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Small bowel adenocarcinoma (SBA) is a rare malignancy with a poor prognosis and limited treatment options. Despite prior studies, molecular characterization of this disease is not well defined, and little is known regarding Chinese SBA patients. In this study, we conducted multigene next‐generation sequencing and 16S ribosomal RNA gene sequencing on samples from 76 Chinese patients with surgically resected primary SBA. Compared with colorectal cancer and Western SBA cohorts, a distinctive genomic profile was revealed in Chinese SBA cohorts. According to the levels of clinical actionability to targetable alterations stratified by OncoKB system, 75% of patients harbored targetable alterations, of which ERBB2, BRCA1/2, and C‐KIT mutations were the most common targets of highest‐level actionable alterations. In DNA mismatch repair–proficient (pMMR) patients, significant associations between high tumor mutational burden and specific genetic alterations were identified. Moreover, KRAS mutations/TP53 wild‐type/nondisruptive mutations (KRASmut/TP53wt/non‐dis ) were independently associated with an inferior recurrence‐free survival (hazard ratio [HR] = 4.21, 95% confidence interval [CI] = 1.94‐9.14, P, Molecular characterization of small bowel adenocarcinoma (SBA) is not well defined, and little is known regarding Chinese SBA patients. Compared with colorectal cancer and Western SBA cohorts, a distinctive genomic profile was revealed in Chinese SBA cohorts. A molecular subtype of KRAS mutations/TP53 wild‐type/nondisruptive mutations was found to be associated with an inferior recurrence‐free survival in SBA.

The genomic profiles of osteosarcoma (OS) patients have been extensively investigated; however, the genetic prognostic biomarkers still remain unclear. In the present study, we analyzed the mutational profiles of pre-treatment primary tumor samples from 33 OS patients using whole exome sequencing (WES). These 33 OS patients were divided into two groups according to clinical outcomes: a good prognosis group involving 21 patients with tumor free survival, and a poor prognosis group involving the remaining12 patients who had lung metastases at initial diagnosis. Overall we found that the MAPK signaling pathway may play an important role in determining a good prognosis, while the PI3K-Akt signaling pathway may be an important factor leading to a poor prognosis. Significant differences were observed in the number of somatic copy number alterations, including del (3p), amp (4q), del (7p) and amp (8q), between the two groups. Moreover, significant differences were observed in mutation sites and frequencies between these two groups. The good prognosis group exhibited a significantly higher mutation frequency in somatic JAK-STAT and germline base excision repair pathways than the poor prognosis group. Furthermore, significant difference was also observed in the frequency of potentially actionable alterations between the two groups, suggesting that patients with a poor prognosis potentially have access to a larger number of treatment options. These findings highlight the importance of evaluating genomic disparities in OS, and provide a novel insight into the potential prognostic biomarkers.

Background Occurrence at a young age is known to be associated with unique clinical features in colorectal cancer (CRC). However, the genomic differences between young and old patients with CRC are not well elucidated and, to the best of our knowledge, have never been investigated in a Chinese population. Methods Tumor tissue samples from 29 young (age ≤50 years) and 46 old (age >50 years) patients with CRC were collected. Targeted sequencing of 808 cancer‐related genes was conducted to characterize the genomic landscape for Chinese CRC. Results Overall, mutational profiles exhibited notable differences between the two groups. In particular, APC and PIK3CA mutations were more frequently observed in old patients (p = 0.009 and p = 0.012, respectively), while SMAD4 mutations tended to occur in young patients (p = 0.054). Mutation loci distributions of KRAS in the young cohort differed from those in the old cohort, and a higher frequency of KRAS codon 12 mutations was potentially associated with a young age (p = 0.076). The frequencies of clinically actionable alterations were analyzed by defined age categories, which unveiled a distinctive targeted genomic profile in the young group. Furthermore, among patients with mismatch repair‐proficient (pMMR) CRC, tumor mutation burden (TMB) was positively correlated with age (Pearson's r = 0.306, p = 0.011), and genomic alterations associated with high TMB in young patients differentiated from those in old patients. Conclusions These findings revealed different molecular characterization between young and old Chinese patients with CRC, which may provide novel insights for the personalized treatment of CRC., This is the first NGS study to compare genomic profiles between young and old Chinese cohorts with CRC. The results demonstrated that young patients with CRC presented with some unique molecular features. Moreover, the comprehensive investigation of clinically actionable mutations and TMB will contribute to the improvement of personalized therapy and clinical management in CRC.

Background The identification of NTRK fusions in tumours has become critically important due to the actionable events predictive of response to TRK inhibitor. It is not clear whether the NTRK breakpoint location is different for response to targeted therapy and NTRK fusions affects the efficacy of immunotherapy. Case presentation Here we reported a 60-year-old female diagnosed with advanced lung adenocarcinoma. NGS-based molecular profiling identified a novel NCOR2-NTRK1 fusion and high tumor mutational burden (TMB) (58.58 mutations/Mb) in this case. Additionally, program death-ligand 1 (PD-L1) expression was detected in 20–30% of the tumor cells by immunohistochemical (IHC) staining. The patient received treatment with anti-PD-1 immune checkpoint inhibitor of camrelizumab. After two cycles of treatment, the CT scan showed some tumor nodules were still enlarged, indicating disease progression. She was then changed to TRK inhibitor larotrectinib. One month later, the CT scan showed the volume of some lesions started to decrease, and no metastasis lesions were found. The patient then continued the administration of larotrectinib, and some lesion sizes were significantly reduced or even disappeared in the next few months. Currently, this patient is still alive. Conclusions Altogether, this report provided a new driver of lung adenocarcinoma expanded the mutational spectrum of NTRK1 fusion variants and suggested using larotrectinib as the targeted therapy is more effective than anti-PD-1 inhibitor in lung adenocarcinoma harboring with NTRK fusion, positive PD-L1 expression, and high TMB simultaneously.

Purpose: Because of their high tumor specificity and immunogenicity, neoantigens have been considered as ultimate targets for cancer immunotherapy. Neoantigen-based vaccines have demonstrated promising efficacy for several cancer types. To further investigate the antitumor potentials for other types of solid tumors, we designed a peptide-based neoantigen vaccine, iNeo-Vac-P01, and conducted a single-arm, open-labeled, investigator-initiated clinical trial (NCT03662815). Patients and Methods: Personalized neoantigen vaccines were designed and manufactured according to our bioinformatics analysis results from the whole-exome sequencing of tumor and peripheral blood cell DNAs. Patients were scheduled to be vaccinated subcutaneously with adjuvant on days 1, 4, 8, 15, and 22 (prime phase), and days 78 and 162 (boost phase). Additional immunizations were administrated every 2–3 months as per patient's potential benefit. The safety and efficacy were assessed through adverse events (AE), progression-free survival (PFS), overall survival (OS), and other parameters. Results: Of the 22 patients enrolled with advanced malignancies, 20 had no or mild AEs, while 2 had grade 3 or 4 acute allergic reactions only after their sixth boost vaccination. The disease control rate was 71.4%. The median PFS was 4.6 months, whereas the median OS was not reached (12-month OS = 55.1%). Around 80% of individual peptides or peptide pools elicited measurable specific immune response. In addition, our findings revealed several potential biomarkers for the prediction of better response. Conclusions: iNeo-Vac-P01 as monotherapy is feasible and safe for patients with advanced solid tumors. It could elicit T-cell–mediated immune response targeting tumor neoantigens, and might have promising antitumor efficacy. See related commentary by Filderman and Storkus, p. 4429

Purpose: Enhanced lipogenesis and mitochondrial function are two critical metabolic characteristics in melanoma, but their crosstalk involved in tumor biology and targeted therapy remains unknown. ATP-citrate lyase (ACLY) is a crucial lipogenic enzyme that is greatly implicated in tumor development, but its role in mitochondrial function and melanoma pathogenesis has not been elucidated. Experimental Design: In vitro and in vivo functional experiments were performed to determine the effect of ACLY on melanoma growth. mRNA expression profile analysis and a panel of biochemical assays were used to investigate the role of ACLY in mitochondrial oxidative phosphorylation and the underlying mechanism. The effect of combined ACLY inhibition on the efficacy of MAPK inhibition therapy was also examined. Results: We first found that ACLY expression was increased in melanoma and facilitated cell proliferation and tumor growth both in vitro and in vivo. Subsequent mRNA expression profile analysis and functional studies unveiled that ACLY specifically activated MITF–PGC1α axis to promote mitochondrial biogenesis and melanoma growth. Mechanistically, ACLY enhanced the activity of acetyltransferase P300, increasing the histone acetylation at MITF locus to promote MITF–PGC1α axis transcription. More importantly, the combined inhibition of ACLY sensitized BRAF-mutant melanoma to MAPK inhibition by suppressing MITF–PGC1α axis. Conclusions: We demonstrate that ACLY epigenetically potentiates oxidative phosphorylation to promote melanoma growth and MAPK inhibition adaptive resistance. Our study discovers the novel crosstalk between lipogenesis and mitochondrial function in tumor biology and highlights targeting ACLY as a potent therapeutic approach via simultaneously impairing tumor growth and MAPK inhibition resistance in melanoma.

