Meng Ma

Analysis of mTOR Gene Aberrations in Melanoma Patients and Evaluation of Their Sensitivity to PI3K-AKT-mTOR Pathway Inhibitors.

Purpose: mTOR is a validated target in cancer. It remains to be determined whether melanoma patients bearing mTOR mutation could be selected for treatment with PI3K–AKT–mTOR pathway inhibitors. Experimental Design: A total of 412 melanoma samples were included. Gene aberrations in all exons of mTOR were detected by Sanger sequencing and confirmed by using Agilents SureSelect Target Enrichment System. HEK293T cells stably expressing mTOR mutants were constructed by using transcription activator-like effector nucleases technique. Function of mTOR mutants and in vitro sensitivity of gain-of-function mTOR mutations to PI3K–AKT–mTOR pathway inhibitors were analyzed. Results: The overall incidence of somatic nonsynonymous mutations of mTOR was 10.4% (43/412). mTOR nonsynonymous mutations were relatively more frequent in acral (11.0%) and mucosal (14.3%) melanomas than in chronic sun-induced damage (CSD; 6.7%) and non-CSD (3.4%) melanomas. Of the 43 cases with mTOR mutations, 41 different mutations were detected, affecting 25 different exons. The median survival time for melanoma patients with mTOR nonsynonymous mutation was significantly shorter than that for patients without mTOR nonsynonymous mutation (P = 0.028). Transient expression of mTOR mutants in HEK293T cells strongly activated the mTOR–p70S6K pathway. In HEK293T cells with stable expression of H1968Y or P2213S mTOR mutants, LY294002 and AZD5363 showed higher potency than temsirolimus or BYL719 in inhibiting the PI3K–AKT–mTOR pathway and cell proliferation. Conclusions: mTOR nonsynonymous mutations are frequent in melanoma patients. mTOR nonsynonymous mutation may predict a worse prognosis of melanoma. Clinical trials with PI3K–AKT–mTOR pathway inhibitors may be beneficial for melanoma patients with specific mTOR mutations. Clin Cancer Res; 22(4); 1018–27. ©2015 AACR.

Frequent Genetic Aberrations in the CDK4 Pathway in Acral Melanoma Indicate the Potential for CDK4/6 Inhibitors in Targeted Therapy

Purpose: Effective therapies for the majority of metastatic acral melanoma patients have not been established. Thus, we investigated genetic aberrations of CDK4 pathway in acral melanoma and evaluated the efficacy of CDK4/6 inhibitors in targeted therapy of acral melanoma. Experimental Design: A total of 514 primary acral melanoma samples were examined for the copy number variations (CNV) of CDK4 pathway-related genes, including Cdk4, Ccnd1, and P16INK4a, by QuantiGenePlex DNA Assay. The sensitivity of established acral melanoma cell lines and patient-derived xenograft (PDX) containing typical CDK4 aberrations to CDK4/6 inhibitors was evaluated. Results: Among the 514 samples, 203 cases, 137 cases, and 310 cases, respectively, showed Cdk4 gain (39.5%), Ccnd1 gain (26.7%), and P16INK4a loss (60.3%). The overall frequency of acral melanomas that contain at least one aberration in Cdk4, Ccnd1, and P16INK4a was 82.7%. The median overall survival time for acral melanoma patients with concurrent Cdk4 gain with P16INK4a loss was significantly shorter than that for patients without such aberrations (P = 0.005). The pan-CDK inhibitor AT7519 and selective CDK4/6 inhibitor PD0332991 could inhibit the cell viability of acral melanoma cells and the tumor growth of PDX with Cdk4 gain plus Ccnd1 gain, Cdk4 gain plus P16INK4a loss, and Ccnd1 gain plus P16INK4a loss. Conclusions: Genetic aberration of CDK4 pathway is a frequent event in acral melanoma. Acral melanoma cell lines and PDX containing CDK4 pathway aberrations are sensitive to CDK4/6 inhibitors. Our study provides evidence for the testing of CDK4/6 inhibitors in acral melanoma patients. Clin Cancer Res; 23(22); 6946–57. ©2017 AACR.

