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Background In a pilot study using both cannabidiol (CBD) and tetrahydrocannabinol (THC) as single agents in advanced cancer patients undergoing palliative care in Thailand, the doses were generally well tolerated, and the outcome measure of total symptom distress scores showed overall symptom benefit. The current study aims to determine the intensity of the symptoms experienced by breast cancer patients receiving chemotherapy, to explore the microbiome profile, cytokines, and bacterial metabolites before and after the treatment with cannabis oil or no cannabis oil, and to study the pharmacokinetics parameters and pharmacogenetics profile of the doses. Methods A randomized, double-blinded, placebo-controlled trial will be conducted on the metastatic breast cancer cases receiving chemotherapy at King Chulalongkorn Memorial Hospital (KCMH), Bangkok, Thailand. Block randomization will be used to allocate the patients into three groups: Ganja Oil (THC 2 mg/ml; THC 0.08 mg/drop, and CBD 0.02 mg/drop), Metta Osot (THC 81 mg/ml; THC 3 mg/drop), and placebo oil. The Edmonton Symptom Assessment System (ESAS), microbiome profile, cytokines, and bacterial metabolites will be assessed before and after the interventions.

Cisplatin (CDDP) induces senescence characterized by senescence-associated secretory phenotypes (SASP) and the unfolded protein response (UPR). In this study, we investigated the proteins related to the UPR during the senescence cell fate. Strikingly, we found that one of the critical ER-resident proteins, GRP78/BiP, was significantly altered. Here we show that GRP78 levels differentially expressed depending on non-small lung cancer subtypes. GRP78 indeed regulates the evasion of senescence in adenocarcinoma A549 cells, in which the increased GRP78 levels enable them to re-proliferate after CDDP removal. Conversely, GRP78 is downregulated in the senescence H460 cells, making them lacking senescence evasion capability. We observed that the translational regulation critically contributed to the GRP78 protein levels in CDDP-induces senescence. Furthermore, the increased GRP78 level during senescence confers resistance to senolytic drug, Bortezomib, as observed by a twofold increase in IC50 in A549 senescence cells compared to the wild-type. This observation is also consistent in the cells that have undergone genetic manipulation by transfection with pcDNA3.1(+)-GRP78/BiP plasmids and pSpCas9(BB)-2A-Puro containing guide RNA sequence targeting GRP78 exon 3 to induce the overexpression and downregulation of GRP78 in H460 cells, respectively. Our findings reveal a unique role of GRP78 on the senescence evasion cell fate and senolytic drug resistance after cisplatin-based chemotherapy.

Lung cancer remains a leading cause of death in cancer patients, and deregulation of apoptosis is a serious concern in clinical practice, even though therapeutic intervention has been greatly improved. Plants are a versatile source of biologically active compounds for anticancer drug discovery, and aspiletrein A (AA) is a steroidal saponin isolated from Aspidistra letreae that has a potent cytotoxic effect on various cancer cell lines. In this study, we investigated and determined the underlying molecular mechanism by which AA induces apoptosis. AA strongly induced apoptosis in NSCLC cells by mediating ROS generation and thereby activating AMP-activated protein kinase (AMPK) signaling. Consequently, downstream signaling and levels of phosphorylated mTOR and Bcl-2 were significantly decreased. Pretreatment with either an antioxidant, N-acetylcysteine, or an AMPK inhibitor, compound C, could reverse the apoptosis-inducing effect and counteract the effect of AA on the AMPK signaling pathway. Decreased levels of Bcl-2 were due to AA-mediating Bcl-2 degradation via a ROS/AMPK/mTOR axis-dependent proteasomal mechanism. Consistently, the apoptotic-inducing effect of AA was also observed in patient-derived malignant lung cancer cells, and it suppressed an in vitro 3D-tumorigenesis. This study identified the underlying mechanism of AA on lung cancer apoptosis, thereby facilitating potential research and development of this compound for further clinical implications.

The posttranslational modifications (PTMs) of microtubules have been reported to play an important role in cancer aggressiveness, including apoptosis resistance. In this study, we aimed to investigate the biological role of microtubule PTMs in the regulation of paclitaxel responsiveness. The acetylated tubulin (Ace-tub) level was strongly associated with paclitaxel sensitivity, as observed in patient-derived primary lung cancer cells and xenografted immunodeficient mice. We showed that paclitaxel-resistant H460 lung cancer cells, generated by a stepwise increase in paclitaxel, exhibited markedly increased tubulin acetylation and consequently acquired paclitaxel resistance. Upregulation of tubulin acetylation by overexpression of α-tubulin acetyltransferase 1 wild-type (αTAT1wt), an enzyme required for acetylation, or by treatment with trichostatin A (TSA), a histone deacetylase 6 (HDAC6) inhibitor, significantly attenuated paclitaxel-induced apoptosis. Investigation of the underlying mechanism revealed that the levels of antiapoptotic Mcl-1 appeared to increase in αTAT1wt-overexpressing and TSA-treated cells compared to control cells, whereas the levels of other antiapoptotic regulatory proteins were unchanged. On the other hand, decreased tubulin acetylation by αTAT1 RNA interference downregulated Mcl-1 expression in patient-derived primary lung cancer and paclitaxel-resistant lung cancer cells. A microtubule sedimentation assay demonstrated that Mcl-1 binds to microtubules preferentially at Ace-type, which prolongs the Mcl-1 half-life (T1/2). Furthermore, immunoprecipitation analysis revealed that polyubiquitination of Mcl-1 was extensively decreased in response to TSA treatment. These data indicate that tubulin acetylation enhances the resistance to paclitaxel-induced cell death by stabilizing Mcl-1 and protecting it from ubiquitin–proteasome-mediated degradation.

