Lung adenocarcinoma with EGFR L858R-K860I and L858R-L861F doublet mutations from which the L858R mutation is undetectable through the cobas EGFR mutation test v2.

In East Asia, epidermal growth receptor factor (EGFR) mutations are the most prevalent and important biomarkers for treating patients with advanced lung cancer. However, as L858R doublet mutations are rare, commercially available EGFR tests may yield false-negative results. To determine whether the L858R mutation of the L858R-K860I and L858R-L861F doublet mutations could be identified using different types of EGFR detection tests and to describe the clinical response of patients with lung cancer with L858R doublet mutations to EGFR tyrosine kinase inhibitors (TKI). Information and samples from four patients with L858R doublet mutations, including three with L858R-K860I and one with L858R-L861F, were retrospectively collected from the archives of our department. For each case, the clinical response to EGFR-TKI was retrieved from the medical records. Archived formalin-fixed paraffin-embedded blocks were subjected to Sanger sequencing, the cobas and Idylla EGFR tests, the IntelliPlex-LCP-DNA assay, and AmoyDx PLC panel. L858R mutations were all detected by Sanger sequencing and the Idylla EGFR test but missed by the cobas assay. The AmoyDx PLC detected L858R only in cases with L858R-K860I while the IntelliPlex-LCP-DNA assay detected L858R in the case with L858R-L861F. Additionally, three of the patients, who had measurable tumors, showed partial responses to afatinib and osimertinib. The L858R mutation associated with L858R-K860I and L858R-L861F doublet mutations could be detected using Idylla but not cobas EGFR tests. Using next-generation sequencing analysis should be considered after initial negative reports from the cobas test, because patients with L858R doublet mutations may benefit from EGFR-TKIs.

The association of EGFR amplification with aberrant exon 20 insertion report using the cobas EGFR Mutation Test v2.

Determining the exact type of epidermal growth factor receptor (EGFR) exon 20 insertion (ex20ins) mutation in lung cancer has become important. We found that not all ex20ins mutations reported by cobas EGFR test v2 could be validated by Sanger sequencing even using surgical specimens with high tumor contents. This study aimed to validate the ex20ins results reported by the cobas test and to determine whether there were clinicopathological factors associated with aberrant cobas ex20ins report. In total, 123 cobas-reported cases with ex20ins were retrospectively collected and validated by Sanger sequencing and Idylla assay. Clinicopathological features between ex20ins cobas+/Sanger+ group (n = 71) and cobas+/Sanger- group (n = 52) were compared. The Idylla assay detected ex20ins in 82.6% of cobas+/Sanger+ cases but only in 4.9% of cobas+/Sanger- cases. The cobas+/Sanger- group was significantly associated with higher tumor contents, poorly differentiated patterns, tumor necrosis, and a lower internal control cycle threshold value reported by the Idylla which suggesting the presence of increased EGFR gene copy numbers. EGFR fluorescence in situ hybridization (FISH) revealed the majority of cobas+/Sanger- group had EGFR high copy number gain (16%) or amplification (76%) according to the Colorado criteria. Among cases reported to have concomitant classic EGFR and ex20ins mutations by the cobas, the classic EGFR mutations were all detected by Sanger sequencing and Idylla, while the ex20ins mutations were undetected by Sanger sequencing (0%) or rarely reported by Idylla assay (3%). FISH revealed high EGFR copy number gain (17.9%) and amplification (79.5%) in cases reported having concomitant classic EGFR and ex20ins mutations by the cobas. This study demonstrated an unusually high frequency of EGFR amplification in cases with aberrant cobas ex20ins report which could not be validated by Sanger sequencing or Idylla assay. Ex20ins reported by the cobas test should be validated using other methods especially those reported having concomitant ex20ins and classic EGFR mutations.

Increased Tuberculosis Reactivation Risk in Patients Receiving Immune Checkpoint Inhibitor-Based Therapy.

