randomized into the fixed exercise or fixed-interactive exercise group. At 6 weeks, changes in walking distances during 6 minutes (6MWD) and grade of dyspnea (mMRC), and at 12weeks those in QoL and service satisfaction, were observed. Paired T-test were used for analysis of the variables. Results: An mHealth pulmonary rehabilitation app and real-time patient monitoring website were developed. Significant changes were observed in the intervention group at 12 weeks for 6MWT from 433.43±65.60m to 471.25±75.69m (p=0.001), and 0.94±0.66 to 0.61±0.82 (p=0.017) for mMRC. The intervention showed a significant improvement in the quality of life (EQ-VAS) in compared to the baseline (p=0.002) Conclusions: A personalized mHealth-based pulmonary rehabilitation app for recording and monitoring real-time health data of patients with non-small cell lung cancer can plays a supplementary role in traditional center-based rehabilitation programs. Our clinical trial results support the use of this technology to encourage changes in 6MWT and to improve mMRC.