Pitfalls in diagnosing a case of extranodal NK/T-cell lymphoma with CD20 aberrant expression and IGH gene rearrangement.

Extranodal NK/T-cell lymphoma (ENKTL) is a subtype of non-Hodgkin lymphoma mainly derived from NK cells and, uncommonly, T-cells. A diagnostic challenge is presented when an atypical phenotype and gene rearrangement are encountered. Herein, we report a case of ENKTL with CD20 expression and IGH gene rearrangement, which is extremely rare. A 57-year-old female patient was seen in 2021 due to a nodule on her left leg and simultaneously impaired eyesight for 6 months. Skin biopsy and immunohistochemistry were performed. The lymphoid cells were positive for CD3, CD56, granzyme B, and TIA-1, partially positive for CD2, and mildly positive for CD20. In situ hybridization for Epstein-Barr virus was positive. Molecular studies revealed immunoglobulin heavy chain (IGH) gene rearrangement, while no T-cell receptor gene rearrangement was detected. The positron emission tomography scan showed that the lymphoma affected bilateral adrenal glands, pelvic cavity, peritoneal cavity, small intestine, skin, and subcutis of the bilateral lower extremities of the patient. Her disease progressed despite eight cycles of chemotherapy and radiation therapy. The importance of this case lies in the atypical phenotype and IGH gene rearrangements, necessitating comprehensive interpretation of clinicopathological data.

Diagnostic value of IL-22, IL-23, and IL-17 for NK/T cell lymphoma.

NK/T cell lymphoma (NKTCL) is a common blood cancer, and early diagnosis of this disease is crucial. This study is aimed to investigate the roles of IL-17, IL-22 as well as IL-23 for the diagnosis of NKTCL. Sixty-five patients with NKTCL were included and the blood samples were collected, and sixty healthy objectives served as the controls. Serums of the patients and controls were collected. The expression levels of IL-17, IL-22, and IL-23 were examined using enzyme-linked immunosorbent (ELISA) assay. The receiver operator characteristic (ROC) curve was drawn for determining the potential diagnostic value of these cytokines. The serum levels of IL-17 (156.0 ± 67.75 pg/mL), IL-22 (39.98 ± 23.88 pg/mL), and IL-23 (43.05 ± 25.69 pg/mL) were all markedly increased in NKTCL patients (P<0.001); ROC analysis showed the serum level of IL-17, IL-22, and IL-23 could serve as the potential diagnostic biomarker for NKTCL with high sensitivity and specificity. The AUC of IL-17 was 0.9487 (95% confidence interval (CI), 0.9052 to 0.9922). Area under the curve (AUC) of IL-22 was 0.7321 (95% CI, 0.6449 to 0.8192). The AUC of IL-23 was 0.7885 (95% CI, 0.7070 to 0.8699). Our data indicated that IL-17, IL-22, and IL-23 were all increased in NKTCL and may function as potential diagnostic biomarkers for NKTCL.

T-cell dysfunction in natural killer/T-cell lymphoma.

Natural killer/T-cell lymphoma (NKTCL) is an incurable aggressive T-cell lymphoma closely correlated with Epstein‒Barr virus (EBV) infection. Chronic and consistent viral infection induces T-cell exhaustion. Herein, we describe T-cell dysfunction in NKTCL patients for the first time. Peripheral blood mononuclear cells (PBMCs) from age-matched healthy donors (HDs) and NKTCL patients were collected, and lymphocyte distributions, multiple surface inhibitory receptors (IRs), effector cytokine production and cell proliferation were determined by flow cytometry. PBMCs from HDs were cocultured with NKTCL cell lines to verify the clinical findings. IR expression was further assessed in NKTCL tumor biopsies using multiplex immunohistochemistry (mIHC). NKTCL patients have higher frequencies than HDs of inhibitory T regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSCs). T-cell distribution also varies between NKTCL patients and HDs. T cells from NKTCL patients demonstrated higher expression levels of multiple IRs than HDs. Meanwhile, T-cell proliferation and interferon-γ production was significantly reduced in NKTCL patients. More importantly, the number of EBV-specific cytotoxic cells was lower in NTKCL patients, and these cells demonstrated upregulation of multiple IRs and secreted fewer effector cytokines. Interestingly, NKTCL cells caused normal PBMCs to acquire T-cell exhaustion phenotypes and induced generation of Tregs and MDSCs. In line with ex vivo finding, mIHC results showed that CD8+ T cells from NKTCL tumor biopsies expressed much higher level of IRs compared with reactive lymphoid hyperplasia individuals. The immune microenvironment of NKTCL patients exhibited T-cell dysfunction and accumulation of inhibitory cell components, which may contribute to impaired antitumor immunity.

