Natural killer/T cell lymphoma (NKTCL) is a rare and aggressive malignancy with a higher prevalence in Asia and South America. However, the molecular genetic mechanisms underlying NKTCL remain unclear. Here, we identify somatic mutations of GNAQ (encoding the T96S alteration of Gαq protein) in 8.7% (11/127) of NKTCL patients, through whole-exome/targeted deep sequencing. Using conditional knockout mice (Ncr1-Cre-Gnaqfl/fl), we demonstrate that Gαq deficiency leads to enhanced NK cell survival. We also find that Gαq suppresses tumor growth of NKTCL via inhibition of the AKT and MAPK signaling pathways. Moreover, the Gαq T96S mutant may act in a dominant negative manner to promote tumor growth in NKTCL. Clinically, patients with GNAQ T96S mutations have inferior survival. Taken together, we identify recurrent somatic GNAQ T96S mutations that may contribute to the pathogenesis of NKTCL. Our work thus has implications for refining our understanding of the genetic mechanisms of NKTCL and for the development of therapies.

Nasal-type natural killer/T-cell lymphoma (NKTCL) is a subtype of non-Hodgkin lymphoma (NHL) that is clinically aggressive and has a poor prognosis. Platelet-derived growth factor receptors (PDGFRs) and their ligands (PDGFs) play important roles in angiogenesis, cancer cell proliferation, survival, migration and poor prognosis in various tumours. However, the significance of PDGFRs in NKTCL remains unknown. Herein, the present study aimed to investigate the important role of PDGFRα in pathogenesis, progression and prognisis of NKTCL. Firstly, we performed immunohistochemical staining, qRT-PCR and western blotting to determine PDGFRα expression in formalin-fixed, paraffin-embedded tissue sections from 78 NKTCL cases and in cell lines. Secondly, correlations between PDGFRα expression and NKTCL clinical parameters and prognosis were analysed. Moreover, a biological assessment of PDGFRα blockade in two NKTCL cell lines was conducted through proliferation assay, cell-cycle evaluation and apoptosis detection by flow cytometry analyses. Furthermore, we detected in vivo activity of imatinib in mouse model of NKTCL. We found that the expression of PDGFRα was significantly higher in NKTCL tissues compared to the reactive lymphoid hyperplasia of the nasopharynx (P = 0.028). High PDGFRα expression was strongly associated with a high LDH level (P = 0.028) and III-IV stage (P = 0.013). NKTCL patients with high PDGFRα expression displayed a reduced median overall survival time and progression-free survival time when compared with those with low PDGFRα expression (P = 0.011, P = 0.005, respectively). Cox multivariate analysis showed that III-IV stage (P = 0.024) and high PDGFRα expression (P = 0.003) were independent prognostic factors in NKTCL patients. Biological assessment assays in two NKTCL cell lines revealed that a specific PDGFR antagonist, imatinib, inhibited cell viability, blocked cell cycle progression at G0/G1 stage and induced apoptosis. Similarly, the in vivo assay showed that imatinib delayed mouse model tumour growth. In conclusion, NKTCL tumour cells have prominent PDGFRα expression, which can serve as a candidate prognostic marker. PDGFR antagonists have significant biological effect on NKTCL and may be useful therapeutic agents for treatment of NKTCL.