OBJECTIVE: Reports of tuberculosis (TB) during anticancer treatment with immune checkpoint inhibitors (ICIs) are increasing. However, it is not clear whether the use of ICIs is a significant risk factor for TB, including reactivation or latent TB infection (LTBI). METHODS: To determine the risk of TB reactivation in patients with lung cancer who use ICIs or tyrosine kinase inhibitors (TKIs), we conducted a retrospective study using a hospital-based cancer registry. In addition, we monitored patients with cancer using ICI or TKI in a multicenter prospective study to check the incidence of LTBI. RESULTS: In the retrospective study, several demographic factors were imbalanced between the ICI and TKI groups: the ICI group was younger, had more males, exhibited more squamous cell carcinoma in histology rather than adenocarcinoma, had fewer EGFR mutations, and received more chemotherapy. Propensity score matching was used to control for confounding factors, and we found that the incidence of TB was higher among patients with lung cancer who received ICIs than among those who received TKIs (2298 vs 412 per 100 000 person-years, P = .0165). Through multivariable analysis, group (ICI vs TKI) was the independent risk factor for TB development (adjusted hazard ratio (aHR): 6.29, 95% CI, 1.23-32.09, P = .0269). In the prospective cohort, which included 72 patients receiving ICIs and 50 receiving TKIs, we found that the incidence of positive seroconversion of LTBI by interferon gamma release assay (IGRA) was significantly higher in patients receiving ICIs (18% vs 0%, aHR: 9.88, P = 0.035) under multivariable Cox regression. CONCLUSION: The use of ICIs may be linked to a higher likelihood of TB reactivation and LTBI than individuals solely receiving TKIs as anticancer therapy. Consequently, the implementation of a screening program for TB reactivation and LTBI among patients undergoing ICI treatment could prove advantageous by enabling early detection and prompt treatment of the infection.

Comprehensive Genomic Analysis of Patients With Non-Small-Cell Lung Cancer Using Blood-Based Circulating Tumor DNA Assay: Findings From the BFAST Database of a Single Center in Taiwan.

PURPOSE: The Blood First Assay Screening Trial (BFAST) is a prospective study using next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) in treatment-naïve advanced/metastatic non-small-cell lung cancer (NSCLC). We compared liquid biopsy to tissue testing and analyzed genomic alterations in Taiwanese patients with NSCLC using the BFAST database. MATERIALS AND METHODS: A total of 269 patients underwent FoundationOne Liquid Companion Diagnostic (F1LCDx) assay at the National Taiwan University Hospital, of whom 264 underwent tissue-based genetic testing also. We analyzed the actionable mutations and the concordance between tissue-based genetic testing, which was limited to EGFR, ALK, ROS1, and BRAF, in a real-life clinical setting and blood-based NGS in the clinical trial. Additionally, we analyzed the co-occurring genomic alterations from the blood-based ctDNA assay. RESULTS: A total of 76.2% patients showed actionable mutations. Standard tissue testing did not detect known driver alterations in about 22.7% of the patients (sensitivity, 70.24%). Liquid NGS detected additional mutations (RET, KRAS, MET, and ErbB2) in 14% of the patients, which went undetected by the standard-of-care testing. The complementary use of ctDNA NGS increased the detection rate by 42%. The F1LCDx assay had a sensitivity of 83.41%. Lower tumor and metastasis stages predicted nondetected blood-based NGS ctDNA results. Common co-occurring mutations in the blood-based NGS ctDNA assay were TP53, DNMT3A, TET2, PIK3CA, CTNNB1, and RB1. Among the patients with EGFR-mutated NSCLC, TET2 co-occurring alterations correlated with shorter progression-free survival of EGFR tyrosine kinase inhibitor treatment. CONCLUSION: NGS ctDNA analysis in comprehensive genetic testing improves actionable mutation identification, vital for treating Asian NSCLC cases with high actionable mutation rates. Lower stages correlated with undetected blood-based NGS ctDNA assay results.

Effects of Early Short-Course Corticosteroids on Immune-Related Adverse Events in Non-Small Cell Lung Cancer Patients Receiving Immune Checkpoint Inhibitors.

INTRODUCTION: In real-world practice, most non-small cell lung cancer (NSCLC) patients receiving combined immunochemotherapy are exposed to short-course corticosteroids following immune checkpoint inhibitor (ICI) infusion to prevent chemotherapy-related adverse events. However, whether this early short-course corticosteroid use prevents immune-related adverse events (irAEs) remains unknown. METHODS: Between January 1st, 2015, and December 31st, 2020, NSCLC patients who received at least one cycle of ICI with or without chemotherapy were enrolled. Early short-course corticosteroids were defined as corticosteroids administered following ICI injection and before chemotherapy on the same day and no longer than 3 days afterward. The patients were categorized as either "corticosteroid group" or "non-corticosteroid group" depending on their exposure to early short-course corticosteroid. The frequencies of irAEs requiring systemic corticosteroid use and irAEs leading to ICI discontinuation were compared between the two groups, and exploratory survival analyses were performed. RESULTS: Among 252 eligible patients, 137 patients were categorized as "corticosteroid group" and 115 patients as "non-corticosteroid group." The corticosteroid group enriched patients in the first-line setting (n = 75, 54.7%), compared to the non-corticosteroid group (n = 28, 24.3%). Thirty patients (21.9%) in the corticosteroid group and 35 patients (30.4%) in the non-corticosteroid group developed irAEs requiring systemic corticosteroid use (odds ratio [OR], 0.64; 95% confidence interval [CI], 0.35-1.18; p = 0.15). Eight patients (5.8%) in the corticosteroid group, as compared with 18 patients (15.7%) in the non-corticosteroid group, permanently discontinued ICI due to irAEs (OR, 0.34; 95% CI, 0.12-0.85; p = 0.013). CONCLUSION: Early short-course corticosteroids following each ICI injection may reduce the rate of irAEs that lead to ICIs discontinuation, warranting further investigation of its prophylactic use to mitigate clinically significant irAEs.

