Circulating tumor DNA (ctDNA)-molecular residual disease (MRD) has been recognized as a robust prognostic biomarker post-lung cancer surgery. However, its application in adjuvant therapy guidance is limited by the insufficient sensitivity of single time-point landmark MRD detection. A total of 163 patients with undetectable landmark MRD within 90 days post-surgery were included in the training set. We used perioperative features and baseline ctDNA characteristics, analyzed with the LASSO algorithm to select features correlated with disease-free survival (DFS). The final model was built using the cox model in the XGBoost algorithm. Blood samples were processed by MRD_Navigator assay. The validation set comprised 92 patients from the TRACERx cohort who showed negative ctDNA at the landmark time point. We developed a model incorporating seven prognostic factors, with baseline ctDNA level as the top contributor (gain index 0.44, Table). The model classified patients into high and low-risk groups; in the training set, 64 were high-risk and 99 low-risk. A significant difference in 2-year DFS rates was found between these two groups (p<0.001, HR=8.276, 95%CI: 3.722-18.4), at 59% and 98%. Similarly, in the validation set, 63 were high-risk and 29 low-risk, with significant difference in 2-year DFS rates, 53% and 86% respectively (p=0.009, HR=2.245, 95%CI: 1.204-4.186). Table: 111PSeven prognostic features extracted by LASSO for model establishmentClinical featuresGain indexBaseline ctDNA level0.4404TNM Stage0.1783Landmark cfDNA concentration0.1473Baseline cfDNA concentration0.1254Baseline oncoKB mutant gene detected0.0677Baseline ctDNA detected0.0378Histology0.0031 Open table in a new tab This XGBoost model could enhance the negative predictive value of landmark undetectable MRD and needs further validation for its effectiveness in guiding de-escalation of adjuvant therapy.

Tumor cells frequently suffer from excessive endoplasmic reticulum (ER) stress resulted from both increased accumulation of misfolded proteins and stressful tumor microenvironment characterized by hypoxia, acidosis and nutrients deprivation. Previous studies have elucidated the role of ER stress and thereby unfolded protein response (UPR) in melanoma pathogenesis. However, the effect of ER stress on tumor-infiltrating lymphocytes and anti-cancer immunosurveillance, as well as the underlying mechanism, remains far from understood in melanoma. Herein, we report that ER stress re-shapes tumor microenvironment and enhances the efficacy of anti-PD-1 immunotherapy via the regulation of chemokines, pro-inflammatory factors and PD-L1 expression. We firstly found that the activation of UPR was positively associated with the feature of tumor-infiltrating lymphocytes in TCGA SKCM database and melanoma tissues. Then, pharmacological induction of ER stress exerted better anti-tumor effect in vivo and was highly dependent on CD8+ T cells. In parallel, the profile of immune cells in tumor microenvironment was significantly re-shaped. Subsequent mechanistic studies revealed that ER stress facilitated the expression and secretion of multiple chemokines and cytokines via IRE1α-NF-κB pathway, which was related to increased infiltration and anti-tumor capacity of lymphocytes. Furthermore, induction of ER stress promoted the expression of PD-L1 via IRE1α-NF-κB axis, which helped to bring synergized therapeutic effect along with anti-PD-1 antibody. Taken together, our results demonstrate that ER stress facilitates anti-cancer immunosurveillance and improves immunotherapy efficacy via the regulation of chemokines, pro-inflammatory factors and PD-L1 expression. The combination of ER stress inducer and anti-PD-1 antibody could be a potent approach for melanoma immunotherapy.