There are numerous types of B-cell lymphoma including Burkitt lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma/mucosa-associated lymphoid tissue lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma and other less frequent subtypes. However, the molecular pathogenesis of B-cell lymphoma remain largely unknown. Here we chose three different B-cell lymphoma subtype cell lines to establish mouse models, which could be useful to determine the efficacy and mechanisms of novel therapies.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare form of non-Hodgkin lymphoma accounting for approximately 1% of all primary cutaneous lymphomas (Willemze et al, 2005). To date, little is known about the pathogenesis of this lymphoma, partly due to its rarity. To obtain a genome-wide view of the molecular genetic alterations underlying SPTCL pathogenesis, we performed whole-exome sequencing (WES) and targeted sequencing for a series of SPTCL cases. Eighteen SPTCL cases with available formalin-fixed paraffin-embedded (FFPE) tumour tissue from four cancer centres between 2005 and 2016 were collected (see Supplementary Information). The demographics, clinical and immunohistochemical features of the patient cohort are summarized in Table S1. To identify somatic genomic variants associated with SPTCL, we first performed WES on 10 tumour samples and 3 matched whole blood samples from our cancer centre. The mean sequencing depth was 140·99× (ranging from 109·4 to 193·2×), and a mean of 98·10% of the target sequence covered a depth of at least 10× (Table S2). The mutation frequencies in the 10 patients with SPTCL varied remarkably, with a median of 345 somatic non-synonymous mutations per case (ranging from 104 to 2383, Figure S1A). We observed a preference for C>T/G>A alterations analogous to the somatic single-nucleotide variation (SNV) spectrum in other cancers (Figure S1B). Forty SNVs/Indels (insertion-deletions) were selected based on the variant frequency for validation by Sanger sequencing, and 92·5% of the variants were confirmed (Table S3). We identified substantial heterogeneity in the SPTCL somatic mutation landscape. There were 7032 somatic non-synonymous mutations affecting 4714 genes identified in the 10 SPTCL subjects. Interestingly, most common mutations described previously in other T-cell lymphomas (Forbes et al, 2015), such as TP53, DNMT3A, PLCG1, STAT3 and CDKN2A, were not frequently observed in SPTCL. To efficiently identify the potential driver mutations contributing to SPTCL pathogenesis, we selected genes included in the Cancer Gene Census (Futreal et al, 2004) and/or in the lists of cancer genes reported by Kandoth et al (2013); Vogelstein et al (2013) and Tamborero et al (2013) for further analysis. We detected 284 non-synonymous mutations affecting 202 genes in the 10 SPTCL patients (Table S4). Pathway analysis of these 202 mutated genes revealed that there was a significant enrichment of genes involved in the phosphoinositide 3-kinase (PI3K)/AKT/mechanistic/mammalian target of rapamycin (mTOR) and JAK-STAT signalling pathways (P < 0·001; Table S5). We further applied a comprehensive targeted sequencing approach to validate the above findings. The panel, comprising 560 genes selected for targeted sequencing, is listed in Table S6. Tumour and matched normal DNA samples extracted from 8 independent SPTCL cases in three other cancer centres (Data S1) were subjected to targeted sequencing. The mean sequencing depth was 674× (ranging from 505–1044×); 21 non-synonymous mutations were identified (Tables S7, S8). Collectively, mutations in epigenetic modifiers were found in 13 of 18 (72%) SPTCL cases (Fig 1). These modifiers are involved in the epigenetic machinery at almost every level, including key players in chromatin organization (ARID1B, 3/18; SMARCA4, 3/18; and CHD4, 3/18), DNA methylation (MBD1, 1/18), and histone modification (CREBBP, 2/18; KMT2D, 2/18; and DOT1L, 2/18). Recurrent mutations were identified in genes encoding components of the PI3K/AKT/mTOR signaling pathway in 8 of 18 (44%) SPTCL cases. Within the PI3K/AKT/mTOR cascade, most frequent mutations were noted in the MTOR and TSC1 genes (3/18 cases), followed by PIK3CA (2/18), PIK3CD (2/18) and PIK3CB, TSC2 and AKT2 (1/18 each) (Fig 1). These gene mutations have not been previously reported, except for the MTOR (p.A1124V) mutations, which have been reported in the Catalogue of Somatic Mutations in Cancer database (COSMIC, http://cancer.sanger.ac.uk/cosmic). Immunohistochemical staining showed that tumours with PI3K/AKT/mTOR mutations exhibited higher AKT1 and 4E-BP1 (also termed EIF4BP1) phosphorylation levels than that of tumours without the mutations (P < 0·05; Figure S2), suggesting that these gene mutations might participate in the activation of PI3K signalling. In addition, the mutations in the PI3K/AKT/mTOR cascade appeared to be mutually exclusive despite the low number of patients. Dysregulation of the JAK-STAT pathway is commonly implicated in the pathogenesis of T-cell lymphomas (Kiel et al, 2015). In this study, we identified somatic mutations affecting JAK3 (3/18), STAT3 (1/18) and IL7R (1/18), presenting in 28% (5 of 18) of our cases (Fig 1). We identified three novel mutations (p.V152M, p.G24S and p.G153D) in JAK3, an activating D661Y hotspot mutation in STAT3, and a p.T179M mutation in IL7R. All of these mutations appeared to be mutually exclusive. In our study, TP53 mutation was identified in only one SPTCL case, which is less frequent than that of other T-cell lymphomas. NAV3 (neuron navigator 