Tissue or liquid rebiopsy? A prospective study for simultaneous tissue and liquid NGS after first-line EGFR inhibitor resistance in lung cancer.

INTRODUCTION: According to current International Association for the Study of Lung Cancer guideline, physicians may first use plasma cell-free DNA (cfDNA) methods to identify epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-resistant mechanisms (liquid rebiopsy) for lung cancer. Tissue rebiopsy is recommended if the plasma result is negative. However, this approach has not been evaluated prospectively using next-generation sequencing (NGS). METHODS: We prospectively enrolled patients with lung cancer with first-line EGFR-TKI resistance who underwent tissue rebiopsy. The rebiopsied tissues and cfDNA were sequenced using targeted NGS, ACTDrug®+, and ACTMonitor®Lung simultaneously. The clinicopathological characteristics and treatment outcomes were analyzed. RESULTS: Totally, 86 patients were enrolled. Twenty-six (30%) underwent tissue biopsy but the specimens were inadequate for NGS. Among the 60 patients with paired tissue and liquid rebiopsies, two-thirds (40/60) may still be targetable. T790M mutations were found in 29, including 14 (48%) only from tissue and 5 (17%) only from cfDNA. Twenty-four of them were treated with osimertinib, and progression-free survival was longer in patients without detectable T790M in cfDNA than in patients with detectable T790M in cfDNA (p = 0.02). For the 31 T790M-negative patients, there were six with mesenchymal-epithelial transition factor (MET) amplifications, four with ERBB2 amplifications, and one with CCDC6-RET fusion. One with MET amplification and one with ERBB2 amplification responded to subsequent MET and ERBB2 targeting agents respectively. CONCLUSIONS: NGS after EGFR-TKI resistance may detect targetable drivers besides T790M. To do either liquid or tissue NGS only could miss patients with T790M. To do tissue and liquid NGS in parallel after EGFR-TKI resistance may find more patients with targetable cancers.

Regulation of ROS/inflammasome Axis is Essential for Cardiac Regeneration in Aging Rats Receiving Transplantation of Mesenchymal Stem Cells.

BACKGROUND: Aging is a biological and gradual deterioration of function in living organisms. Aging is one of the risk factors for heart disease. OBJECTIVE: Although mesenchymal stem cell transplantation shows potential in heart disease treatment, the relationship between stem cell-based therapy and oxidative stress/inflammasome axis regulation remains unclear. This study hypothesized that intervention of stem cells showed a protective effect on heart aging induced by D-galactose through regulation of oxidative stress/inflammasome axis. METHODS: An aging animal model was designed to test the above hypothesis. Experimental animals were divided into three groups, including Sham, D-gal (aging rats induced by d-galactose), and D-gal+WJSC (aging rats receiving mesenchymal stem cells). RESULTS: Compared to the Sham, the experimental results indicate that structural alteration (HE stain and Masson's Trichrome stain), oxidative stress elevation (increase of TBARS level, expression of gp-91 and suppression of Sirt-1 as well as SOD2), increase of aging marker p53, suppression of cardiogenesis marker Troponin T, and inflammasome related protein markers expression (NLRP3, caspase-1 and IL-1 beta) were significantly observed in D-gal. In contrast, all pathological pathways were significantly improved in D-gal+WJSC when compared to D-gal. In addition, migration of stem cells to aging heart tissues was observed in the D-gal+WJSC group. CONCLUSION: These findings suggest that mesenchymal stem cell transplantation effectively ameliorates aging hearts through oxidative stress/inflammasome axis regulation. The results from this study provide clinical potential for stem cell-based therapy in the treatment of aging hearts.

Upfront liquid next-generation sequencing in treatment-naïve advanced non-small cell lung cancer patients: A prospective randomised study in the Taiwanese health system.

