Development and validation of serological dynamic risk score to predict outcome in gastric cancer with adjuvant chemotherapy: a multicentre, longitudinal, cohort study.

Background: Baseline serological biomarkers have the potential to predict the benefits of adjuvant chemotherapy in patients with gastric cancer. However, the fluctuating nature of postoperative recurrence risk makes precise treatment challenging. We aimed to develop a risk score in real-time predicting outcomes for postoperative GC patients using blood chemistry tests. Materials and methods: This was a retrospective, multicentre, longitudinal cohort study from three cancer centres in China, with a total of 2737 GC patients in the pTNM stage Ib to III. Among them, 1651 patients with at least two serological records were assigned to the training cohort. Model validation was carried out using separate testing data with area under curve (AUC). The least absolute shrinkage and selection operator (LASSO) and random forest-recursive feature elimination (RF-RFE) algorithm were used to select the parameters. Results: The Cox regression model derived six risk factors to construct a composite score (low-risk: 0-2 score; high risk: 3-6 score), including CEA, CA125, CA199, haemoglobin, albumin, and neutrophil to lymphocyte ratio. The risk score accurately predicted mortality in 1000-time bootstrap (AUROCs:0.658; 95% CI: 0.645, 0.670), with the highest AUROC (0.767; 95% CI: 0.743, 0.791) after 1 year since the gastrectomy. In validation dataset, the risk score had an AUROC of 0.586 (95% CI 0.544, 0.628). Furthermore, patients with high risk at 1 month derived significant clinical benefits from adjuvant chemotherapy (P for interaction <0.0001). Compared with the low-low-low risk group, the low-low-high risk group of the long-term state chain (risk state at baseline, 6 months, 1 year) had the worse OS (HR, 6.91; 95%CI: 4.27, 11.19) and DFS (HR, 7.27; 95%CI: 4.55, 11.63). Conclusion: The dynamic risk score is an accurate and user-friendly serological risk assessment tool for predicting outcomes and assisting clinical decisions after gastrectomy.

Association of survival with adjuvant chemotherapy in patients with stage IB gastric cancer: a multicentre, observational, cohort study.

Background: Recurrence following radical resection in patients with stage IB gastric cancer (GC) is not uncommon. However, whether postoperative adjuvant chemotherapy could reduce the risk of recurrence in stage IB GC remains contentious. Methods: We collected data on 2110 consecutive patients with pathologic stage IB (T1N1M0 or T2N0M0) GC who were admitted to 8 hospitals in China from 2009 to 2018. The survival of patients who received adjuvant chemotherapy was compared with that of postoperative observation patients using propensity score matching (PSM). Two survival prediction models were constructed to estimate the predicted net survival gain attributable to adjuvant chemotherapy. Findings: Of the 2110 patients, 1344 received adjuvant chemotherapy and 766 received postoperative observation. Following the 1-to-1 matching, PSM yielded 637 matched pairs. Among matched pairs, adjuvant chemotherapy was not associated with improved survival compared with postoperative observation (OS: hazard ratio [HR], 0.72; 95% CI, 0.52-1.00; DFS: HR, 0.91; 95% CI, 0.64-1.29). Interestingly, in the subgroup analysis, reduced mortality after adjuvant chemotherapy was observed in the subgroups with elevated serum CA19-9 (HR, 0.22; 95% CI, 0.08-0.57; P = 0.001 for multiplicative interaction), positive lymphovascular invasion (HR, 0.32; 95% CI, 0.17-0.62; P < 0.001 for multiplicative interaction), or positive lymph nodes (HR, 0.17; 95% CI, 0.07-0.38; P < 0.001 for multiplicative interaction). The survival prediction models mainly based on variables associated with chemotherapy benefits in the subgroup analysis demonstrated good calibration and discrimination, with relatively high C-indexes. The C-indexes for OS were 0.74 for patients treated with adjuvant chemotherapy and 0.70 for patients treated with postoperative observation. Two nomograms were built from the models that can calculate individualized estimates of expected net survival gain attributable to adjuvant chemotherapy. Interpretation: In this cohort study, pathologic stage IB alone was not associated with survival benefits from adjuvant chemotherapy compared with postoperative observation in patients with early-stage GC. High-risk clinicopathologic features should be considered simultaneously when evaluating patients with stage IB GC for adjuvant chemotherapy. Funding: National Natural Science Foundation of China; the National Key R&D Program of China.