Systemic Immune-Inflammation Index and Circulating T-Cell Immune Index Predict Outcomes in High-Risk Acral Melanoma Patients Treated with High-Dose Interferon

High-dose interferon alfa-2b (IFN-α-2b) improves the survival of patients with high-risk melanoma. We aimed to identify baseline peripheral blood biomarkers to predict the outcome of acral melanoma patients treated with IFN-α-2b. Pretreatment baseline parameters and clinical data were assessed in 226 patients with acral melanoma. Relapse-free survival (RFS) and overall survival (OS) were assessed using the Kaplan-Meier method, and multivariate Cox regression analyses were applied after adjusting for stage, lactate dehydrogenase (LDH), and ulceration. Univariate analysis showed that neutrophil-to-lymphocyte ratio ≥2.35, platelet-to-lymphocyte ratio ≥129, systemic immune-inflammation index (SII) ≥615 × 109/l, and elevated LDH were significantly associated with poor RFS and OS. The SII is calculated as follows: platelet count × neutrophil count/lymphocyte count. On multivariate analysis, the SII was associated with RFS [hazard ratio (HR)=1.661, 95% confidence interval (CI): 1.066-2.586, P=.025] and OS (HR=2.071, 95% CI: 1.204-3.564, P=.009). Additionally, we developed a novel circulating T-cell immune index (CTII) calculated as follows: cytotoxic T lymphocytes/(CD4+ regulatory T cells × CD8+ regulatory T cells). On univariate analysis, the CTII was associated with OS (HR=1.73, 95% CI: 1.01-2.94, P=.044). The SII and CTII might serve as prognostic indicators in acral melanoma patients treated with IFN-α-2b. The indexes are easily obtainable via routine tests in clinical practice.

Anti-GD2/4-1BB chimeric antigen receptor T cell therapy for the treatment of Chinese melanoma patients

BackgroundChimeric antigen receptor (CAR)-engineered T cells have demonstrated promising clinical efficacy in patients with B cell lymphoma. However, the application of CAR-T cell therapy in the treatment of other solid tumors has been limited. We incorporated 4-1BB into the anti-GD2 CAR-T cells to test their cytotoxicity in melanoma in vitro and in vivo. Moreover, we reported the expression of ganglioside GD2 in non-Caucasian melanoma populations for the first time, thus providing a basis for future clinical research.MethodsThis study included tumor samples from 288 melanoma patients at the Peking University Cancer Hospital & Institute. Clinical data were collected. Immunohistochemical assays using antibodies against ganglioside GD2 were performed on formalin-fixed, paraffin-embedded specimens. The ability of ganglioside GD2 CAR-T cells to kill ganglioside GD2+ melanoma cells was evaluated in vitro and in a patient-derived xenograft (PDX) model.ResultsAmong the 288 samples, 49.3% of cases (142/288) demonstrated positive staining with ganglioside GD2. The median survival time in patients exhibiting ganglioside GD2 expression was significantly shorter than that in patients without ganglioside GD2 expression (31 vs. 47.1xa0months, Pu2009<u20090.001). In the present study, CAR was constructed using a GD2-specific scFv (14.G2a), T cell receptor CD3ζ chain, and the CD137 (4-1BB) costimulatory motif. In addition, the GD2.BBζ CAR-T cells demonstrated specific lysis of ganglioside GD2-expressing melanoma cells in vitro. In two PDX models, mice that received intravenous or local intratumor injections of GD2.BBζ CAR-T cells experienced rapid tumor regression.ConclusionsThese data demonstrate that the rate of GD2 expression in Chinese patients is 49.3%. GD2.BBζ CAR-T cells can both efficiently lyse melanoma in a GD2-specific manner and release Th1 cytokines in an antigen-dependent manner in vitro and in vivo. Anti-GD2/4-1BB CAR-T cells represent a clinically appealing treatment strategy for Chinese melanoma patients exhibiting GD2 expression and provide a basis for future studies of the clinical application of immunotherapy for melanoma.