Background Although researchers have shown that prodromal symptoms can predict acute coronary events, the ability of patients with acute coronary syndrome (ACS) to identify these symptoms in a timely manner is limited. Objectives We aimed to assess prodromal symptoms in Chinese patients with ACS and their responses to symptoms by sex. Design This cross-sectional, multicenter study involved 5 teaching hospitals in China and included 806 patients admitted for ACS between June 2013 and February 2014. The McSweeney Acute and Prodromal Myocardial Infarction Symptom Survey (Chinese version) was used to gather data. Results Among 806 patients (including 483 women), 688 (85.4%) experienced at least 1 prodromal symptom before ACS onset. Using adjusted logistic regression models, we determined that women were significantly more likely than men to report back pain, between- or under-shoulder blade pain/discomfort, sleep disturbances, anxiousness, or heart racing. The prevalence of generalized chest pain and loss of appetite was higher among men than women. Only 41% of patients attributed their prodromal symptoms to the heart, and women were more likely than men to attribute prodromal symptoms to a heart attack. Conclusions More than two-thirds of patients with ACS reported at least 1 prodromal symptom, with some significant sex differences. Most patients do not attribute their symptoms to an impending ACS event.

BackgroundRenal cell carcinoma (RCC) is a disease of genomic alterations, of which the complete panorama helps in facilitating molecular-guided therapy. Germline mutation profiles and associated somatic and clinical characteristics remains unexplored in Chinese RCC patients.MethodsWe retrospectively profiled the germline and somatic mutations of 322 unselected RCC patients using a panel consisting of 808 cancer-related genes. We categorized patients into three groups based on germline mutation status and compared the somatic mutation spectrum among different groups.ResultsApproximately one out of ten (9.9%) RCC patients were identified to carry pathogenic/likely pathogenic (P/LP) germline variants (PGVs), of which 3.7% were variants in syndromic RCC-associated genes and 6.2% were other cancer-predisposition genes. The most common PGV was found in VHL (2.2%), followed by FH, TSC2, ATM, BRCA1, NBN, and BLM (0.6% each). Young patients (≤46 years) were more likely to harbor PGVs. Variants in syndromic RCC-associated genes were predominant identified in young patients, while variants in other cancer-predisposition genes were found in patients >46 years more frequently. Furthermore, 39.3% (11/28) of patients carrying PGVs were detected to have somatic “second hit” events. Germline and somatic sequencing, including microsatellite instability (MSI) status analysis, provided potentially actionable therapeutic targets in 17.1% of patients in the whole cohort.ConclusionsOur results revealed that approximately 10% of RCC patients carried clinically significant germline mutations. Current guidelines recommendation for genetic testing seemed not sensitive enough to identify patients with hereditary RCC susceptibility. It is rational to promote genetic testing in RCC population.

Background Bladder cancer (BLCA) is one of most common urinary tract malignant tumor and immunotherapy have generated a great deal of interest in BLCA. Immune checkpoint blockade (ICB) therapy has significantly progressed the treatment of BLCA. Multiple studies have suggested that specific genetic mutations may serve as immune biomarkers for ICB therapy. Objective In this study, we aimed to investigate the role of mutations genes and subtypes in prognosis and immune checkpoint prediction in BLCA. Method Mutation information and expression profiles were acquired from The Cancer Genome Atlas (TCGA) database. Integrated bioinformatics analysis was carried out to explore the mutation genes of BLCA. Functional enrichment analysis Gene Ontology (GO) and Gene set enrichment analysis (GSEA) was conducted. The infiltrating immune cells and the prediction of ICB between different subtypes group were explored using immuCellAI algorithm. Results The mutation genes Filaggrin (FLG) gene were identified. Following the study on its subtypes and functional enrichment analysis, Sub2 of FLG-wide type was found to have relationships with poor prognosis and immune infiltration BLCA. What’s more, Sub2 of FLG-wide type may be used as a biomarker to predict the prognosis of BLCA patients receiving ICB. Conclusion This research provides a new basis and ideas for guiding the clinical application of BLCA immunotherapy.

The tumor mutational burden (TMB) calculated by whole-exome sequencing (WES) is a promising biomarker for the response to immune checkpoint inhibition (ICIs) in solid tumors. However, WES is not feasible in the routine clinical setting. In addition, the characteristics of the TMB in Chinese urothelial carcinoma (UC) are unclear. The aim of this study was to demonstrate the reliability of an Acornmed 808 panel and analyze the characteristics of the TMB in Chinese UC. An Acornmed 808 panel was designed and virtually validated using UC data from the cancer genome atlas (TCGA). Comprehensive analysis of sequencing and clinical data was performed to explore the characteristics of the TMB for 143 Chinese UC patients. Compared to the TMB calculated with random 808-, 500-, and 250-gene panels, the TMB calculated with the Acornmed 808 panel was closer to that calculated by WES. There were marked disparities in the mutational landscape and TMB between Chinese and TCGA UC data. The TMB was negatively associated with copy number variation (CNV). In contrast, the TMB was positive correlation with numbers of mutated DDR genes. Exposure to aristolochic acid signature was observed only in the TMB-high groups. The Acornmed 808 panel is a clinically practical method to assess the TMB. The TMB was associated with the DDR gene status and CNV counts and might be a biomarker for further stratification of UC patients. The study suggested that patients with high TMB may have a unique carcinogenic mechanism.

Anaplastic lymphoma kinase (ALK) fusions have been recognized as a therapeutic target in non‐small cell lung cancer (NSCLC). However, molecular signatures and clinical characteristics of the Chinese population with ALK‐rearranged NSCLC are not well elucidated. In the present study, we carried out targeted next‐generation sequencing on tissue and plasma ctDNA samples in 1688 patients with NSCLC. Overall, ALK fusions were detected in 70 patients (4.1%), and the frequencies of ALK fusions detected in tissue and plasma samples were 5.1% and 3.3%, respectively. Additionally, the prevalence of breakpoint locations for EML4‐ALK fusions in ctDNA was significantly correlated with that in tumor tissues (R 2 = .91, P = .045). According to age, the incidence rates of ALK fusions among young (age 70 years) patients were significantly different (P

Mitochondrial dysregulation has been implicated in oxidative stress-induced melanocyte destruction in vitiligo. However, the molecular mechanism underlying this process is merely investigated. Given the prominent role of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase Sirtuin3 (SIRT3) in sustaining mitochondrial dynamics and homeostasis and that SIRT3 expression and activity can be influenced by oxidative stress-related signaling, we wondered whether SIRT3 could play an important role in vitiligo melanocyte degeneration by regulating mitochondrial dynamics. Methods: We initially testified SIRT3 expression and activity in normal and vitiligo melanocytes via PCR, immunoblotting and immunofluorescence assays. Then, cell apoptosis, mitochondrial function and mitochondrial dynamics after SIRT3 intervention were analyzed by flow cytometry, immunoblotting, confocal laser microscopy, transmission electron microscopy and oxphos activity assays. Chromatin immunoprecipitation (ChIP), co-immunoprecipitation (Co-IP), immunoblotting and immunofluorescence assays were performed to clarify the upstream regulatory mechanism of SIRT3. Finally, the effect of honokiol on protecting melanocytes and the underlying mechanism were investigated via flow cytometry and immunoblotting analysis. Results: We first found that the expression and the activity of SIRT3 were significantly impaired in vitiligo melanocytes both in vitro and in vivo. Then, SIRT3 deficiency led to more melanocyte apoptosis by inducing severe mitochondrial dysfunction and cytochrome c release to cytoplasm, with Optic atrophy 1 (OPA1)-mediated mitochondrial dynamics remodeling involved in. Moreover, potentiated carbonylation and dampened peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) activation accounted for SIRT3 dysregulation in vitiligo melanocytes. Finally, we proved that honokiol could prevent melanocyte apoptosis under oxidative stress by activating SIRT3-OPA1 axis. Conclusions: Overall, we demonstrate that SIRT3-dependent mitochondrial dynamics remodeling contributes to oxidative stress-induced melanocyte degeneration in vitiligo, and honokiol is promising in preventing oxidative stress-induced vitiligo melanocyte apoptosis.