BACKGROUND: Next-generation sequencing (NGS) of plasma cell-free DNA identifies driver mutations in advanced non-small cell lung cancer (NSCLC) and may complement routine molecular evaluation. The utility of liquid NGS at the start of tumour workup is undetermined. METHODS: This is a randomised study of patients with suspected advanced NSCLC. All patients received blood liquid NGS testing at their first clinic visit and underwent standard histological diagnosis and tissue genotyping, encompassing polymerase chain reaction based methods for EGFR mutation, immunohistochemical (IHC) staining for ALK fusion and BRAF V600E mutation, and an IHC screening followed by confirmation using fluorescence in situ hybridization confirmation for ROS1 fusion. They were then randomly assigned to receive NGS results either after tissue genotyping (Group A) or as soon as possible after histological diagnosis of advanced NSCLC (Group B). The study measured time to start of systemic treatment as the primary endpoint and secondary endpoints included biomarker discovery rate, objective response rate (ORR), and progression-free survival (PFS). RESULTS: This study enroled 180 patients with suspected advanced NSCLC, randomised into two groups. 63 patients in Group A and 59 in Group B with advanced NSCLC were confirmed as advanced NSCLC and analysed. Most had adenocarcinoma (Group A: 77.8%, Group B: 79.7%). The prevalence of EGFR mutations in the two groups was similar (Group A: 57.1%; Group B: 56.6%). Other driver alterations were rare. The median time to treatment was shorter in Group B (20 days) than in Group A (28 days). ORR and PFS did not differ between groups significantly. Liquid NGS had high concordance with tissue testing and identified driver mutations in 42.6% (20/47) of tissue-negative cases. CONCLUSION: Performing liquid NGS at the initial clinic visit for suspected advanced NSCLC identifies more patients suitable for targeted therapies and shortens time to the start of treatment.

Oral uracil-tegafur compared with intravenous chemotherapy as adjuvant therapy for resected early-stage non-small cell lung cancer patients.

BACKGROUND: Studies comparing the effectiveness of either adjuvant oral uracil-tegafur (UFT) or intravenous chemotherapy on early-stage (stage I and II) non-small cell lung cancer (NSCLC) patients treated with complete surgical treatment remain limited. METHODS: From January 2011 to December 2017, patients with early-stage NSCLC (defined as tumor size >3 cm without mediastinal lymph node involvement or any distant metastasis) receiving either adjuvant oral UFT or intravenous chemotherapy after surgical resection were identified from the Taiwan Cancer Registry. Overall survival (OS) and relapse-free survival (RFS) were the primary and secondary outcomes, respectively. Propensity matching was used for controlling confounders. RESULTS: A total of 840 patients receiving adjuvant therapy after surgery (including 595 oral UFT and 245 intravenous chemotherapy) were enrolled. Before matching, patients using oral UFT had significantly longer OS (HR: 0.69, 95% CI: 0.49-0.98, p = 0.0387) and RFS (HR: 0.79, 95% CI: 0.61-0.97, p = 0.0392) than those with intravenous chemotherapy. A matched cohort of 352 patients was created using 1:1 propensity score-matching. In the Cox regression analysis, the UFT and the matched chemotherapy groups had similar OS (HR: 0.80, 95% CI: 0.48-1.32, p = 0.3753) and RFS (HR: 0.98, 95% CI: 0.72-1.34, p = 0.9149). Among subgroup analysis, oral UFT use was associated with longer RFS among the subgroups of non-drinker (HR: 0.66, 95% CI: 0.34-0.99, p = 0.0478) and patients with stage IB disease (HR: 0.67, 95% CI: 0.42-0.97, p = 0.0341). CONCLUSIONS: This population-based study in the real-world setting of Taiwan demonstrates comparable effectiveness between oral UFT and intravenous chemotherapy in terms of clinical outcomes for early-stage NSCLC patients after surgery.

A 3D culture system for evaluating the combined effects of cisplatin and anti-fibrotic drugs on the growth and invasion of lung cancer cells co-cultured with fibroblasts.

Fibrosis and fibroblast activation usually occur in the tissues surrounding a malignant tumor; therefore, anti-fibrotic drugs are used in addition to chemotherapy. A reliable technique for evaluating the combined effects of anti-fibrotic drugs and anticancer drugs would be beneficial for the development of an appropriate treatment strategy. In this study, we manufactured a three-dimensional (3D) co-culture system of fibroblasts and lung cancer cell spheroids in Matrigel supplemented with fibrin (fibrin/Matrigel) that simulated the tissue microenvironment around a solid tumor. We compared the efficacy of an anticancer drug (cisplatin) with or without pretreatments of two anti-fibrotic drugs, nintedanib and pirfenidone, on the growth and invasion of cancer cells co-cultured with fibroblasts. The results showed that the addition of nintedanib improved cisplatin's effects on suppressing the growth of cancer cell spheroids and the invasion of cancer cells. In contrast, pirfenidone did not enhance the anticancer activity of cisplatin. Nintedanib also showed higher efficacy than pirfenidone in reducing the expression of four genes in fibroblasts associated with cell adhesion, invasion, and extracellular matrix degradation. This study demonstrated that the 3D co-cultures in fibrin/Matrigel would be useful for assessing the effects of drug combinations on tumor growth and invasion.