Association of CDX2 and mucin expression with chemotherapeutic benefits in patients with stage II/III gastric cancer.

BACKGROUND: Better predictors of patients with stage II/III gastric cancer (GC) most likely to benefit from adjuvant chemotherapy are urgently needed. This study aimed to assess the ability of CDX2 and mucin markers to predict prognosis and fluorouracil-based adjuvant chemotherapy benefits. METHODS: CDX2 and mucin protein expressions were examined by immunohistochemistry and compared with survival and adjuvant chemotherapy benefits in a prospective evaluation cohort of 782 stage II/III GC patients. Then, the main findings were validated in an independent validation cohort (n = 386) and an external mRNA sequencing dataset (ACRG cohort, n = 193). RESULTS: In the evaluation cohort, CDX2, CD10, MUC2, MUC5AC, and MUC6 expressions were observed in 59.7%, 26.7%, 27.6%, 55.1%, and 57.7% of patients, respectively. However, only the expression of CDX2 was found to be associated with adjuvant chemotherapy benefits. Most importantly, CDX2-negative patients had a poorer prognosis when treated with surgery only, while the prognosis of CDX2-negative and CDX2-positive patients was similar when receiving postoperative adjuvant chemotherapy. Further analysis revealed that patients with CDX2 negative tumors benefited from chemotherapy (5-year overall survival rates: 60.0% with chemotherapy vs. 23.2% with surgery-only, p < 0.001), whereas patients with CDX2 positive tumors did not (pinteraction = 0.004). Consistent results were obtained in the validation and ACRG cohorts. CONCLUSIONS: Negative expression of CDX2 is an independent risk factor for survival in stage II/III GC, but subsequent adjuvant chemotherapy is able to compensate for this unfavorable effect. Therefore, active chemotherapy is more urgent for patients with negative CDX2 expression than for patients with positive CDX2 expression.

Impact of HER2 on prognosis and benefit from adjuvant chemotherapy in stage II/III gastric cancer patients: a multicenter observational study.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) is a well-developed therapeutic target in breast and gastric cancer (GC). However, the impact of HER2 on survival and benefit from fluorouracil-based adjuvant chemotherapy remains unclear in patients with GC. MATERIALS AND METHODS: This multicenter cohort study involved 5622 consecutive stage II/III GC patients. HER2 expression was assessed prospectively via immunohistochemistry (IHC). The staining intensity was graded on a scale of 0 to 3+. An IHC score of 2+or 3+was defined as high expression, and a score of 3+was defined as overexpression. RESULTS: HER2 overexpression was independently associated with a lower 5-year overall survival (OS) in stage II [hazard ratio (HR), 2.10; 95% CI: 1.41-3.11], but not in stage III GC (HR, 1.00; 95% CI, 0.82-1.20). Further analysis revealed that stage II patients with high HER2 expression showed a poorer response to chemotherapy than stage II patients with low HER2 expression ( Pinteraction =0.024). The HRs for 5-year OS were 0.51 (95% CI, 0.38-0.70) for stage II patients with low HER2 expression, 0.58 (95% CI, 0.51-0.66) for stage III patients with low HER2 expression, 1.13 (95% CI, 0.61-2.09) for stage II patients with high HER2 expression, and 0.47 (95% CI, 0.36-0.61) for stage III patients with high HER2 expression. CONCLUSIONS: Fluorouracil-based adjuvant chemotherapy is insufficient for stage II GC patients with high HER2 expression, indicating that prospective trials are required to validate alternative HER2-targeted adjuvant therapies in the individuals above.