Autophagy plays a protective role in the oxidative stress-induced melanocyte death. Dysregulated autophagy increases the sensitivity of melanocytes in response to oxidative damage and promotes the melanocyte degeneration in vitiligo. However, the molecular mechanism underlying this process is not fully understood. Here, using RNA-Seq technology, we compared the transcriptome change between normal and vitiligo melanocytes with or without treatment of oxidative stress. We found that autophagy-related protein 5 and 12 (Atg5 and Atg12), the critical components for autophagosome formation, were significantly reduced in vitiligo melanocytes under oxidative stress. Mechanistically, heat shock factor 1 (HSF1) is the prime transcription factor for both Atg5 and Atg12, accounting for the reduced level of Atg5 and Atg12 in vitiligo melanocytes. Deficiency of HSF1 led to accumulation of intracellular reactive oxygen species (ROS), imbalance of mitochondrion membrane potential and apoptosis in melanocytes exposure to oxidative stress. Further, overexpression of HSF1 could ameliorate oxidative stress-induced melanocytes death by activation of autophagy through upregulating Atg5 and Atg12. These findings suggested targeting HSF1-Atg5/12 axis could prevent oxidative stress-induced melanocyte death and may be used as therapeutic strategies for vitiligo treatment.

ObjectiveThe incidence of early stage multiple primary lung cancer (MPLC) has been increasing in recent years, while the ideal strategy for its diagnosis and treatment remains controversial. The present study conducted genomic analysis to identify a new molecular classification method for accurately predicting the diagnosis and therapy for patients with early stage MPLC.MethodsA total of 240 tissue samples from 203 patients with multiple-non-small-cell lung cancers (NSCLCs) (n = 30), early stage single-NSCLC (Group A, n = 94), and advanced-stage NSCLC (Group B, n = 79) were subjected to targeted multigene panel sequencing.ResultsThirty patients for whom next-generation sequencing was performed on >1 tumor were identified, yielding 45 tumor pairs. The frequencies of EGFR, TP53, RBM10, ERBB2, and CDKN2A mutations exhibited significant differences between early and advanced-stage NSCLCs. The prevalence of the EGFR L858R mutation in early stage NSCLC was remarkably higher than that in advanced-stage NSCLC (P = 0.047). The molecular method classified tumor pairs into 26 definite MPLC tumors and four intrapulmonary metastasis (IM) tumors. A high rate of discordance in driver genetic alterations was found in the different tumor lesions of MPLC patients. The prospective Martini histologic prediction of MPLC was discordant with the molecular method for three patients (16.7%), particularly in the prediction of IM (91.7% discordant).ConclusionsComprehensive molecular evaluation allows the unambiguous delineation of clonal relationships among tumors. In comparison, the Martini and Melamed criteria have notable limitations in the recognition of IM. Our results support the adoption of a large panel to supplement histology for strongly discriminating NSCLC clonal relationships in clinical practice.

Chun Zeng,1,2 Jing Wang,3 Mingwei Li,4 Huina Wang,4 Feng Lou,4 Shanbo Cao,4 Changyu Lu3 1Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, People’s Republic of China; 2China National Clinical Research Center for Neurological Diseases, Beijing, People’s Republic of China; 3Department of Neurosurgery, Peking University International Hospital, Beijing, People’s Republic of China; 4Acornmed Biotechnology Co., Ltd, Beijing, People’s Republic of ChinaCorrespondence: Changyu LuDepartment of Neurosurgery, Peking University International Hospital, Beijing, People’s Republic of ChinaTel +8669006144Email luchangyu2020@163.comShanbo CaoAcornmed Biotechnology Co., Ltd, Floor 18, Block 5, Yard 18, Kechuang 13 Road, Beijing, 100176, People’s Republic of ChinaTel +8653606156Email shanbocao@acornmed.comBackground: Tremendous efforts have been made to explore biomarkers for classifying and grading glioma. However, the majority of the current understanding is based on public databases that might not accurately reflect the Asian population. Here, we investigated the genetic landscape of Chinese glioma patients using a validated multigene next-generation sequencing (NGS) panel to provide a strong rationale for the future classification and prognosis of glioma in this population.Methods: We analyzed 83 samples, consisting of 71 initial treatments and 12 recurrent surgical tumors, from 81 Chinese patients with gliomas by performing multigene NGS with an Acornmed panel targeting 808 cancer-related hotspot genes, including genes related to glioma (hotspots, selected exons or complete coding sequences) and full-length SNPs located on chromosomes 1 and 19.Results: A total of 76 (91.57%) glioma samples had at least one somatic mutation. The most commonly mutated genes were TP53, TERT, IDH1, PTEN, ATRX, and EGFR. Approximately one-third of cases exhibited more than one copy number variation. Of note, this study identified the amplification of genes, such as EGFR and PDGFRA, which were significantly associated with glioblastoma but had not been previously used for clinical classification (P< 0.05). Significant differences in genomic profiles between different pathological subtypes and WHO grade were observed. Compared to the MSKCC database primarily comprised of Caucasians, H3F3A mutations and MET amplifications exhibited higher mutation rates, whereas TERT mutations and EGFR and CDKN2A/B copy number variations presented a lower mutation rate in Chinese patients with glioma (P< 0.05).Conclusion: Our multigene NGS in the simultaneous evaluation of multiple relevant markers revealed several novel genetic alterations in Chinese patients with glioma. NGS-based molecular analysis is a reliable and effective method for diagnosing brain tumors, assisting clinicians in evaluating additional potential therapeutic options, such as targeted therapy, for glioma patients in different racial/ethnic groups.Keywords: glioma, molecular pathology, molecular biomarker, copy number variations, multigene NGS panel

Background Different genomic characterization in urothelial carcinoma (UC) by site of origin may imply contrasting therapeutic opportunities and pathogenetic mechanisms. The aim of this study was to investigate whether differences between upper tract UC (UTUC) and UC of the bladder (UCB) result from intrinsic biological diversity. Materials and Methods We prospectively sequenced 118 tumors and matched blood DNA from Chinese patients with UC using next-generation sequencing techniques, including 45 UTUC and 73 UCB. Two hundred twenty-six patients with UTUC and 350 patients with UCB for The Cancer Genome Atlas were acquired from the cbioportal. Results There were marked disparities in the mutational landscape for UC according to race and site of origin. Signature 22 for exposure to aristolochic acid was only observed in the UTUC cohort. Conversely, signature 6 for defective DNA mismatch repair only existed in the UCB cohort. Compared with UCB, UTUC had higher clonal and subclonal mutation numbers. TP53, PIK3CA, and FGFR3 mutations may be the driver genes for UTUC, whereas for UCB, the driver gene may be BRCA1. Patients with UTUC had lower PD-L1 than those with UCB. There was no significant difference in the number of DDR mutations, copy number variation counts, and tumor mutational burden between UTUC and UCB. Conclusion UTUC and UCB exhibit significant differences in the prevalence of genomic landscape and carcinogenesis. Consequently, molecular subtypes differ according to location, and these results may imply the site-specific management of patients with urothelial carcinoma. Mutational signature may be used as a screening tool to assist clinical differential diagnosis between UTUC and UCB. Implications for Practice This study's findings lay the foundation for a deeper understanding of distinct molecular mechanisms and similar treatment opportunities between upper tract urothelial carcinoma (UTUC) and urothelial carcinoma of the bladder (UCB) and had important implications for the site-specific management of patients with urothelial carcinoma. A comprehensive understanding of the biology of UTUC and UCB is needed to identify new drug targets in order to improve clinical outcomes.