3) deletion has been reported in approximately 40% of STCL samples (Hahtola et al, 2008). We also detected NAV3 mutations in two cases of SPTCL. These results therefore implicate that NAV3 might play a vital role in SPTCL pathogenesis, but further investigation is needed. To test the therapeutic potential of targeting the PI3K/AKT/mTOR pathway, one primary SPTCL sample (sample SP1) harbouring mutations in MTOR was subjected to PI3K and/or mTOR inhibition. Immunohistochemical analysis indicated that the PI3K/AKT/mTOR signalling pathway was activated (Fig 2A). Tumour cells (CD4−, CD8+ and CD56−) were enriched and purified from the primary SPTCL sample with magnetic beads, and the purity was approximately 85% (Fig 2B). Two inhibitors targeting mTOR (rapamycin and everolimus) and one inhibitor targeting mTOR and PI3K (apitolisib) reduced cell viability in a dose-dependent manner (Fig 2C). Moreover, these three inhibitors could inhibit the downstream signalling cascades by suppressing p70S6K, 4E-BP1 and AKT phosphorylation (Fig 2D). Primary SPTCL cells were resistant to an inhibitor for a non-relevant pathway, the NF-κB inhibitor BAY 11-7082; therefore, the observed effects were probably not attributable to general cytotoxicity. The primary tumour cells from SPTCL samples also exhibited high sensitivity to the histone deacetylase (HDAC) inhibitor romidepsin (Fig 2C), this is consistent with an earlier report that romidepsin was effective in SPTCL patients (Bashey et al, 2012). In summary, pharmacological targeting of PI3K/AKT/mTOR or HDAC signalling could inhibit the growth of primary SPTCL tumour cells. In conclusion, to the best of our knowledge this is the first study to comprehensively characterize the genome-wide mutational landscape of SPTCL. We identified frequent gene mutations in epigenetic modifiers and the PI3K/AKT/mTOR pathway in SPTCL. Targeting these two pathways may represent a promising therapeutic strategy in SPTCL. This study was supported by the Youth Innovation Funds Project of The First Affiliated Hospital of Zhengzhou University. The authors would like to thank Xinfeng Chen and Shuai Liu for their excellent technical assistance, and Virender Sachdeva, M.S. from Department of Paediatric Ophthalmology of Nimmagada, Prasad Children's Eye Care Centre, L V Prasad Eye Institute, GMRV Campus, India and Tian Tian from the Department of Neurology, the First Affiliated Hospital of Zhengzhou University, China for language editing. Z.L., L.L., and M.Z. designed the study, interpreted results, and wrote the manuscript; Z.L., L.L., Y.W., W.X., Z.Z., M.J., and Y.Q. performed the experiments; W.S. and X.F. performed the bioinformatics analysis; Y.C., F.N., J.Y., G.W., Z.S., X.F., L.L., X.L., X.W., J.W., and L.Z. provided samples and clinical data. Authors declare no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Pegaspargase combined with gemcitabine have greatly improved the outcomes of advanced extranodal NK/T cell lymphoma (ENKL). However, patients frequently undergo recurrent disease due to chemoresistance, and few predictive parameters are available. The present study explored potential biomarkers to predict the therapeutic response of advanced ENKL treated with pegaspargase/gemcitabine and evaluate the prognostic significance. Through serum proteomic analysis, we identified 61 upregulated and 22 downregulated proteins in nonresponders compared with responders. We further validated that patients with unfavourable treatment outcomes displayed higher levels of S100A9 and ORM1 via enzyme-linked immunosorbent assay (ELISA). Moreover, the sensitivity and specificity for detecting refractory patients were 81.5% and 71.4% for S100A9 > 62.0 ng/ml, 85.2% and 77.1% for ORM1 > 1436 ug/ml, 100% and 57.1% for S100A9 combined with ORM1. Furthermore, in multivariate analysis elevated levels of S100A9 were associated with poor 2-year OS (40.2% vs. 76.6%, RR = 2.92, p = 0.005) and 2-year PFS (33.1% vs. 61.1%, RR = 2.61 p = 0.011). High ORM1 also predicted inferior 2-year OS (38.7% vs. 76.1, RR = 2.46, p = 0.023) and 2-year PFS (18.4% vs. 73.2%, RR = 2.86, p = 0.009). Our results indicated that S100A9 and ORM1 could serve as reliable predictors of therapeutic response and independent prognostic factors of survival in advanced ENKL patients treated with pegaspargase/gemcitabine.

The aim of the present study was to identify the potential relevant biomarkers to predict the therapeutic response of advanced extranodal natural killer/T cell lymphoma(ENKTL) treated with asparaginase-based treatment. Proteomic technology is used to identify differentially expressed proteins between chemotherapy-resistant and chemotherapy-sensitive patients. Then enzyme-linked immunosorbent assay is used to validate the predictive value of selective biomarkers. A total of 61 upregulated and 22 downregulated proteins are identified in chemotherapy-resistant patients compared with chemotherapy-sensitive patients. Furthermore, they validated that pretreatment high level 14-3-3 epsilon(ε)(≥61.95 ng/mL, 84.0 and 95.2% for sensitivity and specificity, respectively) is associated with poor 2-year overall survival (OS) (5.3 vs 68.8%, p <0.0001) and PFS (4.5 vs 76.9%, p <0.0001). In multivariate survival analysis, pretreatment high level 14-3-3 epsilon significantly is correlated with both inferior OS ( p = 0.033) and PFS ( p = 0.005). These findings indicate that pretreatment high level 14-3-3 epsilon is an independent predictor of chemotherapy-resistance and poor prognosis for patients with advanced ENKTL in the era of asparaginase.