BACKGROUND: Despite advances in systemic therapy and improvements in survival for advanced epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC), brain metastasis (BM) remains a poor outcome. Previous studies on risk factors for BM occurrence included unselected patients and biomarker prediction of BM in these populations were not well studied. We aimed to identify the role of epithelial mesenchymal transition (EMT) marker and clinical factors predicting BM in EGFR: mutant NSCLC patients. METHODS: Advanced EGFR-mutant NSCLC patients in the King Chulalongkorn Memorial Hospital from January 2013 to December 2017 were included. Vimentin expression was assessed by immunohistochemistry. The correlation between vimentin expression and factors associated with BM occurrence was analyzed by univariate and multivariate analyses. RESULTS: 304 patients were enrolled. Of these, 149 patients (49%) developed BM. In multivariate analysis, the occurrence of BM was associated with age

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+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, pp=0.0005], and 1585 [478-5224] vs. 758.5 [318-1976, p+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.

Background Calcium is an essential signal transduction element that has been associated with aggressive behaviours in several cancers. Cell motility is a prerequisite for metastasis, the major cause of lung cancer death, yet its association with calcium signalling and underlying regulatory axis remains an unexplored area. Methods Bioinformatics database analyses were employed to assess correlations between calcium influx channels and clinical outcomes in non-small cell lung cancer (NSCLC). Functional and regulatory roles of influx channels in cell migration and invasion were conducted and experimental lung metastasis was examined using in vivo live imaging. Results High expression of TRPM7 channel correlates well with the low survival rate of patients and high metastatic potential. Inhibition of TRPM7 suppresses cell motility in various NSCLC cell lines and patient-derived primary cells and attenuates experimental lung metastases. Mechanistically, TRPM7 acts upstream of O-GlcNAcylation, a post-translational modification and a crucial sensor for metabolic changes. We reveal for the first time that caveolin-1 and c-Myc are favourable molecular targets of TRPM7/O-GlcNAc that regulates NSCLC motility. O-GlcNAcylation of caveolin-1 and c-Myc promotes protein stability by interfering with their ubiquitination and proteasomal degradation. Conclusions TRPM7/O-GlcNAc axis represents a potential novel target for lung cancer therapy that may overcome metastasis.

Aberrant c-Myc is a common feature in the majority of human cancers and has been linked to oncogenic malignancies. As a profound activator of aggressive lung cancer, targeting c-Myc would lead to the better clinical outcome. Here we develop a novel c-Myc targeted compound N, N-bis (5-ethyl-2-hydroxybenzyl) methylamine (EMD) and present evidence demonstrating its effectiveness in targeting c-Myc for degradation in human lung carcinoma. EMD exhibited strong cytotoxicity towards various human lung cancer cell lines ae well as chemotherapeutic-resistant patient-derived lung cancer cells through apoptosis induction, in comparison to chemotherapeutic drugs. Mechanistically, EMD eliminates c-Myc in the cells and initiates caspase-dependent apoptosis cascade. Cycloheximide chase assay revealed that EMD significantly shortened c-Myc half-life by approximately two-fold. Cotreatment of EMD with proteasome inhibitor MG132 reversed its c-Myc targeting effect, suggesting the involvement of ubiquitin-mediated proteasomal degradation in the process. We further verified that EMD strongly induced the ubiquitination of c-Myc that prompted it for protein degradation. c-Myc inhibition and apoptosis induction were additionally shown in hematologic malignant K562, indicating the generality of the observed EMD effects. Altogether, we have identified EMD as a novel potent compound targeting oncogenic c-Myc, which may offer new opportunities for lung cancer treatment. SIGNIFICANCE STATEMENT Deregulation of c-Myc is frequently associated with cancer progression. This study examined the effect of a new compound EMD in targeting c-Myc in several lung cancer cell lines and drug-resistant primary lung cancer cells. EMD induced dramatic c-Myc degradation through ubiquitin-proteasomal mechanism. The promising anti-cancer and c-Myc targeted activities of EMD support its potential new approaches to treat c-Myc driven cancer.