Monitoring of T790M in plasma ctDNA of advanced EGFR-mutant NSCLC patients on first- or second-generation tyrosine kinase inhibitors.

BACKGROUND: The T790M mutation is the major resistance mechanism to first- and second-generation TKIs in EGFR-mutant NSCLC. This study aimed to investigate the utility of droplet digital PCR (ddPCR) for detection of T790M in plasma circulating tumor DNA (ctDNA), and explore its impact on prognosis. METHODS: This prospective study enrolled 80 advanced lung adenocarcinoma patients treated with gefitinib, erlotinib, or afatinib for TKI-sensitizing mutations between 2015 and 2019. Plasma samples were collected before TKI therapy and at tri-monthly intervals thereafter. Genotyping of ctDNA for T790M was performed using a ddPCR EGFR Mutation Assay. Patients were followed up until the date of death or to the end of 2021. RESULTS: Seventy-five of 80 patients experienced progressive disease. Fifty-three (71%) of 75 patients underwent rebiopsy, and T790M mutation was identified in 53% (28/53) of samples. Meanwhile, plasma ddPCR detected T790M mutation in 23 (43%) of 53 patients. The concordance rate of T790M between ddPCR and rebiopsy was 76%, and ddPCR identified 4 additional T790M-positive patients. Ten (45%) of 22 patients who did not receive rebiopsy tested positive for T790M by ddPCR. Serial ddPCR analysis showed the time interval from detection of plasma T790M to objective progression was 1.1 (0-4.1) months. Compared to 28 patients with rebiopsy showing T790M, the overall survival of 14 patients with T790M detected solely by ddPCR was shorter(41.3 [95% CI, 36.6-46.0] vs. 26.6 months [95% CI, 9.9-43.3], respectively). CONCLUSION: Plasma ddPCR-based genotyping is a useful technology for detection and monitoring of the key actionable genomic alteration, namely, T790M, in patients treated with gefitinib, erlotinib, or afatinib for activating mutations, to achieve better patient care and outcome.

Real-world treatment patterns and outcomes among patients with advanced non-small-cell lung cancer with spindle cell and/or giant cell carcinoma.

Objectives: A definitive diagnosis of pulmonary sarcomatoid carcinoma cannot be made with small biopsies. In clinical practice, a diagnosis of advanced non-small-cell lung cancer with spindle cell and/or giant cell carcinoma (NSCLCsg), or possible sarcomatoid carcinoma, is acceptable. Therefore, we aimed to investigate the treatment patterns and outcomes of advanced NSCLCsg. Materials and methods: Between 01 January 2012 and 01 April 2021, patients with pathologically proven advanced NSCLCsg were enrolled. The choice of treatment was based on clinician discretion. Results: In all, 101 patients with advanced NSCLCsg were enrolled. In total, 77 (76.2%) patients received at least one line of systemic therapy; 44 patients (43.1%) had received platinum doublet chemotherapy; 27 (26.7%) patients had been treated with targeted therapies; and 23 patients (22.8%) had been given an immune checkpoint inhibitor (ICI). The median overall survival (OS) was 6.3 months [95% confidence interval (CI): 3.6-9.0 months]. Excluding patients without systemic therapy, patients who had received an ICI had better OS (median: 18.2 months) than those who had not (median 3.8 months, log-rank test p = 0.002). No significant difference in OS was detected between patients who had or had not received platinum doublet chemotherapy (log-rank test p = 0.279), or targeted therapy (log-rank test p = 0.416). Having received any systemic therapy [hazard ratio (HR): 0.33, 95% CI: 0.18-0.61, p < 0.0001) and ICI (HR: 0.38, 95% CI: 0.19-0.78, p = 0.008) were independent factors for better OS. Patients with programmed death ligand-1 (PD-L1) expression ⩾50% had better OS than those with PD-L1 expression <50% (HR: 0.51, 95%: 0.30-0.86, p = 0.012). Conclusion: Although advanced NSCLCsg has a poor survival outcome, our results showed that ICI may prolong OS in patients with advanced NSCLCsg. Further prospective studies are warranted to gain more understanding of the role of ICI in this specific patient population.