Acetyl-CoA is a key metabolic intermediate with an indispensable role in sustaining tumor cell growth as well as in regulating genes transcription. However, its role in immune checkpoint regulation and tumor immune evasion remains unclear. Herein, we demonstrate that ACLY, the enzyme controlling cytosolic acetyl-CoA biogenesis, drives CD8+T cells mediated tumor immune evasion through upregulating endogenous and IFN-r induced PD-L1 expression. We first used integrative analysis to explore the association of acetyl-CoA metabolic pathway and ACLY with immune features across multiple cancer types. We further investigated the efficacy of ACLY inhibitor, SB204990 or combinatory treatment of SB204990 and anti-PD-1 antibody in mouse models. In vitroassays were carried out to examine the regulating roles of ACLY on PD-L1. In addition, multiple interventions of AcCoA metabolism pathways including siRNA and small molecular inhibitors were employed to assess the role of cytosolic acetyl-CoA in regulating PD-L1 expression. Lastly, ChIP assays was performed to examine the mechanism of AcCoA and ACLY regulated PD-L1 expression. Our bioinformatics analysis revealed a strong association between acetyl-CoA metabolic pathway and immune features. We further demonstrated that blockade of ACLY significantly reduced tumor burden by promoting anti-tumor immunity. In addition, knockdown of ACLY rendered tumors more sensitive to anti-PD-1 treatment. Importantly, perturbing cytosolic acetyl-CoA level by knockdown of ACSS2 and PDC inhibitor caused down-and up-regulated PD-L1 levels, respectively, while MPC inhibitor treatment had no effect on PD-L1 expression. Mechanistically, ACLY maintains cytosolic acetyl-CoA level, which promoted histone acetylation at the promoter of CD274 mediated by P300, leading to downstream PD-L1 expression on tumors. Collectively, these data delineate an acetyl-CoA metabolism dependent epigenetic mechanism contributing to tumor immune evasion, and the combination of ACLY inhibitor and anti-PD-1 antibody provides potential therapeutic strategies for melanoma treatment.

Oxidative stress promotes the secretion of chemokine CXCL16 from keratinocytes, leading to epidermal migration of CD8+T cells and subsequent melanocyte destruction, thus leading to vitiligo. However, the upstream regulatory mechanism of CXCL16 secretion induced by oxidative stress is still unclear. In the present study, we initially found that inhibited autophagy in keratinocytes promote CXCL16 secretion under oxidative stress. In addition, the results showed that oxidative stress induced Atg5 cleavage and arrested the formation of ATG12-ATG5 complex, thereby inhibiting autophagy and promoting CXCL16 secretion. Furthermore, we illustrated that TRPM2-mediated calcium influx activates calpain1 as a downstream of oxidative stress, leading to Atg5 shearing. More importantly, down-regulation of Atg5 expression enhanced the binding of IRF3 to CXCL16 promoter region by activating TBK1 and downstream IRF3, and promoted CXCL16 transcription and secretion. In summary, our study suggested that TRPM2-mediated calcium influx activate calpain1, which lead to Atg5 shearing and then promote CXCL16 secretion via the Atg5-TBK1-IRF3 signaling pathway. Therefore, inhibition of TRPM2 may as a potential target for inhibiting CXCL16 secretion by keratinocytes under oxidative stress.

Anti-programmed cell death protein 1 (PD-1) and anti-programmed death-ligand 1 (PD-L1) immune checkpoint inhibitors (ICIs) has provided clinical benefit for lung cancer (LC). Plasma cell-free DNA (cfDNA) could be used to characterize the genomic profiles of LC. Here, we examined cfDNA to explore mutational features related to the effectiveness of immunotherapy for LC.