Lymph node involvement is one of the important prognostic factors for early-stage lung cancer. However, in lymph node-negative (N0) lung cancer the recurrent rate may be as high as 30%. We aimed to study potential prognostic factors including clinicopathological factors and epidermal growth factor receptor (EGFR) mutation status in this lung cancer population.We retrospectively reviewed the medical records and pathological examinations of patients with completely resected N0 pulmonary adenocarcinoma treated in our institute between 2009 and 2016. We used CobasWe recruited 220 patients with median duration of follow up 5 years. Majority of these patients were in stage I (80%) and did not receive adjuvant therapy (86%). There were 53% with EGFR mutations which comprised of exon 19 deletion 51% and L858R 43%. Recurrence occurred in 64 out of 220 patients (29%). The median time to recurrence was 2.1 years. Statistically significant prognostic factors in both univariate and multivariate analyses included tumor size ≥4 centimeter (cm) (HR: 1.94; 95% CI: 1.03-3.67), visceral pleural invasion (HR: 2.53; 95% CI: 1.34-4.79), tumor necrosis (HR: 2.45; 95% CI: 1.13-5.31) and bronchial resection margin2 cm (HR: 1.96; 95% CI: 1.10-3.51). However, presence of sensitizing EGFR mutation was not found to be a significant prognostic factor (HR: 1.20; 95% CI: 0.66-2.18; P=0.56).In N0 surgically resected lung adenocarcinoma, there were significant pathological prognostic factors including tumor 4 cm or more, visceral pleural invasion, tumor necrosis and bronchial resection margin less than 2 cm. Mutation of EGFR is not a significant prognostic factor to determine the risk of recurrence in this population and their risks shall be determined by the other poor prognostic factors.

Metal nanomaterials can enhance the efficacy of current cancer therapies. Here, we show that Ti0.8O2 nanosheets cause cytotoxicity in several lung cancer cells but not in normal cells. The nanosheet-treated cells showed certain apoptosis characteristics. Protein analysis further indicated the activation of the p53-dependent death mechanism. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses revealed the cellular uptake of the nanosheets and the induction of cell morphological change. The nanosheets also exhibited a substantial apoptosis effect on drug-resistant metastatic primary lung cancer cells, and it was found that the potency of the nanosheets was dramatically higher than standard drugs. Ti0.8O2 nanosheets induce apoptosis through a molecular mechanism involving peroxynitrite (ONOO−) generation. As peroxynitrite is known to be a potent inducer of S-nitrosylation, we further found that the nanosheets mediated the S-nitrosylation of p53 at C182, resulting in higher protein-protein complex stability, and this was likely to induce the surrounding residues, located in the interface region, to bind more strongly to each other. Molecular dynamics analysis revealed that S-nitrosylation stabilized the p53 dimer with a ΔGbindresidue of &lt, −1.5 kcal/mol. These results provide novel insight on the apoptosis induction effect of the nanosheets via a molecular mechanism involving S-nitrosylation of the p53 protein, emphasizing the mechanism of action of nanomaterials for cancer therapy.

Alpha folate receptor (FRα) is currently under investigation as a target for the treatment of patients with non-small-cell lung cancer (NSCLC), since it is highly expressed in tumor cells but is largely absent in normal tissue. In this study, a novel human variable domain of a heavy-chain (VH) antibody fragment specific to FRα was enriched and selected by phage bio-planning. The positive phage clone (3A102 VH) specifically bound to FRα and also cross-reacted with FRβ, as tested by ELISA. Clone 3A102 VH was then successfully expressed as a soluble protein in an E. coli shuffle strain. The obtained soluble 3A102 VH demonstrated a high affinity for FRα with affinity constants (Kaff) values around 7.77 ± 0.25 × 107 M−1, with specific binding against both FRα expressing NSCLC cells and NSCLC patient-derived primary cancer cells, as tested by cell ELISA. In addition, soluble 3A102 VH showed the potential desired property of a targeting molecule by being internalized into FRα-expressing cells, as observed by confocal microscopy. This study inspires the use of phage display to develop human VH antibody (Ab) fragments that might be well suited for drug targeted therapy of NSCLC and other FRα-positive cancer cells.

Background/aim Individualized proper chemotherapy using in vitro drug sensitivity testing has been proposed as a novel therapeutic modality and shown to have better efficacy than empiric chemotherapy. However, issues around establishing a patient-derived cell culture or xenograft, the timing of the testing obtained, and the validity of testing represent major limitations to translating the use of such a technique to clinical practice. Patients and methods In this study, we assessed the feasibility of an in vitro drug sensitivity technique for testing malignant pleural effusion from advanced-stage non-small cell lung cancer. Results Our technique was able to produce a turnaround time for in vitro drug sensitivity testing of less than 1 week, with a success rate of more than 90% of cases. Correlated with the individual clinical outcome, using the area under the dose response curve (AUC) could define the level of in vitro drug sensitivity as: responsive (AUC>0.25), intermediate response (0.1≤AUC≤0.25), or resistance (AUC Conclusion Data obtained from this method of drug testing were correlated with the clinical outcome. The present drug sensitivity evaluation may benefit the development of individual precision chemotherapy.