Mutations in BRAF codons 594 and 596 predict good prognosis in melanoma

B-Raf proto-oncogene serine/threonine kinase (BRAF) V600E is the most common kinase-activating mutation and is associated with poor prognosis in melanoma. However, the clinical significance of kinase-impairing mutations remains unclear. The present study aimed to analyze kinase-impairing mutations in BRAF codons 594 and 596 in non-Caucasian patients with melanoma and to investigate their possible clinical significance. To detect hotspot mutations, exon 15 of the BRAF gene was amplified using polymerase chain reaction in samples from 1,554 patients with melanoma. Among these patients, a total of 912 valid follow-up data were obtained. These patients were divided into three groups according to their BRAF activation status: BRAF wild-type (n=752), BRAF V600E (n=147); and BRAF D594/G596 (n=13). Then the correlation between BRAF activation status, and the clinicopathological features and overall survival (OS) of the patients were analyzed. The prevalence of BRAF mutations in non-Caucasian patients with melanoma was 24.3% (377/1554). Three patients carried two mutations simultaneously. The overall mutation frequencies of kinase-activating mutations, kinase-impairing mutations, and mutations with unknown effects were 93.4 (355/380), 3.4 (13/380), and 3.2% (12/380), respectively. BRAF V600E was identified to be associated with a poor prognosis. Patients with BRAF mutations in codons 594 and 596 had a longer OS time compared with those with a BRAF V600E mutation [median OS, 45 vs. 25 months; HR, 0.45 (95% confidence interval, 0.31–0.97); P=0.043]. To the best of our knowledge, this is the first study to examine a large number of samples from non-Caucasian patients with melanoma and report the characteristics of BRAF mutations according to mutant kinase activity. Melanoma arising from a mutation in BRAF codon 594 or 596 can be differentiated from BRAF V600E-induced melanoma, and mutations in these codons may be good prognostic factors for melanoma. The results of the present study are thus of significance for the development of accurate personalized medicine to treat melanoma.

MNK1/2 inhibition limits oncogenicity and metastasis of KIT-mutant melanoma

Melanoma can be stratified into unique subtypes based on distinct pathologies. The acral/mucosal melanoma subtype is characterized by aberrant and constitutive activation of the proto-oncogene receptor tyrosine kinase C-KIT, which drives tumorigenesis. Treatment of these melanoma patients with C-KIT inhibitors has proven challenging, prompting us to investigate the downstream effectors of the C-KIT receptor. We determined that C-KIT stimulates MAP kinase–interacting serine/threonine kinases 1 and 2 (MNK1/2), which phosphorylate eukaryotic translation initiation factor 4E (eIF4E) and render it oncogenic. Depletion of MNK1/2 in melanoma cells with oncogenic C-KIT inhibited cell migration and mRNA translation of the transcriptional repressor SNAI1 and the cell cycle gene CCNE1. This suggested that blocking MNK1/2 activity may inhibit tumor progression, at least in part, by blocking translation initiation of mRNAs encoding cell migration proteins. Moreover, we developed an MNK1/2 inhibitor (SEL201), and found that SEL201-treated KIT-mutant melanoma cells had lower oncogenicity and reduced metastatic ability. Clinically, tumors from melanoma patients harboring KIT mutations displayed a marked increase in MNK1 and phospho-eIF4E. Thus, our studies indicate that blocking MNK1/2 exerts potent antimelanoma effects and support blocking MNK1/2 as a potential strategy to treat patients positive for KIT mutations.

Increased AURKA Gene Copy Number Correlates with Poor Prognosis and Predicts the Efficacy of High-dose Interferon Therapy in Acral Melanoma

Background: AURKA kinase is an essential serine/threonine kinase for mitosis and chromosome stability. The aberrant amplification and overexpression of AURKA are commonly observed in various types of cancer, including cutaneous melanoma. However, the status and the clinical significance of AURKA copy number (CN) in acral melanoma (AM) have not been fully elucidated. Methods: Four hundred and seventy-two AM samples were included in the study. AURKA CN was examined using the QuantiGenePlex DNA Assay. We analysed the relationship of AURKA CN to clinicopathological characteristics and survival of patients with AM. Results: In this study, AURKA copy gain (set as more than 2.0 copies) was detected in 24.6% (116/472) of the samples. We did not observe any obvious correlation between clinicopathological characteristics and AURKA copy gain of the patients. However, patients with AURKA copy gain had a significantly shorter overall survival time (OS) and progression-free survival time (PFS) than those with normal AURKA CN (OS: P = 0.022; PFS: P < 0.001). Furthermore, multivariate Cox regression analysis showed that AURKA copy gain was an independent poor prognostic factor for patients with AM undergoing adjuvant interferon therapy. Conclusions: This study suggested that AURKA copy gain is an adverse prognostic factor for AM. Furthermore, AURKA copy gain may be a useful biomarker to predict the outcome of interferon therapy in patients with AM.