The aim of the present study was to perform a meta-analysis to assess the diagnostic value of fluorine-18 fluorodeoxyglucose ((18)F-FDG) PET-CT/PET in the pre-operative evaluation of TNM staging in patients with primary colorectal cancer (CRC). The Medline, Embase and Web of Knowledge were searched for studies assessing the diagnostic value of (18)F-FDG PET-CT/PET in the pre-operative evaluation of TNM staging in CRC patients. We pooled the sensitivity, specificity, positive and negative Likelihood ratio (LR+ and LR-) and Diagnostic Odds Ratio (DOR) and constructed summary receiver operating characteristic curves. A total of 28 studies including 2283 CRC patients were analyzed. The pre-operative tumor detecting rate of PET-CT was 95.35%, which was superior to CT (P < 0.05). The pooled sensitivity and specificity of pre-operative T staging by PET-CT/PET was 0.73 (95% CI: 0.65-0.81) and 0.99 (95% CI: 0.98-0.99), which the AUC and Q* were 0.96 and 0.91, respectively. Concerning pre-operative N staging, the pooled sensitivity and specificity of PET-CT/PET were 0.62 and 0.70, which the AUC and Q* were 0.76 and 0.70, respectively. As for M staging, the pooled sensitivity and specificity of PET-CT/PET were 0.91 (95% CI: 0.80-0.96) and 0.95 (95% CI: 0.91-0.98), which the AUC and Q* were 0.96 and 0.91, respectively. (18)F-FDG PET-CT/PET had good performance in the pre-operative tumor detecting rate, T staging and M staging in patients with primary CRC, which might alter the therapeutic strategy. However, the diagnostic value of (18)F-FDG PET-CT/PET in pre-operative N staging in CRC patients was not ideal.

Objective To investigate the expressions of the key moleculesβ-catenin,c-myc,and cyclinD1 of the Wnt/β-catenin signal pathway in the NK/T-cell lymphoma(NKTCL)tissue,and to clarify their relationships with the clinicopathological features of the NKTCL patients.Methods Real-time PCR method was used to detect the expression levels ofβ-catenin,c-myc,and cyclinD1 mRNA in human NKTCL cell lines(SNK-6and YTS)and normal NK cells;the positive expression rates ofβ-catenin and c-myc proteins in the NKTCL tissue and reactive hyperplasia of lymph node tissue were detected by immunohistochemistry SP method;the correlations among the expressions ofβ-catenin and c-myc proteins and the clinicopathological features of the NKTCL patients were analyzed;correlation analysis was used to analyze the relationship between the expressions ofβ-catenin and c-myc proteins of the NKTCL patients;Kaplan-Meier method was used to analyze the relationship between the expression ofβ-catenin and overall survival rate of the NKTCL patients.Results The positive expression rates ofβ-catenin,c-myc,and cyclinD1 mRNA in the SNK-6cell line and YTS cell line were significantly higher than those in the normal NK cells(P0.05);the expressionβ-catenin and c-myc proteins in NKTCL tissue(24% and 56%)were higher than those in reactive hyperplasia of lymph node tissue(0and 25%)(P0.05);the expression ofβ-catenin was correlated with Ann Arbor stage(P0.05),and the expression of c-myc was correlated with Ann Arbor stage and Ki-67(P0.05);there were positive correlations between the expression ofβ-catenin and the expression of c-myc in the NKTCL patients(r=0.770,P0.05);the patients withβ-catenin positive expression had a shorter overall survival time than the patients with negative expression(P 0.05).Conclusion The key moleculesβ-catenin,c-myc,and cyclinD1 of the Wnt/β-catenin signal pathway are highly expressed in the human NKTCL tissue,and they show positive correlations with Ann Arbor stage of the NKTCL patients.