Background There has been a sharp rise in the incidence of human papillomavirus (HPV) associated oropharyngeal squamous cell carcinoma (OPSCC) in many countries. Patients with HPV-positive OPSCC have a more favorable prognosis compared with HPV-negative OPSCC, leading to investigation and adoption of de-escalation treatment protocols. The baseline rate of HPV prevalence in certain populations is of epidemiologic significance. We aimed to evaluate the rate of high-risk HPV in a large cohort of Thai patients, including OPSCC, oral SCC (OSCC) and laryngeal SCC (LSCC). Methods In total, 504 patients with HN cancer (110 OPSCC, 260 OSCC and 134 LSCC) who had been treated in Chulalongkorn University between 2010 and 2016 formed the sample set. All histological slides were reviewed to validate the diagnosis and render the histological type as keratinizing (K), non-keratinizing (NK) or non-keratinizing with maturation (NK-M). Immunohistochemistry with p16 was performed in all cases and scored semiquantatively. Positive and equivocal cases were tested by the high-risk HPV DNA in situ hybridization (ISH). Validation with quantitative polymerase-chain reaction (qPCR) was performed in p16-positive OPSCC. Results The OPSCC were represented by NK (7.3%), NK-M (16.4%) and K (76.4%) types, with an HPV incidence of 100, 22.2 and 4.7%, respectively. The average HPV prevalence in OPSCC was 14.5%. The concordance with p16/ISH was 51.6%, while concordance of the NK morphology with positive HPV ISH was 100%. ISH-qPCR concordance in p16-positive OPSCC was 72.7%. Patients with HPV-positive OPSCC had significantly more tumors with a NK histologic type, tonsillar location, earlier clinical stage, less association with smoking, and, finally, better outcome and longer survival time. In non-OPSCC, p16-positive HPV-associated cancers were found in only 1.5% of OSCC (4/260) and LSCC (2/134). Conclusion A low rate of HPV-related OPSCC was found in Thai patients. The NK morphology was an excellent predictor of high-risk HPV infection in OPSCC. For OPSCC patients, HPV-positive ones had a significantly longer survival time than HPV-negative ones. There was a lack of p16-positive HPV-related OSCC and LSCC. Morphology and p16 status had a poor predictive value for detecting HPV in OSCC and LSCC.

Background: Metal oxide nanomaterials are increasingly being exploited in cancer therapy thanks to their unique properties, which can enhance the efficacy of current cancer therapies. However, the nanotoxicity and mechanism of Ti0.8O2 nanosheets for specific site-targeting strategies in NSCLC have not yet been investigated.Methods: The effects of Ti0.8O2 nanosheets on cytotoxicity in NSCLC cells and normal cells were examined. The apoptosis characteristics, including condensed and fragmented nuclei, as assessed by positive staining with annexin V. The cellular uptake of the nanosheets and the induction of stress fiber were assessed via transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses, respectively. We also evaluated the expression of protein in death mechanism to identify the molecular mechanisms behind the toxicity of these cells. We investigated the relationship between S-nitrosylation and the increase in p53 stability by molecular dynamics.Results: Ti0.8O2 nanosheets caused cytotoxicity in several lung cancer cells, but not in normal cells. The nanosheets could enter lung cancer cells and exert an apoptosis induction. Results for protein analysis further indicated the activation of p53, increased Bax, decreased Bcl-2 and Mcl-1, and activation of caspase-3. The nanosheets also exhibited a substantial apoptosis effect in drug-resistant metastatic primary lung cancer cells, and it was found that the potency of the nanosheets was dramatically higher than that of cisplatin and etoposide. In terms of their mechanism of action, we found that the mode of apoptosis induction was through the generation of cellular ONOO− mediated the S-nitrosylation of p53 at C182. Molecular dynamics analysis further showed that the S-nitrosylation of one C182 stabilized the p53 dimer. Consequently, this nitrosylation of the protein led to an upregulation of p53 through its stabilization.Conclusions: Taking all the evidence together, we provided information on the apoptosis induction effect of the nanosheets through a molecular mechanism involving reactive nitrogen species, which affects the protein stability; thus emphasizing the novel mechanism of action of nanomaterials for cancer therapy.

Focal adhesion kinase (FAK) controls several cancer aggressive potentials of cell movement and dissemination. As epithelial–mesenchymal transition (EMT) and the migratory-associated integrins, known influencers of metastasis, have been found to be linked with FAK activity, this study unraveled the potential pharmacological effect of artocarpin in targeting FAK resulting in the suppression of EMT and migratory behaviors of lung cancer cells. Treatment with artocarpin was applied at concentrations of 0–10 μM, and the results showed non-cytotoxicity in lung cancer cell lines (A549 and H460), normal lung (BEAS-2B) cells and primary metastatic lung cancer cells (ELC12, ELC16, and ELC20). We also found that artocarpin (0–10 µM) had no effect on cell viability, proliferation, and migration in BEAS-2B cells. For metastasis-related approaches, artocarpin significantly inhibited cell migration, invasion, and filopodia formation. Artocarpin also dramatically suppressed anchorage-independent growth, cancer stem cell (CSC) spheroid formation, and viability of CSC-rich spheroids. For molecular targets of artocarpin action, computational molecular docking revealed that artocarpin had the best binding affinity of −8.0 kcal/mol with FAK protein. Consistently, FAK-downstream proteins, namely active Akt (phosphorylated Akt), active mTOR (phosphorylated mTOR), and Cdc42, and EMT marker and transcription factor (N-cadherin, Vimentin, and Slug), were found to be significantly depleted in response to artocarpin treatment. Furthermore, we found the decrease of Caveolin-1 (Cav-1) accompanied by the reduction of integrin-αν and integrin-β3. Taken together, these findings support the anti-metastasis potentials of the compound to be further developed for cancer therapy.