BackgroundThe therapeutic effect of immune checkpoint blockers, especially the neutralizing antibodies of programmed cell death (PD-1) and its ligand programmed death ligand 1 (PD-L1), has been well verified in melanoma. Nevertheless, the dissatisfactory response rate and the occurrence of resistance significantly hinder the treatment effect. Inflammation-related molecules like A20 are greatly implicated in cancer immune response, but the role of tumorous A20 in antitumor immunity and immunotherapy efficacy remains elusive.MethodsThe association between tumorous A20 expression and the effect of anti-PD-1 immunotherapy was determined by immunoblotting, immunofluorescence staining and flow cytometry analysis of primary tumor specimens from melanoma patients. Preclinical mouse model, in vitro coculture system, immunohistochemical staining and flow cytometry analysis were employed to investigate the role of A20 in regulating the effect of anti-PD-1 immunotherapy. Bioinformatics, mass spectrum analysis and a set of biochemical analyzes were used to figure out the underlying mechanism.ResultsWe first discovered that upregulated A20 was associated with impaired antitumor capacity of CD8+T cells and poor response to anti-PD-1 immunotherapy in melanoma patients. Subsequent functional studies in preclinical mouse model and in vitro coculture system proved that targeting tumorous A20 prominently improved the effect of immunotherapy through the invigoration of infiltrating CD8+T cells via the regulation of PD-L1. Mechanistically, A20 facilitated the ubiquitination and degradation of prohibitin to potentiate STAT3 activation and PD-L1 expression. Moreover, tumorous A20 expression was highly associated with the ratio of Ki-67 percentage in circulating PD-1+CD8+T cells to tumor burden.ConclusionsTogether, our findings uncover a novel crosstalk between inflammatory molecules and antitumor immunity in melanoma, and highlight that A20 can be exploited as a promising target to bring clinical benefit to melanomas refractory to immune checkpoint blockade.

Melanoma is the most lethal skin cancer caused by the malignant transformation of epidermal melanocytes. Recent progress in targeted therapy and immunotherapy has significantly improved the treatment outcome, but the survival of patients with advanced melanoma remains suboptimal. Ferroptosis, a cell death modality triggered by iron-dependent lipid peroxidation, reportedly participates in cancer pathogenesis and can mediate the effect of anti-PD-1 immunotherapy in melanoma. However, the detailed regulatory mechanism of ferroptosis remains far from being understood. In this study, we report that CAMKK2 defines the ferroptosis sensitivity of melanoma cells by regulating the AMPK‒NRF2 pathway. We first found that CAMKK2 was prominently activated in ferroptosis. Then we proved that CAMKK2 negatively regulated ferroptosis through the activation of NRF2 and the suppression of lipid peroxidation. Subsequent mechanistic studies revealed that AMPK connected CAMKK2 upregulation to NRF2-dependent antioxidative machinery in ferroptosis. In addition, the suppression of CAMKK2 increased the efficacy of ferroptosis inducer and anti-PD-1 immunotherapy in the preclinical xenograft tumor model by inhibiting the AMPK‒NRF2 pathway and promoting ferroptosis. Taken together, CAMKK2 plays a protective role in ferroptosis by activating the AMPK‒NRF2 pathway. Targeting CAMKK2 could be a potential approach to increase the efficacy of ferroptosis inducers and immunotherapy for melanoma treatment.

Background MicroRNAs are reported to play an important role in tumor growth and metastasis, including squamous cell carcinoma (SCC). Accumulative evidence has revealed that dysregulated miR-340 expression contributed to the carcinogenesis and development of various cancers. Objective The aim of the current study was to investigate the role and the underlying mechanism of miR-340 in SCC cell proliferation, migration and invasion. Methods Quantitative real-time PCR was performed to examine the expression of miR-340 in SCC tissues and cell lines. The function of miR-340 in SCC was investigated through Cell Counting Kit-8, wound healing, transwell migration and invasion assays. Bioinformatics analysis, luciferase reporter assay, western blotting and immunohistochemical analysis were conducted to predict and confirm the target gene of miR-340. Results In the present study, we first found that miR-340 was significantly decreased in both SCC tissues and cell lines. Moreover, ectopic expression of miR-340 remarkably attenuated SCC cell proliferation, migration and invasion, whereas inhibition of endogenous miR-340 promoted SCC cell proliferation, migration and invasion in vitro. Our subsequent bioinformatics analysis and luciferase reporter assay showed that RhoA was a novel direct target of miR-340 in SCC cells, and the knockdown of RhoA expression rescued the effects of miR-340 inhibition on SCC cell proliferation, migration and invasion. More importantly, the expression of RhoA and miR-340 was negatively correlated in SCC tissues. Conclusion Our findings demonstrate the tumor suppressor role of miR-340 in SCC by directly regulating RhoA. Therefore, restoration of miR-340 expression can be a potential therapeutic approach for SCC treatment.

Melanoma is among the most life-threatening cancers. The pathogenesis of melanoma has not been fully elucidated. Recently, dysregulated macroautophagy/autophagy has been found to play a critical but inconsistent role in modulating melanoma growth at different stages, with the regulatory mechanism unclear. The histone deacetylase SIRT6 (sirtuin 6) is a known autophagy regulator, and its involvement in cancer development has been reported. Therefore, we sought to determine the role of SIRT6 in melanoma growth and detect its possible link with autophagy in the current study. We initially observed that the expression of SIRT6 decreased in primary melanoma but increased in metastatic melanoma compared with melanocytic nevus. Notably, the expression of SIRT6 was significantly correlated with the expression of autophagy biomarkers including MAP1LC3/LC3 and SQSTM1/p62. Furthermore, SIRT6 suppressed the growth of primary melanoma but promoted metastatic melanoma development in an autophagy-dependent way in vitro. Moreover, SIRT6 exerted its regulation on melanoma growth via the IGF-AKT signaling pathway, and the intervention of AKT could partly reverse the effects of SIRT6 on melanoma growth by regulating autophagy. At last, we determined the effects of SIRT6 on melanoma development in vivo. Taken together, our findings demonstrate that the bimodal expression of SIRT6 at different melanoma stages plays a critical role in regulating melanoma growth through an autophagy-dependent manner, which indicates the potential of SIRT6 to be a biomarker and a therapeutic target in melanoma.

Psoriasis is an autoimmune skin disease, in which keratinocytes play a crucial pathogenic role. High-mobility group protein B1 (HMGB1) is an inflammatory factor that can be released from keratinocyte nuclei in psoriatic lesions. We aimed to investigate the proinflammatory effect of HMGB1 on keratinocytes and the contribution of HMGB1 to psoriasis development. Normal human keratinocytes were treated with recombinant human HMGB1, and the production of inflammatory factors and the intermediary signalling pathways were examined. Furthermore, the imiquimod-induced psoriasis-like mouse model was used to investigate the role of HMGB1 in psoriasis development in vivo. A total of 11 inflammatory factors were shown to be upregulated by HMGB1 in keratinocytes, among which interleukin (IL)-18 showed the greatest change. We then found that activation of the nuclear factor-κB signalling pathway and inflammasomes accounted for HMGB1-induced IL-18 expression and secretion. Moreover, HMGB1 and downstream IL-18 contributed to the development of psoriasiform dermatitis in the imiquimod-treated mice. In addition, T-helper 17 immune response in the psoriasis-like mouse model could be inhibited by both HMGB1 and IL-18 blockade. Our findings indicate that HMGB1 secreted from keratinocytes can facilitate the production and secretion of inflammatory factors such as IL-18 in keratinocytes in an autocrine way, thus promoting the development of psoriasis. Blocking the proinflammatory function of the HMGB1-IL-18 axis may be useful for psoriasis treatment in the future. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Distant metastases are responsible for the majority of melanoma mortalities. As a critical barrier against metastasis, anoikis is a distinct programmed cell death induced by the integrated stress from extracellular matrix (ECM) detachment. In order to survive, tumor cells employ various strategies for overcoming this barrier. Recently, Sesn2 has been reported to play a protective role against integrated stress. In this study, we found that ECM detachment triggered the upregulation of Sesn2 in metastatic melanoma cells. The knockdown of Sesn2 impaired not only the cell viability but also the tumor sphere formation of melanoma cells in suspension cultures. Moreover, an elevated oxidative stress level was detected in Sesn2-silencing melanoma cells in suspension cultures, accompanied with an increased apoptosis rate. Finally, in vivo studies indicated that the Sesn2-knockdown reduced the formation of distant metastasis remarkably. Taken together, our findings illustrated that the upregulation of Sesn2 in response to suspension stress plays a protective role in melanoma against anoikis by detoxifying oxidative stress.