Genome-wide DNA methylation profile analysis identifies an individualized predictive signature for melanoma immune response.

PURPOSE: The current evaluation methods for tumor infiltrating lymphocytes (TILs), particularly CD8 + TILs, mainly rely on semiquantitative immunohistochemistry with high variability. We aimed to construct an individualized DNA methylation-based signature for CD8 + TILs (CD8 + MeTIL) that may characterize melanoma immune microenvironment and guide therapeutic selection. METHODS: The transcriptome profiles and DNA methylation data of 457 melanoma patients from The Cancer Genome Atlas (TCGA) database were analyzed. Differential methylation analysis between groups with high and low CD8 + TILs was performed to select differentially methylated positions (DMPs) and define CD8 + MeTIL. The prognostic value of CD8 + MeTIL and its predictive value for immunotherapy response were investigated using multiple melanoma cohorts. RESULTS: We successfully constructed the CD8 + MeTIL signature based on four DMPs. The survival analyses showed that higher CD8 + MeTIL score was associated with worse survival outcomes in TCGA-SKCM and GSE144487 cohorts. The ROC curve for the predictive analysis revealed that the survival prediction of CD8 + MeTIL score was superior compared with CD8 + TILs (CIBERSORT) and CD8B mRNA expression. Furthermore, we founded that tumors with higher CD8 + MeTIL score were marked with immunosuppressive characteristics, including low immune score and downregulated immune-related pathways. More importantly, the CD8 + MeTIL score showed a potential predictive value for the benefit from immunotherapy in two published cohorts. When combined CD8 + MeTIL with PD-L1 expression, the patient classification showed significantly different immunotherapy response rates and long-term survival outcomes. CONCLUSIONS: The CD8 + MeTIL signature might be as a novel method to evaluate CD8 + TILs and guide immunotherapy approaches.

DNA damage response-related immune activation signature predicts the response to immune checkpoint inhibitors: from gastrointestinal cancer analysis to pan-cancer validation.

OBJECTIVE: DNA damage response (DDR) deficiency has emerged as a prominent determinant of tumor immunogenicity. This study aimed to construct a DDR-related immune activation (DRIA) signature and evaluate the predictive accuracy of the DRIA signature for response to immune checkpoint inhibitor (ICI) therapy in gastrointestinal (GI) cancer. METHODS: A DRIA signature was established based on two previously reported DNA damage immune response assays. Clinical and gene expression data from two published GI cancer cohorts were used to assess and validate the association between the DRIA score and response to ICI therapy. The predictive accuracy of the DRIA score was validated based on one ICI-treated melanoma and three pan-cancer published cohorts. RESULTS: The DRIA signature includes three genes (CXCL10, IDO1, and IFI44L). In the discovery cancer cohort, DRIA-high patients with gastric cancer achieved a higher response rate to ICI therapy than DRIA-low patients (81.8% vs. 8.8%; P < 0.001), and the predictive accuracy of the DRIA score [area under the receiver operating characteristic curve (AUC) = 0.845] was superior to the predictive accuracy of PD-L1 expression, tumor mutational burden, microsatellite instability, and Epstein-Barr virus status. The validation cohort demonstrated that the DRIA score identified responders with microsatellite-stable colorectal and pancreatic adenocarcinoma who received dual PD-1 and CTLA-4 blockade with radiation therapy. Furthermore, the predictive performance of the DRIA score was shown to be robust through an extended validation in melanoma, urothelial cancer, and pan-cancer. CONCLUSIONS: The DRIA signature has superior and robust predictive accuracy for the efficacy of ICI therapy in GI cancer and pan-cancer, indicating that the DRIA signature may serve as a powerful biomarker for guiding ICI therapy decisions.

BNIP3 Upregulation Characterizes Cancer Cell Subpopulation With Increased Fitness and Proliferation.