The aim was to investigate the function of fibroblast growth factor receptor 4 (FGFR4) in gastric cancer (GC) and explore the treatment value of agent targeted to FGFR4. Function assays in vitro and in vivo were performed to investigate the discrepancy of biological features among the GC cells with different expression of FGFR4. GC cells were treated with the single and combination of PD173074 (PD, an inhibitor of FGFR4) and 5-fluorouracil (5-Fu). The invasion ability were stronger, and the apoptosis rates were lower in MGC803 and BGC823 cells treated with FGFR4-LV5 (over-expression of FGFR4 protein) (P < 0.05). The proliferation ability of GC cells is reduced when treated by the single and combination of 5-Fu and PD while that of the FGFR4-LV5 group was less inhibited compared with control group (P < 0.05). The apoptosis rates are remarkably increased in GC cells treated with the single and combination of 5-Fu and PD (P < 0.05). However, the apoptosis rate obviously is reduced in GC cells treated with FGFR4-LV5 compared with control group (P < 0.05). The expression of PCNA and Bcl-XL is remarkably decreased, and the expression of Caspase-3 and cleaved Caspase-3 is obviously increased in GC cells treated with the single and combination of 5-Fu and PD. The tumor volumes of nude mice in FGFR4-LV5 group were much more increased (P < 0.05). The over-expression of FGFR4 enhanced the proliferation ability of GC in vitro and in vivo. The combination of 5-Fu and PD exerted synergetic effect in weakening the proliferation ability and promoting apoptosis in GC cells, while the over-expression of FGFR4 might inhibit the efficacy of two drugs.

Evaluation of bone marrow involvement (BMI) by conventional bone marrow biopsy (BMB) can generate false-negative results if marrow disease is focal. The sensitivity of 18F-2-fluoro-2-deoxy-d-glucose positron emission tomography/computed tomography (FDG-PET/CT) in assessing BMI in extranodal NK/T cell lymphoma (ENKL) has not been determined. We retrospectively collected clinical data from a series of 55 patients with newly diagnosed ENKL, who have received both FDG-PET/CT and BMB prior to treatment. BMB results were used as reference standard. Twelve patients (21.8 %) were considered positive lymphomatous infiltration by FDG-PET/CT (PET-CT/BM+), and five patients (9 %) were identified positive by BMB (BMB/BM+). There was a discordant result in seven patients who were PET-CT/BM+ but BMB/BM−. The sensitivity and specificity of FDG-PET/CT for identifying BMI were 100 and 86 %, respectively. Then, we analyzed the overall survival (OS) and progression-free survival (PFS) of patients who were PET-CT/BM+ and PET-CT/BM−. The median follow-up time was 16 months (range, 3 to 43 months). PET-CT/BM+ patients possessed worse 2-year OS than PET-CT/BM− patients (84.8 vs 67.9 %, P < 0.05). The estimated 2-year PFS for PET-CT/BM− and PET-CT/BM+ patients were 72.7 and 41.9 % (P < 0.05), respectively. However, it was hard to conclude that patients who were PET-CT/BM+ had similar survivals to advanced-stage patients due to the low number of patients who were PET-CT/BM+. In conclusion, FDG-PET/CT can complementally detect positive BMI patients missed by BMB in ENKL. The utility of FDG-PET/CT for defining bone marrow status has important prognostic value.

The prognosis of extranodal nature killer (NK)/T cell lymphoma (ENKL) is dismal because of its aggressive course and multidrug resistance. Currently, for patients with relapsed/refractory ENKL, l-asparaginase-based regimens such as l-asparaginase, ifosfamide, methotrexate, etoposide, and dexamethasone (SMILE) or l-asparaginase, methotrexate, and dexamethasone (AspaMetDex) are recommended. We retrospectively investigated the efficacy and safety of gemcitabine, pegaspargase, cisplatin, and dexamethasone (DDGP) combination chemotherapy in the treatment of 17 relapsed/refractory ENKL patients. Clinical data from these patients were collected and analyzed. The primary end point was overall response rate (ORR). All patients were subjected to 2 to 6 cycles of DDGP chemotherapy, and the median number of cycles of DDGP regimen administrated was four. The ORR was 88.2 % (15/17), with nine patients (52.9 %) achieved complete response (CR) and six patients (35.3 %) achieved partial response (PR). The median follow-up time was 17 months (range 2-28 months). The 1-year overall survival (OS) rate and 1-year progression-free survival (PFS) were 82.4 and 64.7 %, respectively. For those CR responders, the median PFS was 17 months. Grade 3/4 neutropenia occurred in nine patients (52.9 %) and grade 3/4 thrombocytopenia occurred in six patients (35.3 %). DDGP combination chemotherapy produces favorable outcomes in relapsed/refractory ENKL, and more attention should be paid to treatment-related myelosuppression. Further prospective trials are expected to define the efficacy.