2601 Background: Microsatellite instability (MSI) is a common genomic alteration in several tumors, such as colorectal cancer, endometrial carcinoma, and stomach, which is characterized as microsatellite instability-high (MSI-H) and microsatellite stable (MSS) based on a high degree of polymorphism in microsatellite lengths. MSI is a predictive biomarker for immunotherapy efficacy in advanced/metastatic solid tumors, especially in colorectal cancer (CRC) patients. Several computational approaches based on target panel sequencing data have been used to detect MSI; However, they are considerably affected by the sequencing depth and panel size. Methods: We developed MSIFinder, a python package for automatic MSI classification, using random forest classifier (RFC)-based genome sequencing, which is a machine learning technology. We included 19 MSI-H and 25 MSS samples as training sets. First, RFC model were built by 54 feature markers from the training sets. Second. The software was validated the classifier using a test set comprising 21 MSI-H and 379 MSS samples. Results: With this test set, MSIFinder achieved a sensitivity (recall) of 0.997, a specificity of 1, an accuracy of 0.998, a positive predictive value (PPV) of 0.954, an F1 score of 0.977, and an area under curve (AUC) of 0.999. We discovered that MSIFinder is less affected by low sequencing depth and can achieve a concordance of 0.993, while exhibiting a sequencing depth of 100×. Furthermore, we realized that MSIFinder is less affected by the panel size and can achieve a concordance of 0.99 when the panel size is 0.5 m (million base). Conclusions: These results indicated that MSIFinder is a robust MSI classification tool and not affected by the panel size and sequencing depth. Furthermore, MSIFinder can provide reliable MSI detection for scientific and clinical purposes.[Table: see text]

e17531 Background: Ovarian Cancer (OC) is the most lethal cancer of all gynecological malignancies.Circulating tumor DNA (ctDNA) has received substantial attention in recent years resulting from the non-invasive, safe and effective method with considerable potential for clinical diagnosis and treatment management in patients with OC. Here, we assessed the mutational feature in homologous recombination repair (HRR) using ctDNA in OC. Methods: Plasma ctDNA was isolated from blood of patients and then was analyzed by AcornMed Biotechnology NGS-based assay for 808 genes panel for genomic alterations. The somatic and germline pathogenic mutations were identified in 12 HRR genes (ATM, BRCA1, BRCA2, BRIP1, CHEK1, CHEK2, FANCA, PALB2, RAD51B, RAD51C, RAD51D, RAD54L). Results: At our institution, 85 patients underwent NGS analysis of ovarian cancer specimens. The median age was 57 (range from 26 to 83). Twenty-six patients(42.34%) harbored a mutation in at least 1 of the HRR genes in their tumor. The most commonly altered HRR gene was BRCA1 (18.25%), followed by BRCA2 (8.76%), ATM (5.84%), RAD51D(3.65%), CHEK2 (2.92%), FANCA(2.19%) and RAD51C (0.73%). To determine the difference of mutation landscape in HRR between Chinese and western populations, we compared prevalence and spectrum in cases between our cohort and the cohort of Heeke et al 1(Table). Prevalence of HRR were different from the Western cases(42.34% vs 20%, P＜0.05). Of note, BRCA1, BRCA2, ATM and CHEK2 alterations rate was higher in our cohort, and BRIP1 and PALB2 were only detected in the western cohort. Conclusions: CtDNA can characterize the mutational feature of HRR in OC. Around 42.3% of patients with OC harbour germline or somatic HRR mutations. The expanded use of PARP inhibitors in HRR deficient tumours using a signature of HRR by ctDNA in clinical practice requires validation.[Table: see text]

e15078 Background: Recently, homologous recombination deficiency (HRD) scores are associated with the efficacy of Poly‐(ADP‐Ribose)‐Polymerase (PARP) inhibition and platinum-based chemotherapy in a variety of cancers. Evaluating HRD level in patients with cancers is becoming far more important and influential, so far, there is no standard method to be used in clinical. In this study, we developed an algorithm to detect HRD from next-generation sequencing (NGS) for finding additional patients may potentially benefit from target therapy. Methods: Forty-eight patients were enrolled, including breast cancer, ovarian cancer, prostatic cancer. Fifteen cell lines with breast cancer and endometrial carcinoma were collected from Cobioer biosciences co., LTD. Forty-eight Formalin-fixed, paraffin embedded (FFPE) samples and 15 cell lines were performed by DNA extracting. We developed an HRD score algorithm, termed as AcornHRD algorithm. HRD score was analyzed by whole-genome sequencing, and GATK mutect2 software was used to detect BRCA1/2mutation by deep sequencing. Results: BRCA1/2 deleterious mutations were observed in 20 patients (41.7%). HRD was explained by deficiencies in 17 patients (85.0%) with BRCA mutation, whereas eight HRD-high tumors were non- BRCA related (28.6%). Among BRCA wild-type patients, the corresponding percentage of HRD positive patients in breast cancer, ovarian cancer and prostate cancer were 36.3%, 37.5% and 11.1%, respectively. Similar results were also verified in the cell line datasets. The findings showed that 100% (3/3) BRCA1/2 deficient cell lines are also HRD-high. Furthermore, HRD scores were highly correlated with standard results in the cell line datasets. Conclusions: We here report the NGS-based HRD scores to distinguish similarly well between BRCA mutant and BRCA wild-type cases in a cohort of Chinese population. AcornHRD scores were highly associated with BRCA1/2 deficiency. AcornHRD algorithm can be a useful tool to detect HRD events in clinical settings.

e16140 Background: Hepatocellular carcinoma (HCC) is one of the most common malignancies and leading cause of cancer deaths worldwide. Biomarkers contribute to predict the response of targeted treatments and immunotherapy. Therapy directed toward homologous recombination deficiency (HRD) is now approved in ovarian and breast cancer, but the pattern of HRD is not clear in HCC. The HRD phenotype has been defined as the presence of a non-silent somatic mutation in homologous recombination-related (HRR) genes. Thus, we aimed to analyze the molecular characteristics of resectable HCC, including HRR genes. Methods: Matched tumor/normal DNA from resectable HCC patients (N = 53) were analyzed by whole exome sequencing (WES) or Acornmed panel with 808 cancer-related genes. Results: Overall, 94.3% (50/53) patients exhibited genetic alterations. TP53, ARID1A, PTEN and NBPF1 were the most commonly mutated genes in resectable HCC. In addtion, 35.9% patinets harbored at least one HRR genes mutation. The frequently mutated genes were BRCA2 (4.4%), BRCA1(4.0%), ATM (3.9 %), and RAD50 (3.8%). Interesting, frequency of HRR genes mutation in the Chinese cohort was higher than that in TCGA (35.9 % vs 20.9%). Analysis of carcinogenic pathways shown that ERBB and PI3K-AKT signaling pathway were the common signaling pathway. In resectable HCC, 32.08% (17/53) patients disclosed high TMB (highest 25%). Further analysis found that PTEN mutations were remarkably associated with low TMB (p < 0.05). Conclusions: This study contributes to understand the molecular characteristics of patients with resectable HCC, which will be useful to guide personalized therapy and promote the clinical management in this population. Furthermore, the study suggested that it is feasibility for resectable HCC patients received PARP inhibitors, DNA-damaging chemotherapies, and immune checkpoint blockade therapy.

e20527 Background: Lung cancer is currently a leading cause of cancer-associated mortality worldwide. Germline mutations potentially predisposing patients to lung cancer have not been systematically defined. In this study, we sought to determine the prevalence of pathogenic/likely pathogenic (P/LP) germline mutations in patients with NSCLC. Methods: A total 1562 patients with NSCLC were included in this study. Leukocyte DNA was extracted and detected were using next generation sequencing technology. Allele frequencies were compared with publicly available database. Interpretation of sequence variations was classified according to the American College of Medical Genetics and Genomics guidelines. Results: Overall, median age at testing of individuals was 63 years (range 12 to 91). The majority of individuals tested were male (56.15%, 877/1562). of 1562 NSCLC, P/LP mutations were identified in 94 (6.02%) in 42 Cancer Susceptibility genes. No individuals carried more than 1 germline variant. The most common P/LP mutations in genes were MUTYH (n = 11, 0.70%), TP53 (n = 10, 0.64%), BRCA2 (n = 8, 0.51%), PALB2(n = 5, 0.32%), PTEN(n = 4, 0.26%), BLM(n = 3, 0.19%), WRN(n = 3, 0.19%), and ATR, BRCA1 and BRIP1 (n = 2, 0.13% each). Twenty eight patients were carried germline mutations in homologous recombination repair(HRR), including BRCA2 (8), PALB2 (5), PTEN (4), BRCA1 (2), BRIP1 (2), CHEK2 (2), FANCA (2), ATM (1), RAD51B (1), RAD54L(1). Of note, the clinical features of patients with HRR mutations were not young adult (median age [range], 57 [41-80] years). Conclusions: Of 1562 patients with NSCLC, 94 (6.02%) had germline mutations and 27 (1.73%) had mutations in HRR genes. The presence of HRR germline mutations could guide cancer screening for patients and their families and serve as predictive biomarkers of response to targeted or immunotherapies. More extensive germline testing in NSCLC should be considered.