Identifying valid biomarkers for patient selection impressively promotes the success of anti-PD-1 therapy. However, the unmet need for biomarkers in gastrointestinal (GI) cancers remains significant. We aimed to explore the predictive value of the circulating T-cell receptor (TCR) repertoire for clinical outcomes in GI cancers who received anti-PD-1 therapy. 137 pre- and 79 post-treated peripheral blood samples were included. The TCR repertoire was evaluated by sequencing of complementarity-determining region 3 (CDR3) in the TRB gene. The Shannon index was used to measure the diversity of the TCR repertoire, and Morisita’s overlap index was used to determine TCR repertoire similarities between pre- and post-treated samples. Among all enrolled patients, 76 received anti-PD-1 monotherapy and 61 received anti-PD-1 combination therapy. In the anti-PD-1 monotherapy cohort, patients with higher baseline TCR diversity exhibited a significantly higher disease control rate (77.8% vs. 47.2%; hazard ratio [HR] 3.92; 95% confidence interval [CI] 1.14–13.48; P = 0.030) and a longer progression-free survival (PFS) (median: 6.47 months vs. 2.77 months; HR 2.10; 95% CI 1.16–3.79; P = 0.014) and overall survival (OS) (median: NA vs. 8.97 months; HR 3.53; 95% CI 1.49–8.38; P = 0.004) than those with lower diversity. Moreover, patients with a higher TCR repertoire similarity still showed a superior PFS (4.43 months vs. 1.84 months; HR 13.98; 95% CI 4.37–44.68; P < 0.001) and OS (13.40 months vs. 6.12 months; HR 2.93; 95% CI 1.22–7.03; P = 0.016) even in the cohort with lower baseline diversity. However, neither biomarker showed predictive value in the anti-PD-1 combination therapy cohort. Interestingly, the combination of TCR diversity and PD-L1 expression can facilitate patient stratification in a pooled cohort. The circulating TCR repertoire can serve as a predictor of clinical outcomes in anti-PD-1 therapy in GI cancers.

In the past decade immune-checkpoint inhibitors (ICIs) has revolutionized the treatment of patients with multiple types of cancer, but the predictive biomarkers are limited. SETD2 is an essential gene related to DNA damage repair (DDR) and an IFN-α induced immune response, indicating a predictive role in immunotherapeutic efficacy. In our discovery cohort, we reviewed 6726 sequencing samples, among them 375 samples were detected with SETD2 mutation and 13 patients from 9 centers were ICIs treated. Validation cohort datas included the TCGA, the MSKCC and the POPLAR/OAK cohort and 10 public ICIs treated cohorts. PolyPhen-2 and SIFT were used to distinguish deleterious mutations from tolerated mutations. Comparisons of tumor mutation burden (TMB), MSIsensor, survival, immune gene expression were calculated. Our discovery cohort showed a high ORR for patients with SETD2 mutation which was 53.8% (7/13) for all patients with immunotherapy and 42.9% (3/7) for PD-1/PD-L1 monotherapy. The favored ICIs outcomes was validated by MSKCC-IO cohort (p<0.0001). A significantly higher TMB was found in SETD2 deleterious mutation group in our discovery cohort including colorectal cancer (p<0.0001), non-small cell lung cancer (p<0.0001), melanoma (p=0.0022) and glioma (p=0.0042). And the elevation of TMB and the favored ICIs outcomes were comparable to other molecular features including POLE/D1, MMRS genes deleterious mutation group. A significantly higher MSIsensor was found in SETD2 deleterious mutation group in three cancers with the highest frequency of mismatch repair protein deletions including gastric adenocarcinoma (p=0.0029), endometrial carcinoma (p<0.0001), and colorectal carcinoma (p=0.0006) and SETD2 was an independent factor influencing MSI-H in gastric adenocarcinoma (p=0.003) and colorectal carcinoma (p<0.0001). Transcriptomic analysis in seven solid tumors from the TCGA database showed features of inflammated tumor microenvironment in tumors with SETD2 deleterious mutation group especially in renal cell carcinoma, colorectal adenocarcinoma and endometrial carcinoma. We identified a new tissue agnosticpredictive biomarker for ICIs: SETD2 deleterious mutation.