12099 Background: Hypersensitivity reaction (HSR) is a common toxicity in patients receiving repeated oxaliplatin. Oxaliplatin induced HSR could be severe and results in treatment discontinuation. There is no standard recommendation for prevention of oxaliplatin induced HSR. Methods: We conducted a prospective, single center, open-label, randomized controlled trial comparing the standard premedication and 2-hour infusion protocol to the additional antihistamine premedication, intravenous chlorpheniramine, and 3-hour infusion protocol. After three months of chemotherapy, the randomization was done at 5thand 7th cycles of 3-week and 2-week oxaliplatin based regimens, respectively. The primary endpoint was the incidence of HSR. Results: From July 2020 to March 2021, a total of 160 patients underwent randomization (80 patients in both intervention and control groups). After 11 months follow-up, all patients had completed planned treatment cycles. HSRs occurred in 1 (1.3 %) and 10 (12.5%) patients in intervention and control groups, respectively, p = 0.005). There were 6 (7.5%) patients with more than grade 1 HSRs in control groups, but none in intervention group. In the intervention group, one patient with HSR experienced grade 1 erythema and palpitation. There is no report of significant adverse event from anti-histamine premedication or oxaliplatin infusion prolongation. Conclusions: Additional antihistamine premedication and infusion prolongation started in 2nd half of treatment course can significantly reduced HSR incidence in patients receiving oxaliplatin. Clinical trial information: TCTR20210506001.

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 p-value 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.

IntroductionThe mainstay systemic treatment for non-oncogenic addictive advanced stage non-small cell lung cancer is chemotherapy. Anti-angiogenic agents are additive compounds that enhance disease control and lead to improvement of overall survival benefit. Recently PD-(L)1 blockage, a checkpoint inhibitor, has been adopted as another line of treatment. A sequential strategy to enhance the efficacy of combination docetaxel and nintedanib after immunotherapy, correlated with genomic mutation, has been explored.MethodA retrospective cohort study of 56 patients from 8 centers in Thailand who received combination docetaxel and nintedanib via the Thai nintedanib Named Patient Use program was conducted. Demographic characteristics, treatment details, and treatment responses were retrieved from medical records.ResultsThe majority of patients were male (62.5%) with adenocarcinoma subtype (88%). Thirty-five percent had sensitizing EGFR mutation. Combination docetaxel and nintedanib was given as second to fourth line of treatment. Median PFS of docetaxel/nintedanib was 5.6 months [95% CI 4.8-6.9]. Median OS of the entire cohort was 22.5 months [95% CI 20.2-31.1]. Among them, only four patients received this combination after immunotherapy which limited the validity of efficacy analysis. Median PFS of those four patients was 7.9 months [range 5.2-9.1] which was slightly higher than the remaining cohort (median PFS 4.5 months, 95% CI: 4.0-6.0, p-value 0.09). Among the adenocarcinoma subtype, a relapse-time of platinum-doublet chemotherapy of more than 6 months was solely indicated as a benefit of combination docetaxel/nintedanib treatment compared to the relapse-time of platinum-doublet chemotherapy of less than 6 months by multivariate HR of PFS 0.32 [95% CI: 0.14-0.68, p-value 0.003].ConclusionCombination docetaxel and nintedanib provided more benefit in relapse-time of platinum-doublet chemotherapy of more than 6 months in advanced stage adenocarcinoma lung cancer. Neither EGFR nor ALK alteration influenced the outcome of treatment.

Aberrant cellular Myc (c-Myc) is a common feature in the majority of human cancers and has been linked to oncogenic malignancies. Here, we developed a novel c-Myc-targeting compound

Cancer stem cells (CSCs) are an important therapeutic target. The therapeutic agents targeting CSCs should lead to improved clinical outcomes. Here we have demonstrated the CSC-suppressing activity of pongol methyl ether (PME), a pure compound from Millettia erythrocalyx. Methods: CSC-suppressing effects were evaluated by spheroid formation assay and detection of CSC markers. The related CSC cell signals were evaluated by Western blot, immunofluorescence and molecular docking analysis. Proteins affected by PME treatment were subjected to bioinformatic analysis. Protein–protein interaction (PPI) networks were constructed by the Search Tool for Interactions of Chemicals (STITCH). The Kyoto Encyclopedia of Genes and Genomes (KEGG) mapper were used to confirm the underlying pathways. Results: PME (5–25 µM) significantly suppressed the ability of lung cancer cells to form colonies, grow in an anchorage-independent manner and generate tumour spheroids. PME at 25 µM significantly decreased the CSC markers (CD133 and ALDH1A1) and pluripotent transcription factors (Oct4 and Nanog). Akt, the key upstream signal of CSC control, was significantly decreased by the PME treatment. The molecular docking indicated that PME was bound to Akt-1 with a binding affinity of −9.2 kcal/mol greater than the Akt-1 inhibitor (reference compound, CQW). The STITCH network identified a total of 15 proteins interacted in PPI networks, and Akt-1 was identified as a central protein. The KEGG mapper indicated that the selected CSC markers were mostly involved in the ‘signalling pathways regulating pluripotency of stem cells’ pathway map and Akt, Oct4 and Nanog were the regulatory proteins in the dominant pathway. In addition, PME (10–25 µM) can suppress spheroid formation and reduce CSC-specific marker expression in patient-derived primary lung cancer cells. Conclusions: Our study revealed a novel pharmacological effect and the underlying mechanism of PME that can attenuate CSC phenotypes in lung cancer cells and may be developed for lung cancer therapy.