e13006 Background: Breast cancer (BC) is the most common cancer in women world-wide. Assessments of genomic variants in circulating tumor DNA (ctDNA) have generated great enthusiasm for their potential application as clinically actionable biomarkers in the management of BC. Alterations in homologous recombination repair (HRR), are a determinant of sensitivity to platinum chemotherapy and poly (ADP-ribose) polymerase inhibitors (PARPi) in BC. However, as far as we known, most of HRR genes, except BRCA1/2, have not yet been fully understood to date. In this study, we aimed to elucidate the comprehensive HRR genetic alteration profile of breast tumors among Chinese patients by ctDNA analysis. Methods: Plasma ctDNA from 259 patients with BC were deeply sequenced via next-generation sequencing (NGS) techniques using AcornMed Biotechnology for 808 genes panel. Molecular profiles were reviewed to identify somatic and germline pathogenic mutations in the 14 HRR genes ( ATM, BRCA1, BRCA2, BRIP1, CHEK1, CHEK2, FANCA, PALB2, RAD51B, RAD51C, RAD51D, RAD54L, CDK12, and BARD1) . Results: At our institution, 259 patients underwent NGS analysis of BC specimens. The median age was 46 (range from 31 to 79). Based on the analysis of the genetic alteration profile from our cohort, at least one of the HRR genes was observed from 29.34% of the tumor samples, with BRCA1 (8.11%), BRCA2 (5.79%), RAD51C (5.02%), CDK12 (4.63%), CHEK2 (1.93%), ATM (1.16%), and BARD1 (1.16%) as the most commonly altered genes. As compared with MSKCC dataset (Table), genetic alterations detected from our cohort affected genes involved in HRR (29.34% vs. 14.03%, P＜0.0001) pathways, with statistically different genetic alteration rates. Moreover, BRCA1/2, RAD51C, CDK12 and CHEK2 mutations exhibited higher mutation rates, whereas ATM, BRIP1 and PALB2 mutations presented a lower mutation rate in Chinese patients with breast cancer (P＜0.05). Conclusions: CtDNA can characterize the mutational feature of HRR in BC. our study contributes to the understanding of the HRR pathways and specific genetic alterations harbored by Chinese patients with BC that could potentially be developed as markers of treatment response to targeted therapeutics. Ref: Razavi P, Chang MT, Xu GT, et al. The Genomic Landscape of Endocrine-Resistant Advanced Breast Cancers. Cancer Cell. 2018 Sep 10;34(3):427-438.e6. doi: 10.1016/j.ccell.2018.08.008.[Table: see text]

e15525 Background: Homologous recombination deficiency (HRD) might serve as a biomarker of immunotherapy and chemotherapy response. In addtion, HRD is associated with sensitivity to PARP inhibitor (PARPi) therapy. The HRD phenotype has been defined as the presence of a non-silent somatic mutation in homologous recombination-related (HRR) genes. Here, we focused on mutational landscape of HRR genes and the relationship between HRR-related genes and clinical outcomes in colon cancer. Methods: We prospectively sequenced 784 Chinese patients with colon cancer using next-generation sequencing techniques with 808 cancer-related genes. 290 patients with somatic mutation from the TCGA project were included. The 15 HRR genes were selected on clinical evidence of sensitivity to PARP inhibitors and immunotherapy. TCGA datasets were performed to identify the correlations between HRR gene mutations and clinical outcomes. Results: In the Chinese cohort, 11.5% (90/784) patients exhibited deleterious somatic mutation in HRR genes, including ATM (3.3%), BRCA2 (2.4%), CDK12 (2.4%), BRCA1 (1.0%), BRIP1 (1.0%), CHEK2 (0.5%), BARD1 (0.4%), RAD54L (0.4%), PALB2 (0.3%), and RAD51B (0.3%). In TCGA cohort, 28.6% (83/290) patients had at least one deleterious somatic mutation in HRR genes, including ATM (14.8%), BRCA2 (9.0%), CDK12 (7.9%), BRIP1 (3.8%), BRCA1 (3.4%), FANCL (3.1%), CHEK2 (2.4%), PALB2 (2.1%), PPP2R2A (2.1%), RAD54L (2.1%), CHEK1 (1.7%), BARD1 (1.4%), RAD51C (1.4%), RAD51B (1.0%), and RAD51D (0.3%). Overall, the mutational frequency of HRR genes in the Chinese cohort was lower than that in the TCGA cohort (p < 0.001). In the TCGA cohort, colon patients with HRR-mut had significantly higher tumor mutational burden and incidence of microsatellite instability high and enhanced immune activity than patients with HRR-wt. Furthermore, In the MSK-IMPACT cohort treated by immune checkpoint inhibitor, patients with HRR-mut had significantly better OS (p = 0.009) and increased immune activities, such as infiltration of cytotoxic cells (p < 0.05) and exhausted CD8+ T cells (p < 0.01), and increased the IFN-γ scores (p < 0.05) than patients with HRR-wt. Conclusions: Our data suggest that detection of somatic mutations in HRR genes might contribute to identify patients who might benefit from immune checkpoint blockade therapy and PARP inhibitors. In addition, the molecular features of HRR provide new opportunities to predict the tumor response to multiple treatments. Exploring other biomarkers of HRD to predict the response to PARPi and immunotherapeutic in colon cancer is necessary.

e15073 Background: Homologous recombination deficiency (HRD) can regulate the tumor immune microenvironment based on increasing tumor-infiltrating lymphocytes and therefore might serve as a biomarker of immunotherapeutic response. The HRD phenotype has been defined as the presence of a non-silent somatic mutation in homologous recombination-related (HRR) genes. Here, we focused on frequency of HRR genes mutation and the relationship between HRR genes mutation and clinical immunotherapy responses in melanoma. Methods: We prospectively sequenced 130 Chinese melanoma patients using next-generation sequencing techniques with Acornmed panel with 808 cancer-related genes. 208 patients with WES data from the TCGA project were included. Correlations between HRR gene mutations and clinical outcomes were identified via bioinformatic analysis using TCGA datasets. Results: In the Chinese cohort, 40% (52/130) patients exhibited genomic alterations in HRR genes. The frequently mutated genes were ATM (14%), BRCA2 (13%), CHEK2 (6%), PALB2 (6%), BRIP1 (4.6%), BARD1 (3%), BRCA1 (2%), CDK12 (2%), CHEK1 (1%), FANCL (1%), and RAD54L (1%). In TCGA cohort, 26% (53/208) patients had at least one somatic mutation in an HRR gene. The frequently mutated genes were BRCA2 (9%), BRCA1 (7%), ATM (6%), BRIP1 (4%), PALB2 (4%), BRAD1 (1%), CHEK2 (1%), PPP2R2A (1%), RAD51B (1%), RAD51C (1%), RAD54L (1%), CHEK1 (1%), and RAD51D. Chinese melanoma patients had higher frequently mutated HRR genes compared to that in TCGA patients (40% vs 26%, p=0.005). In addition, compared with TCGA cohort, the Chinese cohort had higher frequency for ATM (14% vs 6%, p=0.019) and CHEK2 (6% vs 2%, p=0.006). The TCGA cohort had a higher frequency of BRCA1 mutations than the Chinese cohort, although the difference was not statistically significant. Unfortunately, we did not find a significant correlation between HRR mutation and immune cells. Interestingly, in the TCGA cohort, patients with HRR-mut had significantly better OS than patients with HRR-wt (log-rank p=0.027). Conclusions: Our data suggest that detection of somatic mutations in HRR genes might contribute to identify patients who might benefit from immune checkpoint blockade therapy. In addition, the molecular features of HRD provide new opportunities to predict the tumor response to multiple treatments.[Table: see text]

Melanoma is among the most malignant cancers with notorious aggressiveness, and its prognosis is greatly influenced by progression status. Serum microRNAs are small noncoding RNAs with high stability and easy accessibility in human blood. Their expression profiles are frequently dysregulated in cancers; hence, levels of serum microRNAs may reflect progression status and thus predict melanoma prognosis. In a hospital based case-control study, we found a significant reduction of serum miR-16 level in melanoma patients compared with cancer-free controls (P0.001). In addition, serum miR-16 level markedly decreased in melanoma patients with increased tumor thickness, occurrence of ulceration, and advanced American Joint Committee on Cancer stages, and was highly correlated with tissue Ki-67 expression (r = -0.521, P0.0001). Kaplan-Meier analysis and Cox proportional hazards regression analysis revealed a prognostic role of serum miR-16 (hazard ratio 2.49, 95% confidence interval 1.10-5.63, P = 0.028), which independently evaluated patients' survival outcome. Finally, the suppressive role of miR-16 in melanoma growth was validated both in vitro and in vivo. In conclusion, we demonstrated that serum miR-16 is a potential biomarker for predicting melanoma prognosis.