Mesenchymal Stem Cell Transplantation Increases Antioxidant Protein Expression and Ameliorates GP91/ROS/Inflammasome Signals in Diabetic Cardiomyopathy.

BACKGROUND: Cardiomyopathy is one of the complications associated with diabetes. Due to its high prevalence, diabetic cardiomyopathy has become an urgent issue for diabetic patients. Various pathological signals are related to diabetic cardiomyopathy progress, including inflammasome. Mesenchymal stem cell transplantation is full of potential for the treatment of diabetic cardiomyopathy because of stem cell cardiac regenerative capability. This study investigates whether mesenchymal stem cell transplantation shows therapeutic effects on diabetic cardiomyopathy through inflammasome signaling regulation. METHODS: Wistar male rats were divided into three groups including Sham, T1DM (rats with type 1 diabetes) and T1DM + WJSC (T1DM rats receiving 1 × 106 stem cells per rat). RESULTS: Compared to the Sham, experimental results indicated that several pathological conditions can be observed in heart tissues with T1DM, including structural change, fibrosis, oxidative stress elevation and inflammasome related protein expression. All of these pathological conditions were significantly improved in T1DM rats receiving mesenchymal stem cell transplantation (T1DM + WJSC). Furthermore, the experimental findings suggest that mesenchymal stem cell transplantation exerted antioxidant protein expression in diabetic heart tissues, resulting in a decrease in oxidative stress and inflammasome signaling blockage. CONCLUSION: These findings imply that mesenchymal stem cell transplantation shows therapeutic effects on diabetic cardiomyopathy through inflammasome regulation induced by oxidative stress.

Paracrine IGF-1 Activates SOD2 Expression and Regulates ROS/p53 Axis in the Treatment of Cardiac Damage in D-Galactose-Induced Aging Rats after Receiving Mesenchymal Stem Cells.

Aging is one of the causative agents associated with heart failure. Cell-based therapies show potential in the treatment of cardiac aging due to the characteristics of stem cells, including differentiation and the paracrine effect. This study aimed to investigate in detail the mechanism related to biomolecules released from mesenchymal stem cells in the treatment of cardiac aging. In vitro and in vivo models were designed to explore the above hypothesis. Experimental results from the in vitro model indicated that the elevation of oxidative stress, the expression of aging marker p53, and the suppression of antioxidant marker SOD2 could be found in D-galactose-stressed H9c2 cardiomyoblasts. The co-culture of D-galactose-stressed H9c2 with mesenchymal stem cells significantly improved the above pathological signaling. An animal model revealed that the change in cardiac structure, the accumulation of fibrotic collagen, and the activation of the above pathological signaling could be observed in heart tissues of D-galactose-stressed rats. After the rats had received mesenchymal stem cells, all the pathological conditions were significantly improved in D-galactose-stressed hearts. Further evidence indicated that the release of the survival marker IGF-1 was detected in a stem-cell-conditioned medium. Significant increases in cell viability and the expression of SOD2, as well as a reduction in oxidative stress and the suppression of p53, were found in D-galactose-stressed H9c2 cells cultured with a stem-cell-conditioned medium, whereas the depletion of IGF-1 in stem-cell-conditioned medium diminished the antiaging effect on H9c2 cells. In conclusion, the paracrine release of IGF-1 from mesenchymal stem cells increases the expression of antioxidant marker SOD2, and the expression of SOD2 reduces oxidative stress as well as suppresses p53, leading to a reduction in cardiac senescence in D-galactose-stressed rats.

Adipose-Derived Stem Cells Preincubated with Green Tea EGCG Enhance Pancreatic Tissue Regeneration in Rats with Type 1 Diabetes through ROS/Sirt1 Signaling Regulation.

Type 1 diabetes stem-cell-based therapy is one of the best therapeutic approaches for pancreatic damage treatment due to stem cell tissue regeneration. Epigallocatechin gallate (EGCG) is one of the active components found in green tea. Experimental results suggest that EGCG shows beneficial effects on cell protection. This study explores whether a better pancreatic regeneration therapeutic effect could be found in mesenchymal stem cells pretreated with EGCG compared to stem cells without EGCG pretreatment. A cell model confirmed that adipose-derived stem cells (ADSC) incubated with EGCG increase cell viability under high-glucose (HG) stress. This is due to survival marker p-Akt expression. In an animal model, type 1 diabetes induced the activation of several pathological signals, including islet size reduction, extracellular fibrotic collagen deposition, oxidative stress elevation, survival pathway suppression, apoptosis signaling induction, and Sirt1 antioxidant pathway downregulation. Ordinary ADSC transplantation slightly improved the above pathological signals. Further, EGCG-pretreated ADSC transplantation significantly improved the above pathological conditions. Taken together, EGCG-pretreated ADSCs show clinical potential in the treatment of patients with type 1 diabetes through the regeneration of damaged pancreatic tissues.