Individualized proper chemotherapy using in vitro drug sensitivity testing has been proposed as a novel therapeutic modality and shown to have better efficacy than empiric chemotherapy. However, issues around establishing a patient-derived cell culture or xenograft, the timing of the testing obtained, and the validity of testing represent major limitations to translating the use of such a technique to clinical practice.In this study, we assessed the feasibility of an in vitro drug sensitivity technique for testing malignant pleural effusion from advanced-stage non-small cell lung cancer.Our technique was able to produce a turnaround time for in vitro drug sensitivity testing of less than 1 week, with a success rate of more than 90% of cases. Correlated with the individual clinical outcome, using the area under the dose response curve (AUC) could define the level of in vitro drug sensitivity as: responsive (AUC0.25), intermediate response (0.1≤AUC≤0.25), or resistance (AUC0.1).Data obtained from this method of drug testing were correlated with the clinical outcome. The present drug sensitivity evaluation may benefit the development of individual precision chemotherapy.

Non-tumor-derived circulating DNA (nt-cirDNA) of advanced non-small cell lung cancer (NSCLC), with unclear origination, is associated with prognosis. We hypothesized that a part of nt-cirDNA release from CD3 or CD8 tumor-infiltrating lymphocytes (TILs) could have clinical implications. To investigate the feasibility of T-cell-derived circulating DNA (T-cirDNA) detection, real-time PCR with Taqman assay-specific rearranged TCRβ CDR3 region was conducted in plasma of 103 advanced NSCLC. CD3 and CD8-specific immunohistochemistry from biopsy specimen, was reviewed by one blinded pathologist to the T-cirDNA results. Prognostic impact including demographic characteristics was integrated into the model. Circulating DNA was detectable in 100 patients with median of 4 ng ml−1, while median of plasma T-cirDNA was 1.71 pg ml−1. Median %ratio of T-cirDNA/cirDNA was 0.02%. T-cirDNA was categorized by %ratio of T-cirDNA/cirDNA as undetectable, low (≤ 1%) and high (> 1%). Paradoxical prognostic impact of T-cirDNA/cirDNA was observed. Undetectable and high T-cirDNA/cirDNA translated to independent favorable prognostic outcome, HR of 0.54 [95% CI 0.30–0.96] and 0.41 [95% CI 0.21–0.80], respectively. 43 patients were assessed for CD3/CD8 TILs and PD-L1. High intratumoral CD3/CD8 TILs but not stromal CD3 TILs was correlated with high T-cirDNA/cirDNA representing active T-lymphocyte activity to eliminate cancer cells. While the prognosis of undetectable T-cirDNA/cirDNA, represents inactivated naive T-cell, was determined by the presence of EGFR mutation and had long durable response of EGFR inhibitors. T-cirDNA could be a novel biomarker representing adaptive immune resistance in NSCLC patients. Further exploration as a predictive biomarker for EGFR inhibitors in setting of EGFR mutation might be warranted.

9039 Background: Despite the development of predictive biomarkers to shape treatment paradigms and outcomes, 10-20% of de novo EGFR TKI resistance advanced non-small cell lung cancer (NSCLC) in the presence of EGFR mutation remains the issue of concern. Methods: 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 year). Results: Uncommon EGFR mutation subtype was a significant variable in de novo resistance vs. IRs and LTRs with odd ratios of 6.83 ([95% CI 2.36 – 19.80], p-value < 0.001) and 16.84 ([95%CI 1.66 – 171.45, p-value 0.02), respectively. The remaining sensitizing EGFR mutation subtype (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]. Conclusions: The presence of CDK4 /6 amplification in pretreatment specimen serve as a predictive biomarker for de novo resistance in sensitizing EGFR mutation.

12100 Background: Prevention of chemotherapy-induced nausea and vomiting (CINV) is vital in cancer treatment. Here, we compared the efficacy of netupitant-containing regimen; composed of NEPA, dexamethasone, and olanzapine (NEPAs), which is recommended for preventing CINV from high-emetogenic chemotherapy (HEC) to standard 3-drugs; ondansetron, dexamethasone, olanzapine for preventing CINV from high-dose cisplatin (≥75 mg/m2). Methods: This randomized, double-blind, placebo-control trial randomly assigned untreated patients who were received high-dose cisplatin in a 1:1 ratio to either NEPAs or standard 3-drugs combination regimen. Dose of dexamethasone in NEPAs regimen was modified after preplanned interim safety analysis to increase from 4 to 8 mg per day on days 2-4. The stratification factors were concurrent treatment with radiation and sex. The primary endpoint was the overall complete response (CR) rate defined as no vomiting and no use of rescue antiemetic drugs. The protocol allowed crossover to NEPAs for those who received standard 3-drugs and did not reach CR in the first cycle. We collected outcome in the first 2-cycle of treatment. Results: Between January 2019 and December 2020, hundred patients were randomly assigned to either NEPAs (n = 51) or in-house standard 3-drugs (n = 49). Demographic characteristics were well-balanced in both arms. Total events in both arms were 101 events for NEPAs and 78 events for standard 3-drug. Overall CR rate were 70% and 69% in NEPAs and standard 3-drugs, ( p-value 0.87) respectively. According to emesis phase, CR in acute (0- 24 hrs.) and delay phase (24-120 hrs.) were not different in both arms; 91% vs. 89% and 72% vs. 71% in NEPAs and standard 3-drugs respectively. However, mean nausea VAS score was significantly lower in NEPAs (1.63 vs. 2.02, p-value = 0.001). The ad hoc subgroup analysis shown similar efficacy between before and after protocol amendment of NEPAs regimen in term of delay emesis CR rate; 70.9% vs. 73.9% ( p-value 0.73). Conclusions: The NEPA-containing regimen did not show superiority compare to standard 3-drug in terms of complete response rate for CINV prevention among patients receiving high-dose cisplatin. Furthermore, the dexamethasone dosage of 4 vs. 8 mg per day might not affect the efficacy of delay emesis of the NEPAs regimen. Clinical trial information: TCTR20190508001. [Table: see text]