The therapeutic effect of immune checkpoint blockade (ICB) therapy, especially the inhibition of programmed cell death protein 1 (PD-1) and its ligand PD-L1, has been verified in melanoma treatment. However, the dissatisfied response rate and therapeutic efficacy of anti-PD-1/PD-L1 therapy remains a major challenge for melanoma treatment. Here, we reported A20 as a critical regulator that determines the therapeutic effect of anti-PD-1 immunotherapy in melanoma. Through the un-targeted MS-based proteomic analysis, we first found that high expression of A20 was significantly associated with therapeutic resistance to anti-PD-1 immunotherapy in melanoma patients. We then proved that the suppression of tumoral A20 expression potentiated the anti-tumor activity of infiltrated CD8+T cells and increased the efficacy of anti-PD-1 antibody treatment in pre-clinical mice model. A20 promoted PD-L1 expression in tumor cell to impair infiltrated CD8+T cell cytotoxicity and contributed to melanoma immune escape and therapeutic resistance to anti-PD-1 immunotherapy. Moreover, up-regulated A20 expression facilitated PD-L1 transcription via the ubiquitination and degradation of PHB and thereby ameliorating its inhibition of STAT3. Our findings reveal a previously un-recognized regulatory role of A20 in anti-tumor immunity, and demonstrate that A20 can be exploited as a promising target to overcome immune escape and improve the effect of anti-PD-1 immunotherapy in melanoma.

Efficient peptide and protein identification from data-independent acquisition mass spectrometric (DIA-MS) data typically rely on an experiment-specific spectral library with a suitable size. Here, we report a computational strategy for optimizing the spectral library for a specific DIA dataset based on a comprehensive spectral library, which is accomplished by a priori analysis of the DIA dataset. This strategy achieved up to 44.7% increase in peptide identification and 38.1% increase in protein identification in the test dataset of six colorectal tumor samples compared with the comprehensive pan-human library strategy. We further applied this strategy to 389 carcinoma samples from 15 tumor datasets and observed up to 39.2% increase in peptide identification and 19.0% increase in protein identification. In summary, we present a computational strategy for spectral library size optimization to achieve deeper proteome coverage of DIA-MS data.

BACKGROUND:Adenosquamous carcinoma (ASC) of the lung is a heterogeneous disease that is composed of both adenocarcinoma components (ACC) and squamous cell carcinoma components (SCCC). Their genomic profile, genetic origin, and clinical management remain controversial. PATIENTS AND METHODS:Resected ASC and metastatic tumor in regional lymph nodes (LNs) were collected. The ACC and SCCC were separated by microdissection of primary tumor. The 1021 cancer-related genes were evaluated by next-generation sequencing independently in ACC and SCCC and LNs. Shared and private alterations in the two components were investigated. In addition, genomic profiles of independent cohorts of adenocarcinomas and squamous cell carcinomas were examined for comparison. We have also carried out a retrospective study of ASCs with known EGFR mutation status from 11 hospitals in China for their clinical outcomes. RESULTS:The most frequent alterations in 28 surgically resected ASCs include EGFR (79%), TP53 (68%), MAP3K1 (14%) mutations, EGFR amplifications (32%), and MDM2 amplifications (18%). Twenty-seven patients (96%) had shared variations between ACC and SCCC, and pure SCCC metastases were not found in metastatic LNs among these patients. Only one patient with geographically separated ACC and SCCC had no shared mutations. Inter-component heterogeneity was a common genetic event of ACC and SCCC. The genomic profile of ASC was similar to that of 170 adenocarcinomas, but different from that of 62 squamous cell carcinomas. The incidence of EGFR mutations in the retrospective analysis of 517 ASCs was 51.8%. Among the 129 EGFR-positive patients who received EGFR-TKIs, the objective response rate was 56.6% and the median progression-free survival was 10.1 months (95% confidence interval: 9.0-11.2). CONCLUSIONS:The ACC and SCCC share a monoclonal origin, a majority with genetically inter-component heterogeneity. ASC may represent a subtype of adenocarcinoma with EGFR mutation being the most common genomic anomaly and sharing similar efficacy to EGFR TKI.