BNIP3 is a BH3-only protein with both pro-apoptotic and pro-survival roles depending on the cellular context. It remains unclear how BNIP3 RNA level dictates cell fate decisions of cancer cells. Here, we undertook a quantitative analysis of BNIP3 expression and functions in single-cell datasets of various epithelial malignancies. Our results demonstrated that BNIP3 upregulation characterizes cancer cell subpopulations with increased fitness and proliferation. We further validated the upregulation of BNIP3 in liver cancer 3D organoid cultures compared with 2D culture. Taken together, the combination of in silico perturbations using public single-cell datasets and experimental cancer modeling using organoids ushered in a new approach to address cancer heterogeneity.

An Immune-Related Gene Pair Index Predicts Clinical Response and Survival Outcome of Immune Checkpoint Inhibitors in Melanoma.

The durable responses and favorable long-term outcomes are limited to a proportion of advanced melanoma patients treated with immune checkpoint inhibitors (ICI). Considering the critical role of antitumor immunity status in the regulation of ICI therapy responsiveness, we focused on the immune-related gene profiles and aimed to develop an individualized immune signature for predicting the benefit of ICI therapy. During the discovery phase, we integrated three published datasets of metastatic melanoma treated with anti-PD-1 (n = 120) and established an immune-related gene pair index (IRGPI) for patient classification. The IRGPI was constructed based on 31 immune-related gene pairs (IRGPs) consisting of 51 immune-related genes (IRGs). The ROC curve analysis was performed to evaluate the predictive accuracy of IRGPI with AUC = 0.854. Then, we retrospectively collected one anti-PD-1 therapy dataset of metastatic melanoma (n = 55) from Peking University Cancer Hospital (PUCH) and performed the whole-transcriptome RNA sequencing. Combined with another published dataset of metastatic melanoma received anti-CTLA-4 (VanAllen15; n = 42), we further validated the prediction accuracy of IRGPI for ICI therapy in two datasets (PUCH and VanAllen15) with AUCs of 0.737 and 0.767, respectively. Notably, the survival analyses revealed that higher IRGPI conferred poor survival outcomes in both the discovery and validation datasets. Moreover, correlation analyses of IRGPI with the immune cell infiltration and biological functions indicated that IRGPI may be an indicator of the immune status of the tumor microenvironment (TME). These findings demonstrated that IRGPI might serve as a novel marker for treating of melanoma with ICI, which needs to be validated in prospective clinical trials.

Clinical significance of CCNE1 copy number gain in acral melanoma patients.

Copy number variations are frequently observed in cell cycle-related genes in acral melanoma. However, the clinical significance of copy number gain of CCNE1 in acral melanoma has not been fully elucidated. In this study, 490 acral melanoma samples were examined for CCNE1 copy number using the QuantiGenePlex DNA Assay. Correlation between CCNE1 copy number and acral melanoma patients' clinicopathologic features were analyzed using Chi-squared test. The impact of CCNE1 copy number on patients' progression-free survival (PFS) and overall survival (OS) probability were analyzed using Kaplan-Meier analysis. The impact of CCNE1 copy number on patients' median PFS after receiving chemotherapy was also evaluated. The results showed that CCNE1 copy number gain was observed in 28.30% of patients, with 3.16% of patients carrying both CCNE1 copy number gain and BRAF mutation and 4.34% of patients carrying both CCNE1 copy number gain and NRAS mutation. The median PFS time for patients with CCNE1 copy number gain was shorter than that of patients without CCNE1 copy number gain (17.0 vs. 27.0 months, P = 0.002).In the cohort that received chemotherapy (n = 82), the median PFS time for patients with CCNE1 copy number gain was shorter than that of patients without CCNE1 copy number gain (4.8 vs. 7.4 months, P = 00.006). CCNE1 copy number gain was an independent prognostic marker for acral melanoma patients' PFS. Our study indicates that CCNE1 copy number gain is frequent in acral melanoma and may be a biomarker to predict acral melanoma patients' outcomes after receiving chemotherapy.