e15586 Background: Next-generation sequencing (NGS) typically requires greater quantities of DNA than traditional molecular testing. Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration (EBUS-TBNA) is a minimally invasive technique with high sensitivity in the mediastinal staging of lung cancer. This study aimed to evaluate the adequacy of EBUS-TBNA in providing adequate size specimens for genetic mutations and immunotherapy biomarkers analysis in patients with lung cancer. Methods: Tissue samples from patients with advanced lung cancer were collected by EBUS-TBNA and were formalin-fixed paraffin-embedded. NGS assay was carried on with acornmed panel including 808 genes. PD-L1 expression through immunohistochemistry was assessed. Results: A total of 98 patients was enrolled, of which 74 (76%) were adenocarcinoma and 20 (20%) were squamous cell carcinomas. Among the patients, 108 samples (including multipoint puncture of different location and retest of the same patient) were obtained. NGS assay was completed successfully on 106 of the 108 samples (98.14%), and 97.96% of patients had successful testing, identifying an average of 11.3 mutations. With a multi-gene panel comprising up to 808 tumor related genes, actionable variations were found in 93 (86%) samples. Of these, the positive rates of actionable alterations in lung adenocarcinoma samples and lung squamous cell carcinoma samples were 90% and 79%, respectively. The tumor mutation burden (TMB), Microsatellite instability (MSI) and PD-L1 expression were found High in 20 (41%), 1 (2%), and 37 (72%) patients respectively. Conclusions: NGS assay can be successfully conducted with tissue samples obtained from EBUS-TBNA. NGS assay provides more comprehensive information on genetic mutations in tumors, which greatly assists therapeutic decision making for advanced lung cancer.

This paper established the random forest and Bayesian classification prediction models and aims to find out the risk factors of Hepatitis B virus (HBV) reactivation after the precise radiotherapy in patients with primary liver cancer (PLC). Using the identified risk factors we can provide the reference to the doctor to reduce the incidence of the disease. Firstly, we proposed random forest method to select key features and then establish classification prediction models with the key subset. All the features are sorted according to the importance. We select 5 most important features which would be combined into a brand new feature subset and with the new subset we establish random forest and Bayesian classification prediction model. We find that HBV DNA level, TNM tumor staging, V10, V20, outer margin of radiotherapy is the risk factors of HBV reactivation. The classification accuracy of random forest can be reached to 85.15% by using 5 fold cross validation under 200 decision trees, meanwhile, the accuracy of Bayesian classifier reached to 84.57% by using 10 fold cross validation. The experimental results showed that the random forest can be used to evaluate the importance of variables and select the key features. And also, it is a better method to solve the classification prediction problem of HBV reactivation.

This article aims at finding the risk factors for hepatitis B virus (HBV) reactivation after the precise radiotherapy in patients with primary liver cancer (PLC). We use sequential forward selection and sequential backward selection to extract features which would be combined into an optimal feature subset, and then establish Bayesian and support vector machine (SVM) classification model. We use sequential forward selection to select the key features and find that the AFP, HBV DNA levels, outer margin of radiotherapy, times of radiotherapy, split method were the risk factors of HBV reactivation. The accuracy of the key features with Bayesian classifier reached to 80.65% by using 5 fold cross validation and with SVM reached to 79.84% by using 10 fold cross validation. Besides, we use sequential backward selection find that KPS score, HBV DNA level, outer margin of radiotherapy, tumor stage TNM, equivalent biometric were the risk factors of HBV reactivation, meanwhile, the accuracy of Bayesian classifier can be reached to 85.77% by using fold cross validation and with SVM classifier can be reached to 87.31% by using 10 fold cross validation. The accuracy of original feature with Bayesian classifier reached to 71.42% by using 10 fold cross validation and with SVM classifier reached to 78.10% by using 5 fold cross validation. The experimental results showed that the key feature subset has a better classification performance than the initial feature set clearly.

As an optical-acoustic hybrid imaging technology, photoacoustic imaging uniquely combines the advantages of rich optical contrast with high ultrasonic resolution in depth, opening up many new possibilities not attainable with conventional pure optical imaging technologies. To perform photoacoustic molecular imaging, optically absorbing exogenous contrast agents are needed to enhance the signals from specifically targeted disease activity. In this work, we designed and developed folate receptor targeted, indocyanine green dye doped poly(d,l-lactide-co-glycolide) lipid nanoparticles (FA-ICG-PLGA-lipid NPs) for molecular photoacoustic imaging of tumor. The fabricated FA-ICG-PLGA-lipid NPs exhibited good aqueous stability, a high folate-receptor targeting efficiency, and remarkable optical absorption in near-infrared wavelengths, providing excellent photoacoustic signals in vitro. Furthermore, after intravenous administration of FA-ICG-PLGA-lipid NPs, mice bearing MCF-7 breast carcinomas showed significantly enhanced photoacoustic signals in vivo in the tumor regions, compared with those using non-targeted ICG-PLGA-lipid NPs. Given the existing wide clinical use of ICG and PLGA, the developed FA-ICG-PLGA-lipid NPs, in conjunction with photoacoustic imaging technology, offer a great potential to be translated into the clinic for non-ionizing molecular imaging of breast cancer in vivo.

Melanoma is among the most aggressive tumors, and the occurrence of metastasis leads to a precipitous drop in the patients' survival. Therefore, identification of metastasis-associated biomarkers and therapeutic targets will contribute a lot to improving melanoma theranostics. Recently, microRNAs (miRNAs) have been implicated in modulating cancer invasion and metastasis, and are proved as potential non-invasive biomarkers in sera for various tumors. Here, we reported miR-23a as a novel metastasis-associated miRNA that played a remarkable role in modulating melanoma invasive and metastatic capacity and was of great value in predicting melanoma metastasis and prognosis. We found that serum miR-23a level was significantly down-regulated in metastatic melanoma patients and highly correlated with poor clinical outcomes. In addition, miR-23a level was also remarkably decreased in metastatic melanoma tissues and cell lines. Furthermore, overexpressed miR-23a suppressed the invasive and migratory property of melanoma cells by abrogating autophagy through directly targeting ATG12. Specially, miR-23a-ATG12 axis attenuated melanoma invasion and migration through autophagy-mediated AMPK-RhoA pathway. Finally, the overexpression of miR-23a prevented melanoma metastasis in vivo. Taken together, our findings demonstrate that the metastasis-associated miR-23a is not only a potential biomarker, but also a valuable therapeutic target for melanoma.

e14650 Background: Gene fusions, resulting from chromosomal breakage and rearrangement, play a critical role during oncogenesis. Recently, a growing number of gene fusions are being considered as important therapeutic targets in oncology, for instance, Anaplastic lymphoma receptor tyrosine kinase (ALK) fusion, which confer exquisite sensitivity to ALK tyrosine kinase inhibitors (TKIs). However, available methods are inadequate for unbiased identification of potentially targetable fusions. Hence, we developed a novel fusion gene analyzing method named AcornFusion by DNA-seq data. Methods: The method is purely based on the mapping results of soft-clipped reads and the distribution of the positions where the candidate breakpoints locate, which is significantly different from the traditional methods with assembly of short reads and efficient way to do the detection. 309 patients with advanced non-small cell lung cancer (NSCLC) were enrolled. Formalin-fixed, paraffin embedded (FFPE) samples were used to DNA extracting and analyzed by AcornFusion method. Immunohistochemistry (IHC) and Reverse transcriptase polymerase chain reaction (RT-PCR) platform were also applied. Results: Of 309 pts, 17 patients were identified as fusion-positive cases by AcornFusion. 15 patients were EML4-ALK fusion positive and 1 patient was HIP-ALK fusion positive, which were all confirmed by IHC. 1 patient was ROS1 rearrangement positive, which was validated by Reverse transcriptase polymerase chain reaction (RT-PCR) method. Conclusions: With this advantage of high consistency with IHC and RT-PCR, AcornFusion can be a useful tool for investigating gene fusion events in clinical settings.