e13500 Background: Pulmonary sarcomatoid carcinoma (PSC), composed of sarcomatous component (SaC) and carcinomatous component (CaC), is a rare and generally aggressive subtype of non-small-cell lung cancer (NSCLC). Little is known about the genetic origin, TMB, and PD-L1 status of PSC. Methods: 31 immunohistochemically (IHC) confirmed PSCs were enrolled and tumor samples were subjected to microdissection to obtain SaC and CaC. Different components were subjected to targeted sequencing with a 1021-gene-panel. Somatic mutations were used to assess TMB and construct phylogenetic tree. PD-L1 expression level was determined by IHC. Independent cohorts of 90 lung adenocarcinomas (LUAD) from the Geneplus database and 167 sarcomas from TCGA were used for genomic comparison. Results: 87% of patients (pts) were male, with a median age of 57.0 years. 52% were smokers. The most recurrently mutated genes were TP53 (80%), MET (24%), NF1 (21%), EGFR (21%), and KRAS (21%) in CaC, while TP53 (76%), MAP3K1 (21%), NF1 (21%), MET (21%), and KRAS (21%) in SaC. 55.4% of the variations (SNV, small indels, SV, and CNV) were shared within SaC and CaC. TP53, MET, NF1, and EGFR were mostly frequently found in shared mutations. Overwhelming majority of PSCs (29/30) had common mutations between SaC and CaC implying a monoclonal origin. EGFR were mutated significantly less in adenocarcinoma components (AdC) than LUAD (Fisher Exact test, 4/24 vs 52/90, p < 0.001). High-frequency mutated driver genes in SaC, MET, EGFR, NF1, and ALK, were rarely detected in leiomyosarcomas and uterine sarcomas. The median of TMB in SaC and CaC were both 12.9 mutations/Mb (range, SaC 2.88-33.1, CaC 2.88-47.5), with high correlation between the two components (p < 0.001). PD-L1 expression levels in the two components also had high correlation (p < 0.001). More pts showed high PD-L1 expression ( > = 50%) in SaC rather than CaC (8 versus 3). Conclusions: A majority of PSCs share a monoclonal origin. PSCs with AdC exhibited lower mutated frequency of EGFR than LUAD. SaC and CaC had similar higher TMB status than LUAD. SaC and CaC had similar level of PD-L1expression.

Background: Recently, neoadjuvant targeted therapy and immunotherapy have presented a promising clinical effect for localized stage non-small cell lung cancer. However, the characteristics and relationship of genomic and immune profiling in surgical lung adenocarcinomas has been poorly delineated to date. Methods: We prospectively enrolled 100 consecutive patients with pulmonary nodule who intended to undergo curative lung resection during June 2017 to July 2018 (NCT03320044). We investigated the TCR repertoire using next-generation deep sequencing of the complementarity determining region 3 (CDR3) of the TCR β chain in the tissue and WBC samples. Target-capture deep sequencing of 1021 genes was used to detect genomic variations in both tissue and paired plasma samples. Results: EGFR (65.4%), TP53 (36.5%) and KRAS (11.5%) mutated most frequently. KRAS (84.6%) was enriched in mucinous adenocarcinoma. High invasive subtype and no ground-glass opacity status is more likely to be increasing tissue TMB (p = 0.0016, p = 0.0012), higher T-cell clonality (p = 0.05, p = 0.05), and more HLA-LOH event (p = 0.038, p = 0.035). A median of TNB was 2 neoantigens/Mb and showed positive relation with TMB (r = 0.97, p < 0.0001). EGFR-mutant co-occurring mutations were enriched TGF-beta, PI3K-Akt, hippo and Leukocyte trans-endothelial migration pathway (q < 0.05). Lower TCR clonality was shown in patients with EGFR mutation and co-occurring hippo or Leukocyte trans-endothelial migration pathway. Alterations in DNA damage response (DDR) pathways were observed in 15.8% patients, and these patients showed higher clonality (p = 0.03). 35.3%(24/68) ctDNA-positive were found in Stage I with a mean ctDNA abundance of 0.18% (ranged from 0.012 to 3.3%), which suggested the challenge of bTMB in biomarker study of early-stage LUAD. Analysis of TCR repertoire from available paired WBC showed no obvious characteristics. Conclusions: This is the first prospective study integrating the correlation of genomic alteration and T-cell receptor in localized surgical LUAD. Analysis of co-altered pathway of EGFR-mutant LUAD and immune microenviroment will provide a better understanding in choosing biomarkers and optimal benefit patients for neoadjuvant therapy. Clinical trial identification: NCT03320044. Legal entity responsible for the study: Jun Wang. Funding: National Natural Science Foundation of China (No.81602001). Disclosure: All authors have declared no conflicts of interest.