Prognostic value of prognostic nutritional index on extranodal natural killer/T-cell lymphoma patients: A multicenter propensity score matched analysis of 1022 cases in Huaihai Lymphoma Working Group.

Prognostic nutritional index (PNI), comprised of serum albumin level and lymphocyte count, is associated with the prognosis of several malignant diseases, while the prognostic value of PNI in extranodal natural killer/T cell lymphoma, nasal type (ENKTL) remains unclear. This retrospective multicenter study aimed to investigate the value of PNI in predicting the prognosis of newly diagnosed ENKTL patients by using propensity score matched analysis (PSM). A total of 1022 newly diagnosed ENKTL patients were retrieved from Huaihai Lymphoma Working Group and clinicopathological variables were collected. MaxStat analysis was used to calculate the optimal cut-off points of PNI and other continuous variables. The median age at diagnosis was 47 years and 69.4% were males, with the 5-year OS of 71.7%. According to the MaxStat analysis, 41 was the optimal cut-off point for PNI. The Pseudo R2 before matching was 0.250, and it decreased to less than 0.019 after matching. Confounding factors of the two groups were well balanced after PSM. Multivariable analysis revealed that PNI, Korean Prognostic Index (KPI), eastern cooperative oncology group performance status (ECOG PS), the prognostic index of natural killer lymphoma (PINK) and hemoglobin were independent prognostic factors for ENKTL. The results of subgroup analysis demonstrated that patients with low PNI could predict worse prognosis and re-stratify patients in ECOG PS ≥ 2, EBER-positive, the International Prognostic Index (IPI) (HIR + HR), and PINK (HR) groups. PNI combined with IPI, PINK and KPI could improve the prediction efficiency. In conclusion, PNI could accurately stratify the prognosis of ENKTL by PSM analysis and patients with low PNI had poorer prognosis.

Comprehensive Analysis and Summary of the Value of Immunophenotypes of Mature NK Cell Tumors for Differential Diagnosis, Treatment, and Prognosis.

Background: Few studies have been performed to comprehensively analyze and summarize the immunophenotype and differential diagnosis of mature NK cell tumors, and there is often overlap between tumorigenic and reactive NK cell phenotypes. Furthermore, the impact of different phenotypes on patient prognosis has rarely been reported. Methods: The degree of expression of extracellular and intracellular markers of NK cells in each group was compared by FCM, and the differences in expression of various markers among different disease groups and their impact on prognosis have been analyzed and summarized. Results: Compared with normal NK cells, tumor cells of ANKL and ENKTL had characteristics of being more activated and progressive with larger FSC, in contrast to NK-CLPD and RNKL. Differential diagnoses with RNKL, ANKL, and ENKTL have broader FCM clues. In contrast, the phenotypes of NK-CLPD and RNKL are not significantly different, and consistent phenotypic abnormalities require ongoing monitoring to confirm malignant clones. The sensitivity of differentiating malignant NK cells from reactive NK cells by KIRs alone was poor. The clustering results showed that CD5, CD16, CD56, CD57, CD94, CD45RA, CD45RO, HLA-DR, KIRs, Granzyme B, Perforin and Ki-67 were differentially distributed in the expression of three NK cell tumors and reactive NK cell hyperplasia, so a comprehensive judgment using a wide range of antibody combinations is required in disease staging diagnosis. The tumor cell loads in BM and PB were also compared, and there was a clear correlation between the two. Moreover, the sensitivity of PB for monitoring tumor cells was up to 87.10%, suggesting that PB could be used as an alternative to BM for the diagnosis and screening of NK cell tumors. Analysis of the phenotypic impact of ENKTL patients on prognosis showed that those with CD7 and CD45RO expression had a poor prognosis, while those with positive KIRs had a better prognosis. Conclusion: This study systematically characterized the FCM of mature NK cell tumors, emphasizing the importance and clinical value of accurate immunophenotyping in diagnosing, classifying, determining prognosis, and guiding treatment of the disease.