Analysis of NRAS gain in 657 patients with melanoma and evaluation of its sensitivity to a MEK inhibitor

BACKGROUNDnNeuroblastoma rat-sarcoma (NRAS) mutations have been described in Chinese patients with melanoma. However, the status and the clinical significance of NRAS gain have not been investigated on a large scale.nnnMETHODSnA total of 657 melanoma samples were included in the study. NRAS copy number was examined using the QuantiGene Plex DNA assay. The sensitivities of cell lines and patient-derived xenograft (PDX) models containing NRAS gain to a MAP/ERK kinase (MEK) inhibitor (binimetinib) were also evaluated.nnnRESULTSnThe overall incidence of NRAS gain was 14.0% (92 of 657). Incidence of NRAS gain in acral, mucosal, chronic sun-induced damage (CSD) and non-CSD melanomas was 12.2%, 15.8%, 9.5% and 19.4%, respectively. NRAS gain was mutually exclusive to NRAS mutations (Pxa0=xa00.036). The median survival time for melanoma patients with NRAS gain was significantly shorter than that for patients with normal NRAS copy number (Pxa0=xa00.006). For patients containing NRAS gain, the median survival time for higher copy number (>4 copies) was significantly shorter than those with lower copy number (2-4 copies; Pxa0=xa00.002). The MEK inhibitor (binimetinib) inhibited the proliferation of melanoma cells and the tumour growth of PDX models with NRAS gain.nnnCONCLUSIONSnNRAS gain is frequent in patients with melanoma and may predict a poor prognosis of melanoma. The melanoma cells and PDX models containing NRAS gain are sensitive to MEK inhibitor (binimetinib), indicating that NRAS gain might be a new therapeutic target for melanoma.

cMET-N375S germline mutation is associated with poor prognosis of melanoma in Chinese patients

Background: cMET has been known to play an essential role in malignant melanoma tumor progression. Recent studies have shown that a germline missense variant in exon 2 of the cMET , N375S (rs33917957 A>G) occurred at a much higher frequency in East Asian patients with lung cancer. However, the status of this mutation in melanoma is unclear. In this study, we examined the mutation frequency of cMET-N375S in 181 melanoma samples and analyzed its clinicopathological significance. Methods: Tissue samples (n=181) were analyzed for cMET-N375S, BRAF, NRAS , and CKIT mutation in genomic DNA by polymerase chain reaction amplification and Sanger sequencing. The levels of p-cMET protein were determined by immunohistochemistry. Clinical data of patients were also collected. Results: We identified a highly frequent variant (N375S) of cMET in 13.26% (24/181) of the patients with melanoma. This mutation was relatively more frequent in acral (22.8%) and mucosal (12.7%) melanomas (the two most common melanoma subtypes in Asian, but not in Caucasians) than in non-chronic sun induced damage (non-CSD; 5.9%) and CSD (8.3%) melanomas. Among the 24 cases with cMET-N375S mutations, six cases were found to harbor the BRAF mutation, four cases harbored NRAS mutations, and two cases harbored CKIT mutations. And there was no correlation between this mutation and p-cMET levels. Kaplan- Meier analysis showed significant differences in overall survival (OS) and disease-free survival (DFS) between the N375S mutation group and non-N375S mutation group (P=0.039; P=0.030). The Cox proportional hazards model revealed that cMET-N375S mutation was an independent adverse prognostic factor for OS [P=0.003, hazard ratio (HR) =3.577]. Conclusions: Our findings suggested that the cMET-N375S mutation was an adverse prognostic factor for melanoma in a Chinese cohort.

Methylation of O6-methylguanine DNA methyltransferase promoter is a predictive biomarker in Chinese melanoma patients treated with alkylating agents

Background: The O6-methylguanine-DNA methyltransferase gene ( MGMT ) promoter methylation status can be used to predict the prognosis of patients with various cancers following treatment with alkylating agents. Moreover, MGMT promoter methylation often coexists with TP53 gene mutation. However, MGMT has not been identified as a biomarker of melanoma. Therefore, this study systematically analyzed the prognostic role of MGMT and the correlation between MGMT methylation and TP53 mutation in non-Caucasian patients with melanoma. Methods: This study involved tumor samples and clinical data collected from 205 melanoma patients treated with alkylating agents at Peking University Cancer Hospital & Institute. The MGMT promoter methylation and TP53 mutation status were analyzed respectively using methylation-specific polymerase chain reaction and polymerase chain reaction followed by Sanger sequencing. Additionally, MGMT protein expression in tumor samples was assessed via immunohistochemistry. n Results: MGMT promoter methylation was detected in 97 (47%) of the 205 tumor samples, and was significantly associated with a loss of MGMT protein expression (P=0.021). MGMT promoter methylation was also significantly associated with the presence of TP53 mutation (P=0.004). Regarding prognosis, patients without MGMT promoter methylation exhibited worse overall survival outcomes, compared to those with methylation (hazard ratio: 1.443; 95% confidence interval: 0.731–2.342; P=0.015). Conclusions: MGMT promoter methylation appears to coexist frequently with TP53 mutation. Patients harboring MGMT promoter methylation within tumors may benefit from therapy with alkylating agents.