Lung adenosquamous carcinoma (ASC) is a heterogeneous disease that comprises of both adenocarcinoma (AC) and squamous cell carcinoma (SCC) components. Their genomic profile, evolutionary origin, and clinical management remain controversial. Objective of this study is to define the genomic origin of this heterogeneous tumor by independent genomic analyses of the AC and SCC components. Surgical ASCs were collected. AC component and SCC component were obtained separately by microdissection, and Lymph node (LN) metastases were gathered. Targeted sequence was performed for the two components using a 1021-gene panel, independently. Evolutionary relationship of the two components was analyzed. The independent cohorts of adenocarcinoma (n=170) and squamous cell carcinomas (n=62) were used for comparison. EGFR and concomitant mutations with response to EGFR-TKI were analyzed. Retrospective 517 ASCs underwent EGFR detections were collected from 11 centers. Objective response rate (ORR), disease control rate (DCR) and progression free survival (PFS) were analyzed in EGFR-positive patients received EGFR-TKIs. 28 ASCs were collected. NGS was performed on AC component and SCC component samples, respectively. The most frequent alterations in 28 ASCs were EGFR mutation (79%), TP53 mutation (68%), MAP3K1 mutation (14%), EGFR amplification (32%), and MDM2 amplification (18%). 27 patients had trunk variations in the both components suggesting the monoclonal origin of ASCs. The prevalence of trunk mutations was correlated to those of AC, indicating that ASC might originate from AC. Only one patient did not carry any trunk variations between AC and SCC components, which were clearly and geographically distinguishable under the microscope. 22 had AC component or/and SCC component specific variations suggesting the common event of branch evolution. The 23 LNs of 13 patients mainly contained AC and ASC components (AC, SCC, and ASC: 11, 1, and 11, respectively), and each of the LNs carried the trunk mutations of the primary ASC. Like pure AC, the alterations of L858R and Exon 19 Dels of EGFR were common in the 28 ASCs. Unfortunately, these patients have not been treated with TKIs. Further, of 517 retrospective ASCs from 11 centers, 51.8% were EGFR-positive. For the 129 EGFR-positive ASCs who had received TKIs, the ORR and DCR were 56.6% and 89.1%, respectively. The median PFS was 10.1 months (95% CI: 9.0-11.2). The AC and SCC components share a monoclonal origin, and a majority have branching evolution. ASC may represent a subtype of adenocarcinoma with EGFR mutation being the most common genomic anomaly and sharing similar efficacy to EGFR-TKIs.