Patient Stratification of Clear Cell Renal Cell Carcinoma Using the Global Transcription Factor Activity Landscape Derived From RNA-Seq Data.

Clear cell renal cell carcinoma represents the most common type of kidney cancer. Precision medicine approach to ccRCC requires an accurate stratification of patients that can predict prognosis and guide therapeutic decision. Transcription factors are implicated in the initiation and progression of human carcinogenesis. However, no comprehensive analysis of transcription factor activity has been proposed so far to realize patient stratification. Here we propose a novel approach to determine the subtypes of ccRCC patients based on global transcription factor activity landscape. Using the TCGA cohort dataset, we identified different subtypes that have distinct up-regulated biomarkers and altered biological pathways. More important, this subtype information can be used to predict the overall survival of ccRCC patients. Our results suggest that transcription factor activity can be harnessed to perform patient stratification.

Prognostic Role of Tumor Mutation Burden Combined With Immune Infiltrates in Skin Cutaneous Melanoma Based on Multi-Omics Analysis.

Tumor mutation burden (TMB) and tumor infiltrating lymphocytes have been well-recognized as molecular determinants of immunotherapeutic responsiveness in many types of cancer. However, the relationship between TMB with immune infiltrates and their prognostic role are reported occasionally in skin cutaneous melanoma (SKCM). We obtained the somatic mutation data and transcriptome profiles of 454 SKCM patients from The Cancer Genome Atlas (TCGA) database, and analyzed the mutation profiles using "maftools" package. Correlation analysis revealed that lower TMB levels conferred poor survival outcomes, associated with lower age and advanced pathological stage. Differential analysis was conducted to the genome expression between two TMB groups using "limma" package, and we identified four hub TMB-related immune genes including CNTFR, CRABP2, GAL, and PAEP. We further analyzed the underlying relationships of the copy number variations (CNVs) of four hub genes with immune infiltrates in SKCM microenvironment through TIMER database. The results indicated that diverse forms of CNVs carried by hub genes could commonly inhibit immune infiltrates. Based on the CIBERSORT method, we compared the proportions of 22 immune cells in two TMB groups and assessed their prognostic value. The data revealed that infiltrations levels of regulatory T (Treg) cell and dendritic activated cells in high-TMB group were lower than that in low-TMB group, while M1 and M2 macrophages showed the opposite trend, especially the levels of neutrophil and macrophage correlated positively with prognosis of SKCM. Finally, we constructed a TMB Prognostic Index (TMBPI) to evaluate the predictive accuracy of the four hub TMB-related immune genes. The ROC curve was drawn to assess the predictive accuracy with AUC = 0.664 and higher TMBPI conferred poor survival outcomes, which warranted further investigation and larger samples to validate.

MUC4 isoforms expression profiling and prognosis value in Chinese melanoma patients.

Mucin 4 (MUC4), a type I membrane-bound mucin, blocks apoptosis, promotes invasion, proliferation and migration and causes chemo-resistance in epithelial cancers. However, the expression profiling and clinical implications of MUC4 alternative splicing during cancer pathogenesis, including melanoma, remain obscure. We examined the mRNA expression profiling of MUC4 isoforms in gastrointestinal cancer cell lines, melanoma cell lines, human epidermal melanocyte cells, as well as 138 cases of human melanoma tissues by RT-qPCR. Then we analyzed the relationship of mRNA expression of MUC4 isoforms to clinicopathological characteristics and survival of patients. The dynamic mRNA expression profiling of MUC4 isoforms was found in melanoma. We identified MUC4 isoform f was highly expressed in melanoma cell lines but negative in gastrointestinal cancer cell lines. Clinical analysis based on 138 cases of human melanomas showed that MUC4 isoform d was related with melanoma subtypes (p = 0.028) and TNM stage (p = 0.036). MUC4 isoform e was related with tumor thickness (p = 0.004) and T stage (p = 0.036). The Kaplan-Meier assay showed that the median overall survival (OS) for patients with MUC4 isoform f high expression was significantly shorter than that of patients with low expression (p = 0.024). And the median PFS of the patients with high expression of MUC4 isoform d or e was significantly shorter than that of with low expression (p = 0.012 and 0.035, respectively). Multivariate analysis indicated that high level of MUC4 isoform f was an independent prognostic factor for OS, and MUC4 isoform d was an independent prognostic factor for PFS of patients treated with chemotherapy. In conclusion, our results indicate that the dynamic MUC4 isoforms expressed in melanoma, and MUC4 isoform d and f might be served as a novel prognostic indicator of melanoma patients.