e21033 Background: Prognostic biomarker in early-stage lung cancer is less well defined. Despite early detection, one-third of patients with completely resected early-stage lung cancer without lymph node involvement have a recurrence. Our main objective is to investigate potential biomarkers useful in classification and predicting prognosis of this early-stage lung cancer, particularly in lymph node-negative subgroup. Methods: In discovery phase, we performed proteomic profiling on 60 lung cancer tissues to distinguish solid versus non-solid adenocarcinoma. Relative protein quantification was analyzed using Tandem Mass Tag mass spectrometry. In validation phase, tumor tissues from an additional cohort were analyzed by immunohistochemistry in conjunction with quantitative image analysis to confirm the differential expression of significantly altered proteins, including MCM2, MCM4, MCM6, SCAMP3, DPP4 and MYH11. Prognostic potentials of each biomarker were analyzed with univariate and multivariate analyses. Results: Quantitative proteomic analysis of predominantly solid versus non-solid adenocarcinoma tissues showed 30 differentially expressed proteins consisting of 13 increased and 17 decreased proteins found in solid compared with non-solid adenocarcinoma. Four most increased proteins (MCM2, MCM4, MCM6, and SCAMP3) and two most decreased proteins (DPP4 and MYH11) were selected for immunostaining in the validation cohort of 204 patients with resected adenocarcinoma without lymph node involvement. 57 out of 204 (27.9%) patients had recurrent disease. The high expression level of MCM4 was the strongest signature for disease recurrence (HR 4.85, 95%CI 2.78-8.47, p < 0.0001) and remained significant under multivariate analyses adjusted for TNM stage, histologic subtypes, lymphovascular invasion, tumor necrosis, and adjuvant chemotherapy. Recurrence-free survival (RFS) was significantly shorter in patients with high MCM4 compared with low MCM4 (5-y RFS 50% vs 84%, p < 0.0001) and remained highly significant within stage IA (5-y RFS 69% vs 93%, p < 0.0001), or stage IB and II (5-y RFS 36% vs 69%, p = 0.001). High expression of MCM4 correlated with other poor prognosis factors including smoking (p = 0.001), stage (p = 0.003), lymphovascular invasion (p = 0.036), visceral pleural invasion (p = 0.005), tumor necrosis (p < 0.0001) and solid histology (p < 0.0001). Conclusions: High MCM4 expression serves as an important prognostic biomarker predicting recurrence in early-stage node-negative lung adenocarcinoma.

e19398 Background: NK-1 receptor antagonist (NK1RA) is a standard anti-emetic agent in chemotherapy-induced nausea and vomiting (CINV) prevention in patients receiving high-dose (HD) cisplatin. However, in limited-resource settings, three-drug regimen including olanzapine, ondansetron and dexamethasone is affordable and effective in CINV prevention in these patients. Objectives: To evaluate cost effectiveness of additional NK1RA for CINV prevention in Thai patients receiving HD cisplatin in 3-cycle horizon, societal perspective. Methods: During January to December 2019, 20 patient receiving HD cisplatin were prospectively enrolled to receive standard three-drug regimen at King Chulalongkorn Memorial Hospital. Total medical resource utility, non-medical cost and clinical outcomes were actually collected and used for establishing decisional-tree model and cost-effectiveness analysis. Three arms decisional-tree model was constructed. First, standard strategy, a real-world practice in Thailand, patients received three-drug regimen in all cycles of chemotherapy. Second, NK1RA-rescue strategy, patients received three-drug regimen in the first cycle and additional NK1RA in subsequent cycles if the patients experienced CINV. Third, NK1RA upfront strategy, patients received four drug NK1RA-containing regimen in all cycles. Complete response (CR) rate was assumed to be 92.5% in NK1RA-containing regimen based on clinical trials. The study endpoint is to compare cost and QALYs in term of incremental cost-effectiveness ratio (ICER). Results: CR rates were 90% and 71% in first and second cycle in patients receiving standard regimen. The NK1RA upfront strategy produced greatest QALYs of 0.0351, followed by 0.0328 and 0.0292 in NK1RA-rescue strategy and standard strategy respectively. Mean total costs were 10420.46, 3332.19 and 2841.30 Thai baht (THB) respectively. The ICER of NK1RA-rescue strategy relative to standard strategy was 135,774 THB per QALY and the ICER of NK1RA upfront strategy relative to NK1RA-rescue strategy was 3,096,654 THB per QALY. Therefore, the ICER of NK1RA-rescue strategy but not NK1RA upfront strategy is considered to be cost effective at the willingness-to-pay level of 160,000 THB per QALY. Conclusions: Our findings suggested that the additional NK1RA as a rescue strategy is cost-effective in patients receiving HD cisplatin in Thai health care.