TKIs have significantly improved the survival of NSCLC pts carrying sensitive mutations. However, heterozygous responses were observed. We conducted a prospective multicenter clinical trial to explore factors associated with the efficacy of EGFR-TKI, and assess the mutation and TMB concordance between plasma and tissue NGS. Paired tumor and plasma samples were obtained from treatment naïve advanced NSCLC pts whenever applicable. DNA was sequenced by target-capture deep sequencing of 1021 tumor-related genes (pan-cancer panel). PFS was estimated using Kaplan-Meier method and compared using log-rank test. Tissue TMB (tTMB) and plasma TMB (bTMB) analysis interrogated SNVs/Indels with VAF ≥3% and ≥0.5%, respectively. TMB-H pts were identified with ≥9 muts/Mb. From Feb. 2017 to Jan. 2019, 262 advanced NSCLC pts were enrolled from 12 centers. In 224 pts with paired tumor and plasma samples, 144 had EGFR sensitive mutations in tumor samples (L858R, 46%; Ex19Indel, 42%), of whom, 106 (74%) had the identical mutations detected in plasma. The detection rate of tissue EGFR mutations in paired plasma was significantly higher in pts with extrathoracic metastasis (81% vs. 61%, p = 0.03). In 38 pts lacking paired samples, 20 pts had EGFR sensitive mutations detected. Thus, 164 pts were identified as EGFR positive by either plasma or tissue NGS. One hundred of them were treated EGFR TKIs (ORR: 70%, mPFS: 20 mo). The ORR was affected by EGFR subtypes (Ex19Indel vs. L858R: 72% vs. 45%, p = 0.02), concomitant CNV/fusion (with vs. without: 11% vs. 68%, p = 0.002) and CDKN2A mutations (with vs. without: 0% vs. 66%, p = 0.007). Mutations in p53 pathway (p = 0.02), CDK12/13 (p = 0.0002), concomitant CNV/fusion (p = 0.003), and high number of alterations (≥ 5) (p = 0.003) significantly shortened mPFS. tTMB was correlated with bTMB (rPearson = 0.9, p <0.0001), with a concordance rate of 90% for TMB-H and TMB-L classification. Interestingly, 9.8% of the EGFR positive pts were bTMB-H, and mPFS was shorter in bTMB-H pts (6 mo, 95% CI: 5 - NR) than in bTMB-L pts (NR, 95%: 13 - NR) (p = 0.2). Deep sequencing with the pan-cancer panel effectively detected mutations and evaluated TMB in both tissue and plasma with a high consistence. Moreover, the presence of structure variation, high tumor heterogeneity and concomitant mutations in genes such as CDKN2A were associated with worse prognosis. Further studies of predictive factors are ongoing (NCT03059641).

Hyperhomocysteinemia and folic acid deficiencies have been reported in patients with vitligo, while the mechanism underlying this process is unclear. This study aims to investigate the effect of homocysteine (Hcy) on vitiligo pathogenesis and the underlying mechanisms. Our results demonstrated that Hcy was highly expressed in the serum of patients with vitiligo and associated with the degree of disease progression. In vitro study confirmed that oxidative stress can promote the accumulation of Hcy in melanocytes. Hcy treatment can inhibit melanocyte growth and induce cell apoptosis in a dose-dependent manner. Hcy treatment can upregulate glucose-regulated protein 78(GRP78), activate endoplasmic reticulum stress and induce the expression of C/EBP homologous protein(CHOP). Knockout the IRE1 pathway has no effect on apoptosis, whereas, the level of apoptosis is reduced by knockout the PERK pathway. We also observed activation of pPERK in peripheral melanocytes of vitiligo patient. Folic acid serves as cofactors of homocysteine methyltransferase of methionine from homocysteine. Folic acid supplementation can reduce intracellular Hcy levels, inhibit the activation of the perk-eif2a-CHOP pathway, reduce cell apoptosis rate and promote melanocyte surivival. Our study also showed that homosysteine disrupts melanogenesis via inhibition of MITF TYR TYRP1 and melanA experssion, supplementation of folic acid can restore above molecular expression and restore melanin synthesis. Our data suggest that an elevated Hcy level may be a precipitating factor for vitiligo in predisposed individuals. In addition, Hcy facilitate melanocyte apoptosis through perk-eif2a-CHOP pathway, and decrease melanin synthesis. Importantly, folic acid supplementation can reduce the Hcy toxicity to melanocyte and restore melanin synthesis.