Genetic Aberrations in the CDK4 Pathway Are Associated with Innate Resistance to PD-1 Blockade in Chinese Patients with Non-Cutaneous Melanoma.

PURPOSE: PD-1 checkpoint blockade immunotherapy induces long and durable response in patients with advanced melanoma. However, only a subset of patients with melanoma benefit from this approach. The mechanism triggering the innate resistance of anti-PD-1 therapy remains unclear.Experimental Design: Whole-exome sequencing (WES) and RNA sequencing (RNA-Seq) analyses were performed in a training cohort (n = 31) using baseline tumor biopsies of patients with advanced melanoma treated with the anti-PD-1 antibody. Copy-number variations (CNVs) for the genes CDK4, CCND1, and CDKN2A were assayed using a TaqMan copy-number assay in a validation cohort (n = 85). The effect of CDK4/6 inhibitors combined with anti-PD-1 antibody monotherapy was evaluated in PD-1-humanized mouse (C57BL/6-hPD-1) and humanized immune system (HIS) patient-derived xenograft (PDX) models. RESULTS: WES revealed several significant gene copy-number gains in the patients of no clinical benefit cohort, such as 12q14.1 loci, which harbor CDK4. The association between CDK4 gain and innate resistance to anti-PD-1 therapy was validated in 85 patients with melanoma (P < 0.05). RNA-Seq analysis of CDK4-normal cell lines and CDK4-normal tumors showed altered transcriptional output in TNFα signaling via NF-κB, inflammatory response, and IFNγ response gene set. In addition, CDK4/6 inhibitor (palbociclib) treatment increased PD-L1 protein levels and enhanced efficacy (P < 0.05) in the C57BL/6-hPD-1 melanoma cell and the HIS PDX model. CONCLUSIONS: In summary, we discovered that genetic aberrations in the CDK4 pathway are associated with innate resistance to anti-PD-1 therapy in patients with advanced melanoma. Moreover, our study provides a strong rationale for combining CDK4/6 inhibitors with anti-PD-1 antibody for the treatment of advanced melanomas.

miR-let-7b and miR-let-7c suppress tumourigenesis of human mucosal melanoma and enhance the sensitivity to chemotherapy.