IL-10 contributes to gemcitabine resistance in extranodal NK/T-cell lymphoma cells via ABCC4.

Background Chemotherapy resistance is a main reason for treatment failure in extranodal NK/T-cell lymphoma (ENKTL). Interleukin-10 (IL-10) is closely related to the occurrence and prognosis of ENKTL. We intended to study the role and molecular mechanism of IL-10 in ENKTL resistance. Methods Fifty serum samples were collected from patients with a histologically proven diagnosis of ENKTL. Fifty healthy volunteers were enrolled as a control group. The level of serum IL-10 was detected by ELISA. The NK/T-cell lymphoma cell lines YT and NK-92 were divided into the control group (untreated), IL-10 group (treated with IL-10), IL-10 + GEM group (treated with IL-10 and gemcitabine simultaneously) and GEM group (treated with gemcitabine). A CCK8 assay and flow cytometry were employed to detect the effects of IL-10 on each group. Western blotting was applied to detect the expression of ABC membrane transporter family proteins and signaling pathway proteins in each group. Results Serum IL-10 levels were higher in ENKTL patients as well asin patients with ineffective treatment. The IC50 value for gemcitabine in YT and NK-92 cells increased significantly in the presence of IL-10. The effects of gemcitabine resulting in cell killing, cell cycle arrest, and apoptosis promotion were also weakened by IL-10. The expression of ABCC4, STAT1, p-STAT1, Tyk2 and p-Tyk2 was significantly increased by IL-10. Conclusion Our results indicate that IL-10 contributes to the resistance of ENKTL cells via ABCC4 and that IL-10 regulates the JAK/STAT signaling pathway in YT and NK-92 cells.

Clinicopathologic and Genetic Features of Primary T-cell Lymphomas of the Central Nervous System: An Analysis of 11 Cases Using Targeted Gene Sequencing.

Primary central nervous system lymphoma (PCNSL) of peripheral T-cell lineage (T-PCNSL) is rare, and its genetic and clinicopathologic features remain unclear. Here, we present 11 cases of T-PCNSL in immunocompetent individuals from a single institute, focusing on their genetic alterations. Seven cases were subject to targeted panel sequencing covering 120 lymphoma-related genes. Nine of the eleven cases were classified as peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), of which one was of γδT-cell lineage. There was one case of anaplastic lymphoma kinase-positive anaplastic large cell lymphoma and another of extranodal natural killer (NK)/T-cell lymphoma (ENKTL) of αßT-cell lineage. The male to female ratio was 7 : 4 and the age ranged from 3 to 75 years (median, 61 y). Most patients presented with neurological deficits (n=10) and showed multifocal lesions (n=9) and deep brain structure involvement (n=9). Tumor cells were mostly small-to-medium, and T-cell monoclonality was detected in all nine evaluated cases. PTCL-NOS was CD4-positive (n=4), CD8-positive (n=3), mixed CD4-positive and CD8-positive (n=1), or CD4/CD8-double-negative (n=1, γδT-cell type). Cytotoxic molecule expression was observed in 4 (67%) of the 6 evaluated cases. Pathogenic alterations were found in 4 patients: one PTCL-NOS case had a frameshift mutation in KMT2C, another PTCL-NOS case harbored a truncating mutation in TET2, and another (γδT-cell-PTCL-NOS) harbored NRAS G12S and JAK3 M511I mutations, and homozygous deletions of CDKN2A and CDKN2B. The ENKTL (αßT-cell lineage) case harbored mutations in genes ARID1B, FAS, TP53, BCOR, KMT2C, POT1, and PRDM1. In conclusion, most of the T-PCNSL were PTCL-NOS, but sporadic cases of other subtypes including γδT-cell lymphoma, anaplastic lymphoma kinase-positive anaplastic large cell lymphoma, and ENKTL were also encountered. Immunophenotypic analysis, clonality test, and targeted gene sequencing along with clinicoradiologic evaluation, may be helpful for establishing the diagnosis of T-PCNSL. Moreover, this study demonstrates genetic alterations with potential diagnostic and therapeutic utility in T-PCNSL.