Serial Plasma Cell-Free Circulating Tumor DNA Tests Identify Genomic Alterations for Early Prediction of Osimertinib Treatment Outcome in EGFR T790M-Positive NSCLC.

INTRODUCTION: Recent advances in the detection of genomic DNA from plasma samples allow us to follow tumor DNA shedding in plasma during systemic treatment. Osimertinib is the standard of care for patients with NSCLC with acquired EGFR T790M mutations. We assessed changes in serial plasma cell-free circulating tumor DNA (ctDNA) genomic alterations to predict osimertinib efficacy. METHODS: We prospectively collected plasma from patients having EGFR-mutated advanced NSCLC previously treated with EGFR tyrosine kinase inhibitor therapy and with acquired EGFR T790M mutation detected by standard methods. Plasma samples were collected before starting osimertinib treatment, 4 weeks after osimertinib treatment, and on progression. ctDNA was analyzed using the Guardant360 assay. RESULTS: A total of 15 eligible patients received osimertinib. Before starting treatment, EGFR-activating mutations were detected in the ctDNA of all patients, and EGFR T790M was detected in 93% of the cases. Osimertinib treatment was associated with an objective response rate of 53% and a median progression-free survival of 7.3 months. A total of 12 of the 15 patients had undetectable plasma T790M and decreased activating mutation allelic frequency (AF) at week 4. None of the 12 patients had disease progression within 16 weeks. For the remaining three patients, with detectable plasma T790M (n = 2) or increased activating mutation AF (n = 1) at week 4, two had progressive disease within 16 weeks (p = 0.03). CONCLUSIONS: In patients with EGFR-mutated advanced NSCLC, persistent EGFR T790M or increasing activating mutation AF as detected in ctDNA 4 weeks after the start of osimertinib treatment may predict disease progression within 16 weeks.

Prognostic Characteristics and Immunotherapy Response of Patients With Nonsquamous NSCLC With Kras Mutation in East Asian Populations: A Single-Center Cohort Study in Taiwan.

INTRODUCTION: Kras mutation is the most common driver oncogene present in patients with NSCLC. Recently, the precision medicine for patients with Kras-mutated NSCLC has been under investigation, but the best treatment is still unknown. This study aimed to analyze the clinical characteristics, immune checkpoint inhibitor (ICI) response, and prognostic factors of patients with NSCLC with different Kras mutation subtypes. METHODS: From 2005 to 2018, we collected nonsquamous NSCLC tissue samples for Kras mutation analysis using direct Sanger sequencing or MassARRAY genotyping (Agena Bioscience, San Diego, CA) at the National Taiwan University Hospital. Clinical characteristics, ICI treatment effectiveness, time-to-tumor recurrence (TTR), and overall survival (OS) were analyzed using multivariate Cox models, to estimate adjusted hazard ratios (HRs). RESULTS: Among 5278 patients with nonsquamous NSCLC, 246 (4.7%) had Kras mutations. The major Kras mutation subtypes were G12C (32.9%), G12D (23.7%), and G12V (18.9%). Patients with Kras-G12C had a higher proportion of male individuals (p = 0.018) and smokers (p < 0.001). Among the 25 patients treated with ICIs, patients with Kras-G12C had a higher response rate (53.8% versus 8.3%, p = 0.030) and longer progression-free survival (4.8 mo versus 2.1 mo, p = 0.028) than those with Kras-non-G12C. For the 85 patients with early-stage NSCLC, those with G12C had shorter TTR (22.8 mo) than those with Kras-non-G12C (97.7 mo, p = 0.004). For the 143 patients with advanced-stage NSCLC, there was a significant difference in OS between patients with Kras-G12C and Kras-non-G12C (7.7 mo versus 6.0 mo, p = 0.018) and patients with Kras-G12V had the shortest OS (5.2 mo). Multivariate analysis revealed association of shorter OS with Kras-G12V (HR = 2.47, p = 0.002), stage IV disease status (HR = 2.69, p = 0.008), and NSCLC-not otherwise specified histology (HR = 3.12, p = 0.002). CONCLUSIONS: Kras-G12C was associated with favorable ICI treatment effectiveness in patients with NSCLC. Kras-G12C mutation was associated with shorter TTR in patients with early-stage NSCLC, and Kras-G12V mutation was associated with shorter OS in patients with advanced-stage NSCLC when comparing with Kras-G12C.