BACKGROUND: Mucosal melanoma with poor prognosis is a common histopathologic subtype of melanoma among Chinese and other Asian peoples. Regulated microRNAs (miRNAs) have been reported as oncogenes or tumour suppressors in melanoma. However, the roles of specific miRNAs in mucosal melanoma remain largely unknown. Here, we aimed to assess the biological functions, molecular mechanisms and clinical potential of miR-let-7b and miR-let-7c in mucosal melanoma. METHODS: The expression of miR-let-7b and miR-let-7c in mucosal melanoma was determined by quantitative polymerase chain reaction (qPCR). Cutoff scores for miR-let-7b and miR-let-7c expressions were calculated through receiver operating characteristic (ROC) curve analysis in 106 mucosal melanoma patients according to recurrence. Correlations of miR-let-7b and miR-let-7c expression with clinicopathological characteristics, disease-free survival (DFS) and clinical benefits after treatment were then statistically analysed. The biological functions and molecular mechanisms of miR-let-7b and miR-let-7c were studied in vitro and in vivo. RESULTS: The expression of miR-let-7b and miR-let-7c was decreased in 94 cases (88.7%) and 89 cases (84.0%) of 106 mucosal melanoma patients compared with mucosal nevi. A correlation was observed between the expression of miR-let-7b, miR-let-7c and DFS after surgery. In addition, overexpression of miR-let-7b or miR-let-7c inhibited mucosal melanoma cell growth, migration, invasion and metastasis and induced cell apoptosis and cell cycle arrest in vitro and in vivo. Mechanistically, miR-let-7b and miR-let-7c directly targeted metadherin (MTDH) and calumenin (CALU) and suppressed phospho-ERK in mucosal melanoma cells. MTDH and CALU reversed the partial function of miR-let-7b and miR-let-7c in vitro. Furthermore, progression-free survival (PFS) of mucosal melanoma patients upon temozolomide-based and paclitaxel-based chemotherapy was related to miR-let-7b and miR-let-7c expression. Overexpression of miR-let-7b or miR-let-7c in patient-derived xenograft (PDX) models and certain mucosal melanoma cells had better growth inhibition after temozolomide and paclitaxel treatment. MTDH reversed the sensitivity of miR-let-7b and miR-let-7c to paclitaxel in vitro. CONCLUSIONS: Our results suggested that miR-let-7b and miR-let-7c inhibited the recurrence of mucosal melanoma through inhibiting cell growth, migration, invasion and metastasis, inducing cell apoptosis and cell cycle arrest by targeting MTDH and CALU. In addition, miR-let-7b and miR-let-7c increased sensitivity to chemotherapeutic agents by targeting MTDH.

Potential Mutations in Uveal Melanoma Identified Using Targeted Next-Generation Sequencing.

Background/objective: Uveal melanoma (UM) is the most common intraocular malignancy and has a high tendency to metastasize to the liver. Although primary tumours can be successfully treated, there is currently no effective treatment for metastatic UM. To gain insight into the genetics of UM, we performed the targeted next-generation sequencing (NGS) of UM samples from a non-Caucasian population. Methods: This study included tumour samples and blood samples from 107 UM patients at Peking University Cancer Hospital & Institute. Clinical data were collected. DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) specimens. Using the HaloPlex Target Enrichment System (Agilent Technologies), NGS was performed to investigate mutations in a 35-gene panel composed of cancer-related genes. Results: Recurrent coding mutations were found in the known UM drivers GNAQ and GNA11. FOXO1, PIK3R1 and HIF1A were also found to harbour somatic mutations in more than 20% of patients, a result that may indicate previously undescribed associations between these genes and UM pathogenesis. Patients with HIF1A and FOXO1 mutations exhibited worse overall survival (OS). In multivariate analysis, FOXO1 mutation was an independent prognostic factor for OS (P<0.05) that was associated with an increase in the risk ratio by a factor of 1.35. Notably, we found that HIF1A and FOXO1 mutations were associated with metastatic transformation of UM (P<0.05 and P<0.001, respectively). Conclusion: Our findings from analyses of targeted NGS data shed new light on the molecular genetics of UM and facilitate the exploration of mutations associated with metastatic potential.

Prognostic role of NRAS isoforms in Chinese melanoma patients.