e20534 Background: While tumor genotyping is the standard treatment for patients with non-small cell lung cancer (NSCLC), spatial and temporal tumor heterogeneity and insufficient specimens can lead to limitations in the use of tissue-based sequencing. Circulating tumor DNA (ctDNA) fully encompasses tumor-specific sequence alterations and offers an alternative to tissue sample biopsies. The aim of the study was to evaluate whether the frequency of multiple genomic alterations observed following ctDNA sequencing was similar to that observed following tissue sequencing in NSCLC. Methods: A total of 99 NSCLC patients were enrolled in this study, including 40 tissue and 59 plasma samples. All kinds of variants of oncogenic drivers in NSCLC were identified by next-generation sequencing (NGS) with Acornmed panel. Results: The frequencies of genetic alterations detected in ctDNA were significantly correlated to those detected via tissue profiling (Spearman’s r = 0.812, P = 0.022). Genomic data revealed significant mutual exclusivity between alterations in epidermal growth factor receptor ( EGFR) and tumor protein 53 ( TP53; P = 0.020) and between those in EGFR and Kirsten rat sarcoma viral oncogene homolog ( KRAS; P = 0.008), as well as potential mutual exclusivity between alterations in EGFR and Erb-B2 receptor tyrosine kinase 2 ( ERBB2; P = 0.059). Furthermore, the EGFR mutant allele frequency (MAF) was strongly correlated with the TP53 MAF in individual tumors (Spearman’s r = 0.773, P = 0.005), and there was a marked difference in the EGFR MAF between patients with and without the TP53 mutation (P = 0.001). Levels of the tumor serum marker CA242 in patients with ctDNA-detectable mutations were higher than those in patients without ctDNA-detectable mutations. Conclusions: This study provides a better understanding of the spectra of genomic alterations detected by tissue and plasma ctDNA assays in Chinese patients with NSCLC. The present data also highlights the importance of tissue and plasma ctDNA screening by NGS to guide personalized therapy and promote the clinical management of NSCLC patients.

e22501 Background: Osteosarcoma (OS) is characterized by complex genetic features and mysterious biological behavior. Genomic technologies are starting to give more insights. However, recent investigations have focused on patients with the advanced or metastatic disease. This study intended to evaluate the genetic landscape and identify new prognostic biomarkers in OS pts with good prognosis. Methods: This study conducted in-depth genomic profiling in a cohort of 21 patients (MSTS stage II) with long-term follow-up. Whole exome sequencing of primary tumors with matched normal tissues were performed. Sequencing data were analyzed to call tumor specific single nucleotide variants (SNV), small insertions and deletions (Indels), copy number variations (CNV) and chromosomal rearrangements and their prevalence was compared to the pts with metastatic disease reported before (Wang et al, 2018). Tumor mutation burden (TMB) and microsatellite instability (MSI) was analyzed, with additional analysis for mutational signatures. The endpoint was disease free survival (DFS). Results: 21 participants were enrolled between July. 23th, 2008 and Aug. 19th, 2013. 21 pts (M/F 13/8), median age 17y (5-33). Median DFS was 88.4 (95% CI 61.27–111.77) months. The top ranked altered genes were MUC4 (67%), FRG1 (62%), TXNDC2 (62%), ZNF717 (62%), ANKRD20A4 (57%), IGSF3 (52%), MUC16 (52%), NBPF10 (52%), MUC12 (48%) and ARID1A (43%). TMB was evaluated as lower level with a median value of 1.11 muts/MB (range from 0.78 to 3.58), relative to the other solid tumors. None pathogenic and likely pathogenic germline mutations were detected. Mutation signature 5 and 6 were most prevalent. CNV were frequently happened in stage II OS with an incidence of 80.9% (17/21). Among these, The co-occurrence of copy number gain (CNG) and copy number loss (CNL) significantly correlated with DFS (p = 0.0164), comparing with pts of either CNG or CNL . While CNV of driver genes in stage II OS were different from those of stage III published before (Wang et al, 2018), TP53 (0% vs 30% ), RB1 (4.5% vs 60%), CDKN2A (0% vs 30% ), MYC (0% vs 60%) and MDM2 (0% vs 60%). Conclusions: Stage II OS with favorable prognosis has unique genetic characterizes relative to metastatic OS. This study indicates that MUC family could be one of prognostic indicators. Genes with CNV, and the state which CNV occurs could provide clinical significance for the discovery of prognostic markers, which need to be validated by prospective prognostic data.

e16071 Background: The mutation spectrum has been extensively studied in Renal Cell Cancer (RCC), a heterogeneous disease. While major investigations have focused on metastatic RCC (mRCC). This study intended to explore the molecular characteristics between early-stage (stage I/Ⅱ) and advanced-stage (Ⅲ/Ⅳ) of RCC patients (pts). Methods: 36 tumor specimens were obtained from individual pts diagnosed RCC, including 22 tissues paired with blood samples. Somatic mutations were identified via targeted next generation sequencing with Acornmed panel including 808 genes associated with tumor development. Sequencing data were analyzed to call tumor specific single nucleotide variants (SNV), small insertions and deletions (InDels), copy number alterations (CNA) and chromosomal rearrangements. Clinical data of a cohort from TCGA who had RCC was collected and analyzed. Results: Among the 36 pts enrolled, 61% were early-stage group and 39% were advanced-stage group. The most frequently mutated genes in RCC were VHL (50%), PBRM1 (14%), BAP1 (11%), TP53 (11%) and PIK3CA (6%). Truncating mutations were the most frequent alterations accounting for 66% (33/50) of genetic alterations. Mutations in VHL, BAP1, PBRM1 and TP53 were shared by both early and advanced RCC. While mutations in PIK3CA, CDKN2C, EGFR, FAMCA, GRIN2A, KDM6A, SOX17, TSC1 and APC were only detected in early stage group. KNSTRN, KRAS, MYD88, NF2, ATM, PTEN and STAT5B mutations were only detected in advanced group. Among 22 plasma samples, genomic alterations were detected in 40.9% (9/22) of pts postoperatively, such as TP53, PIK3CA, PTEN, and APC. Functional annotation clustering revealed that 4/9 in these genes were tumor suppressors, which negatively regulated apoptosis. Pts with genomic alterations in TP53 (p = 0.0034), PIK3CA (p = 0.0015) or PTEN (p = 0.00017) had worse OS significantly (TCGA). Conclusions: VHL, PBRM1, BAP1 were the most important driver genes mainly detected in all stages of RCC. The heterogeneity between early and advanced-stage may be related with clinical status. Gene alterations in plasma ctDNA postoperatively should have the potential to stratify patients with different prognostic outcome.

e14280 Background: Biomarkers would enrich patients(pts) with responsiveness to both targeted treatments and immunotherapy which have become a priority. However, molecular heterogeneous in actionable gene alterations and mutational loads need to be explored. In this study, based on a real world setting, Cancer Gene Panel (CGP） analysis was performed in a series samples of Chinese pts with advanced solid tumors, to delineate the landscape of actionable mutations and tumor mutational burden (TMB). Methods: 176 advanced pts were enrolled encompassing 19 common tumor types. All tissue samples were analyzed using next-generation sequencing with Acornmed panel including 808 genes. Results: The frequencies of genetic alterations among the most common driver genes were comparable to those reported by MSK-IMPACT (2018) ( TP53: 51.7% vs 44.8%; APC: 9.7% vs 9.8%; EGFR: 12.5% vs 7.7%; KRAS: 16.5% vs 13.6%; PIK3CA: 4.6% vs 12.0%; VHL: 4.0% vs 6.7%). 58.3% of pts with refractory solid tumors had at least one actionable mutation. Among those pts, 16.7% of them including CDK4, CDK6 or CDKN2A/B could benefit from palbociclib, and 32.1% of them including mTOR, PIK3CA, PTEN or STK11 could benefit from everolimus, and 61.9% of them including BRCA2, ARID1A, MSH1/2/6 or ATM could benefit from olaparib. Across all the specimens, the top 20% of TMB was 17.23 mutations/Mb, with a range of 0-48 mutations/Mb. The top 20% of TMB for each tumor type was 21.67(Bladder cancer), 17.23(Lung cancer), 16.45(Colorectal cancer), 11.04 (Renal cell cancer), 16.25 (Prostate cancer) , 19.70 (Hepatobiliary cancer), 14.78 (Esophagogastric cancer), 7.80 (Pancreatic cancer), 25.31 (undefined tumor) mutations/Mb respectively. Conclusions: This study indicates that most of pts with advanced refractory solid tumors have at least one actionable mutation, and matched therapies may confer clinical benefit. TMB fluctuated wildly across different cancer types, illustrating the unequal objective response rate of immunotherapy across various cancer type. Furthermore, CGP analysis in advanced pan-cancer pts can provide opportunities for targeted therapies and immunotherapy, especially in those with refractory cancer.