The relative importance of predictive factors for single first-generation EGFR-TKI use for more than 5 years in patients with advanced non-small cell lung cancer: Taiwan multicenter TIPS-5 study.

BACKGROUND: The relative importance of predictive factors for advanced non-small cell lung cancer (NSCLC) patients on epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) treatment remains unclear. MATERIALS AND METHODS: We retrospectively enrolled advanced NSCLC patients with single first-generation EGFR-TKI treatment for ⩾5 years (Y) in Taiwan. Clinical data was collected and compared with those of another cohort with single first-line EGFR-TKI treatment for <5 Y. Plasma cell-free DNA (cfDNA) samples were collected from patient subsets, pre- and post-TKI, in the >5 Y group. RESULTS: Overall, 128 and 278 patients were enrolled in the ⩾5 Y and <5 Y groups, respectively. Significant factors in the multivariate analysis of patients' characteristics including Eastern Cooperative Oncology Group performance status 0-1, postoperative recurrence, without brain metastasis, oligometastasis (each score of 2), female sex, erlotinib use, and without bone metastasis (each score of 1), were incorporated into a risk scoring system. The area under the receiver operating characteristic curve was 0.82 [95% confidence interval (CI): 0.78-0.86]. Of the plasma cfDNA samples from 33 patients in the ⩾5 Y group, only 1 had a T790M in 25 patients without progressive disease. In 27 patients with single agent use for ⩾96 months, 22 (81.5%) received local treatment (surgery or radiotherapy) for the primary lung tumor before and during TKI treatment. CONCLUSION: For NSCLC patients with single first-generation EGFR-TKI use for ⩾5 Y, factors with different relative importance exist and the risk-scoring model is feasible with modest accuracy. The role of local treatment for primary tumors in patients with long-term TKI use requires further investigation.

Monitoring levels of vimentin-positive circulating cancer stem cells and tumor cells in patients with advanced EGFR-mutated non-small cell lung cancer.

OBJECTIVES: Circulating tumor cells (CTCs) are associated with tumor spread, whereas cancer stem cells may be related to drug resistance. However, few studies have analyzed the levels of circulating cancer stem cells (CCSCs) and CTCs in patients with advanced non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: Treatment-naïve patients with EGFR-mutated NSCLC who received epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapy were recruited prospectively. The cell surface vimentin antibody was used for CTC detection and CD133 antibody for CCSC detection. CCSC and CTC levels were measured as cell count per 4 mL of blood, before treatment, after 2 and 12 weeks of treatment, and at disease progression. Data on clinical characteristics and outcomes were also collected. RESULTS: At diagnosis (n = 29), the median CCSC and CTC levels were 0 (interquartile range, 0-2) and 3 (2-9), respectively. After 12 weeks, the CCSC and CTC levels were lower than those at diagnosis (CCSC: 0 (0-0), p = 0.14; CTC: 1 (0-4), p = 0.048). At disease progression, the median CCSC and CTC levels were 0 (0-1) and 1 (0-2), respectively. Patients with higher CCSC and CTC levels at diagnosis had a numerically shorter progression-free survival. CONCLUSION: In patients with EGFR-mutated NSCLC, CCSC and CTC levels became lower after 12 weeks of EGFR-TKI therapy and remained low at disease progression. High pre-treatment CCSC and CTC levels may be associated with a trend towards poor treatment outcomes.

Autophagy in fibroblasts induced by cigarette smoke extract promotes invasion in lung cancer cells.

We investigated the effects of cigarette smoke extract (CSE) on lung fibroblasts and found that the invasiveness of lung cancer cells was facilitated by the conditioned medium from CSE-treated fibroblasts. CSE induced autophagy in fibroblasts and increased the expression of autophagy-related proteins, including optineurin and Ras-related protein Rab1B. Afterward, the fibroblasts produced high levels of interleukin-8 (IL-8), which promoted cancer cell invasion. The inhibition of either optineurin or Rab1B abrogated a rise in microtubule-associated protein 1 light chain 3 ß and a decrease in p62 protein, as well as the production of IL-8, in CSE-treated fibroblasts. A three-dimensional invasion assay using cancer cell spheroids revealed that the invasion of cancer cells alone and the fibroblast-led cancer cell invasion were both enhanced by the conditioned media from CSE-treated fibroblasts. These results suggest that cigarette smoke may induce autophagy and IL-8 secretion in lung fibroblasts and modify the microenvironment to favor invasion of lung cancer cells.