Neuroblastoma rat-sarcoma viral oncogene homolog (NRAS) isoforms are expressed in melanoma tumor tissues, which have been described in Caucasian melanoma. However, the status and the clinical significance of NRAS isoforms in the Asian population have not been investigated on a large scale. We examined the expression levels of NRAS isoforms of 140 melanoma samples using quantitative real-time PCR. Furthermore, the relationship of mRNA expression of NRAS isoforms to clinicopathological characteristics and survival of patients was analyzed. Statistical analysis showed that NRAS isoform 2 expression was correlated with melanoma subtypes (P=0.007), and NRAS isoform 4 expression was correlated with tumor thickness (P=0.031) and clinical stage (P=0.006). The median overall survival for patients with high expression of NRAS isoform 3 was significantly shorter than that for patients with low expression of NRAS isoform 3 (P=0.007). In addition, high expression of NRAS isoform 5 was associated with a worse prognosis (P=0.049 and 0.002 for overall survival and disease-free survival, respectively). Multivariate Cox regression analysis showed that high expression levels of NRAS isoform 3 and isoform 5 were independent poor prognostic factors for patients. Our results indicated that the mRNA expressions of NRAS isoform 3 and isoform 5 may be novel indicators of the prognosis of Chinese melanoma patients.

Establishment and characterization of melanoma patient-derived xenograft models for preclinical evaluation of novel therapeutics.

Patient-derived xenograft (PDX) models mostly retain the histological and genetic features of their donor tumors, which have been used for investigating various types of cancer. However, PDX models for melanoma, especially acral melanoma, are reported occasionally. We aimed to establish a large panel of melanoma PDX models representing the predominant Asian melanomas. Ninety-three fresh melanoma samples were implanted subcutaneously into nonobese diabetic/severe combined immunodeficiency mice. The histological and genetic characteristics were analyzed in both patient tumors and PDX models using immunohistochemistry, PCR amplification, and Sanger sequencing. Furthermore, the sensitivities of PDX models harboring distinct mutation profiles to binimetinib (a MEK inhibitor), vemubrafenib (a BRAF inhibitor), and imatinib (a KIT inhibitor) were also evaluated. Twenty-five PDX models were established successfully [25/93 (26.9%)] and passaged to maintain tumors in vivo. Clinical stage and origin of tumor sample were correlated with successful establishment rates (P=0.008 and <0.001, respectively). The histological (expression of NRAS, P16, and RB) and genetic (mutation status of NRAS, BRAF, and KIT) characteristics were stably maintained from patient tumors to PDX models. Targeted drugs could inhibit the tumor growth of PDX models harboring the corresponding target gene mutations. These PDX models constitute a pharmacological platform, enabling personalized development of therapeutic strategies for Asian melanomas.

PI3K/AKT/mTOR pathway inhibitors inhibit the growth of melanoma cells with mTOR H2189Y mutations in vitro.

mTOR is an important therapeutic target in many types of cancers. In melanoma, the mTOR nonsynonymous mutation rate is up to 10.4%. However, mTOR inhibitors have shown limited effects in clinical trials of melanoma. Because mTOR mutations are distributed, not selecting patients with specific mTOR mutations may be the main reason for therapeutic failures. Our previous research found that mutations in the mTOR P2213S and S2215Y kinase domains resulted in gain-of-function and were sensitive to specific inhibitors. The purpose of this study was to test the effect of heterozygous/homozygous H2189Y mutations on downstream pathways and sensitivity to inhibitors. mTOR kinase activity was analyzed by western blot and a K-LISA™ mTOR activity kit. The sensitivity of melanoma cells to inhibitors was tested by a proliferation assay. The expression of downstream pathway proteins was also analyzed by western blot. The results showed that heterozygous/homozygous H2189Y mutations were gain-of-function. The heterozygous H2189Y mutation was sensitive to the AKT inhibitor, AZD5363, and the phosphoinositide 3-kinase inhibitor, LY294002. The homozygous H2189Y mutation was sensitive to the mTOR inhibitor, everolimus, and the AKT inhibitors AZD5363 and MK-2206 2HCL,and the phosphoinositide 3-kinase inhibitor, LY294002. These results indicated that homozygous mutations in the kinase domain have a greater effect on protein function than heterozygous mutations. The mTOR kinase domain may play an important role in mTOR kinase activity and may be the target of selective inhibitors. Our study can facilitate the selection of appropriate inhibitors for patients in clinical trials.