Myeloid cell leukemia 1 (Mcl-1) and B-cell lymphoma 2 (Bcl-2) proteins are promising targets for cancer therapy. Here, we investigated the structure&ndash, activity relationships (SARs) and performed molecular docking analysis of renieramycin T (RT) and its analogues and identified the critical functional groups of Mcl-1 targeting. RT have a potent anti-cancer activity against several lung cancer cells and drug-resistant primary cancer cells. RT mediated apoptosis through Mcl-1 suppression and it also reduced the level of Bcl-2 in primary cells. For SAR study, five analogues of RT were synthesized and tested for their anti-cancer and Mcl-1- and Bcl-2-targeting effects. Only two of them (TM-(&ndash, )-18 and TM-(&ndash, )-4a) exerted anti-cancer activities with the loss of Mcl-1 and partly reduced Bcl-2, while the other analogues had no such effects. Specific cyanide and benzene ring parts of RT&rsquo, s structure were identified to be critical for its Mcl-1-targeting activity. Computational molecular docking indicated that RT, TM-(&ndash, )-18, and TM-(&ndash, )-4a bound to Mcl-1 with high affinity, whereas TM-(&ndash, )-45, a compound with a benzene ring but no cyanide for comparison, showed the lowest binding affinity. As Mcl-1 helps cancer cells evading apoptosis, these data encourage further development of RT compounds as well as the design of novel drugs for treating Mcl-1-driven cancers.

Background EGFR mutation is the major driver mutation in East Asian lung cancer. By and large, this group of lung cancer patients has a better prognosis in advanced stage pulmonary adenocarcinoma. However, the prognostic role of these sensitizing EGFR mutations in early stage is uncertain. We aimed to investigate the prognostic value of EGFR mutation and other factors for tumor recurrence in completely resected LNNPA. Methods We recruited 220 patients with LNNPA who were diagnosed and treated at the King Chulalongkorn Memorial Hospital from January 1, 2009 to December 31, 2016. Recurrence included pathological diagnosis, imaging confirmations, or death. Recurrence-free survival was analyzed by univariable and multivariable Cox regression analyses. Results Median follow up time was 4 (2.7-5.6) years. There were more female (62%), never smokers (68%), stage I (86%) and EGFR mutations (54%). The most common EGFR mutations were exon 19 deletion (51%) and L858R (43%). Most patients did not receive adjuvant treatment (85.6%). There were 60 out of 220 (27.3%) patients had recurrent disease. The median time to recurrence was 2.3 years. The rate of loco-regional, distant and both types of recurrence were 27%, 68% and 5% respectively. Univariate analysis revealed smoking ≥ 10 pack-year, performance status ≥ 2, tumor size ≥ 4 cm, histologic grade ≥ 2, lymphovascular invasion, visceral pleural invasion, tumor necrosis and bronchial resection margin (BRM) Table: 1451P . Multivariate analysis Covariate HR 95% CI P-value Tumor size ≥ 4 cm 1.96 1.1 - 3.6 0.03 Visceral Pleural invasion 2.38 1.3 - 4.3 0.004 2.48 1.4 - 4.3 0.002 Tumor necrosis 3.12 1.7 - 5.6 EGFR mutation 1.23 0.6 - 2.7 0.57 Conclusions Among Thai, East Asian, early stage lung cancer, sensitizing EGFR mutation has similar outcomes to those without EGFR mutation. Legal entity responsible for the study The authors. Funding Ratchadapisek Sompoch Endowment Fund. Disclosure All authors have declared no conflicts of interest.

To examine the diversity of somatic alterations and clonal evolution according to aggressiveness of disease, nineteen tumor-blood pairs of 'formerly bronchiolo-alveolar carcinoma (BAC)' which had been reclassified into preinvasive lesion (adenocarcinoma in situ; AIS), focal invasive lesion (minimally invasive adenocarcinoma; MIA), and invasive lesion (lepidic predominant adenocarcinoma; LPA and non-lepidic predominant adenocarcinoma; non-LPA) according to IASLC/ATS/ERS 2011 classification were explored by whole exome sequencing. Several distinct somatic alterations were observed compare to the lung adenocarcinoma study from the Cancer Genome Atlas (TCGA). There were higher numbers of tumors with significant APOBEC mutation fold enrichment (73% vs. 58% TCGA). The frequency of KRAS mutations was lower in our study (5% vs. 32% TCGA), while a higher number of mutations of RNA-splicing genes, RBM10 and U2AF1, were found (37% vs. 11% TCGA). We found neither mutational pattern nor somatic copy number alterations that were specific to AIS/MIA. We demonstrated that clonal cell fraction was the only distinctive feature that discriminated LPA/non-LPA from AIS/MIA. The broad range of clonal frequency signified a more branched clonal evolution at the time of diagnosis. Assessment of tumor clonal cell fraction might provide critical information for individualized therapy as a prognostic factor, however this needs further study.