Virus can induce vitiligo but the mechanism is elusive. Melanoma differentiation-associated gene 5 (MDA5) functions as one of the major sensors of RNA viruses. IFIH1, which encodes the MDA5 protein, has been newly identified as a susceptibility gene in vitiligo. Nevertheless, little is known about the function and mechanism of MDA5 in the onset and progress of vitiligo. We demonstrated here that MDA5 expression increased using immunofluorescence in vitiligo lesions and showed a positive correlation with severity of patients with vitiligo. In addition, as a viral nucleic acid analog, Poly (I:C) can increase the expression of CXCL10, CXCL16 and CCL20 in a MDA5-dependant way. Further more, through knock-down assay using si-RNA, we found poly(I:C)-induced chemokines secretion was due to the activation of MDA5-MAVS-NF-kb pathway. Finally, These chemokines produced by keratinocytes induced migration of autoreactive CD8+ T cells derived from patients with vitiligo, which features the pathology of vitiligo. In summary, we demonstrated that MDA5 mediates CD8+ T-cell skin trafficking under virus infection through the section of chemokines such as CXCL10, CXCL16 and CCL20 in patients with vitiligo. This effect is caused by MDA5-MAVS-NF-kB pathway. This research may provide a new extension of how virus induce the onset and development of vitiligo.

Local consolidative treatment (LCT) following induction systemic therapy for oligometastatic NSCLC has been shown in a randomized phase II trial (NCT01725165) to demonstrate improved PFS and OS compared with no up-front LCT. We present an analysis of longitudinally collected translational correlative markers from this trial including T-cell repertoire and circulating tumor DNA (ctDNA). Peripheral blood obtained from patients enrolled on NCT01725165 were labelled as 1) baseline, 2) early follow-up (FU) if obtained in the 1st or 2nd FU evaluation, and 3) late FU if obtained in the 3rd to 6th FU evaluations. All LCT patients included in this analysis received radiation. The CDR3 variable region in the beta chain of the T cell receptor was sequenced by an analyzer. Collected ctDNA was subjected to next generation sequencing of a 1,021 cancer gene panel using DNA from leukocytes as germline control. T-test and univariate Cox regression were conducted to assess differences in continuous variables and associations with patient outcomes, respectively. Median FU was 38.8 months, at which time 39 and 29 of the 49 randomized patients had progressed and/or died, respectively. Thirty one patients had TCR data analyzed (LCT arm=15 and no LCT arm=16). No significant differences in T-cell metrics were detected between arms when assessing baseline and late FU (all P>0.20). At early FU, the LCT arm exhibited trends towards decreased productive entropy (median 12 vs 14, P=0.12) and increased productive clonality (median 0.10 vs 0.15, P=0.19) compared with the no LCT arm. Assessing LCT arm patients only, at early FU there was a trend towards increased productive clonality (median 0.09 vs 0.15, P=0.15) and cumulative frequency of the top 100 clones (median 20% vs 30%, P=0.15) compared with baseline. ctDNA analysis was performed on 21 patients (LCT arm=10 and no LCT arm=11). At early FU, patients in the LCT arm exhibited a trend towards lower detected mutations compared with patients in the no LCT arm (median: 2 vs 6, P=0.09). Longitudinal tracking of 8 patients who had longterm PFS (median: 10 months, range: 6-26 months) and had at least 3 ctDNA measurements demonstrated <5 detectable mutations at first FU in all patients, with a rise in ctDNA during FU that was antecedent to clinical failure in all patients. No associations between T-cell repertoire and ctDNA metrics were observed with patient outcomes (all P>0.20). LCT for oligometastatic NSCLC was associated with a peripheral T cell oligoclonal expansion with decreased entropy, increased clonality, and expansion among the most prevalent clones. LCT was associated with a lower detectable ctDNA burden at early FU timepoints with subsequent ctDNA increase preceding clinical failure. These analyses were exploratory in nature and statistical tests were limited by small numbers and heterogeneity introduced by outliers.