T and NK cell lymphoma cell lines do not rely on ZAP-70 for survival.

B-cell receptor (BCR) signalling is critical for the survival of B-cell lymphomas and is a therapeutic target of drugs such as Ibrutinib. However, the role of T-cell receptor (TCR) signalling in the survival of T/Natural Killer (NK) lymphomas is not clear. ZAP-70 (zeta associated protein-70) is a cytoplasmic tyrosine kinase with a critical role in T-cell receptor (TCR) signalling. It has also been shown to play a role in normal NK cell signalling and activation. High ZAP-70 expression has been detected by immunohistochemistry in peripheral T cell lymphoma (PTCL) and NK cell lymphomas (NKTCL). We therefore, studied the role of TCR pathways in mediating the proliferation and survival of these malignancies through ZAP-70 signalling. ZAP-70 protein was highly expressed in T cell lymphoma cell lines (JURKAT and KARPAS-299) and NKTCL cell lines (KHYG-1, HANK-1, NK-YS, SNK-1 and SNK-6), but not in multiple B-cell lymphoma cell lines. siRNA depletion of ZAP-70 suppressed the phosphorylation of ZAP-70 substrates, SLP76, LAT and p38MAPK, but did not affect cell viability or induce apoptosis in these cell lines. Similarly, while stable overexpression of ZAP-70 mediates increased phosphorylation of target substrates in the TCR pathway, it does not promote increased survival or growth of NKTCL cell lines. The epidermal growth factor receptor (EGFR) inhibitor Gefitinib, which has off-target activity against ZAP-70, also did not show any differential cell kill between ZAP-70 overexpressing (OE) or knockdown (KD) cell lines. Whole transcriptome RNA sequencing highlighted that there was very minimal differential gene expression in three different T/NK cell lines induced by ZAP-70 KD. Importantly, ZAP-70 KD did not significantly enrich for any downstream TCR related genes and pathways. Altogether, this suggests that high expression and constitutive signalling of ZAP-70 in T/NK lymphoma is not critical for cell survival or downstream TCR-mediated signalling and gene expression. ZAP-70 therefore may not be a suitable therapeutic target in T/NK cell malignancies.

The expression and clinical significance of programmed cell death receptor 1 and its ligand in tumor tissues of patients with extranodal nasal NK/T cell lymphoma.

Appropriate biomarkers may help distinguish the biological behavior of different types of lymphoma and their response to traditional chemotherapy. Extranodal natural killer/T-cell lymphoma (ENKTL) and diffuse large B-cell lymphoma (DLBCL) belong to different subtypes of non-Hodgkin's lymphoma, the biological behavior and prognosis of them are very different, programmed cell death receptor 1 (PD-1) and its ligand (PD-L1) have been investigated in these two types of diseases. However, few studies addressed the difference of PD-1/PD-L1 levels between ENKTL and DLBCL, in order to find out the difference and related clinical application value, the clinical data and tumor tissue paraffin sections of 24 newly diagnosed ENKTL patients and 42 newly diagnosed diffuse large B-cell lymphoma (DLBCL) were collected. PD-1/PD-L1 levels in tumor tissues were detected by immunohistochemical staining. The relationship between the PD-1/PD-L1 levels and clinical data of patients with ENKTL patients was analyzed. Both patient groups showed PD-1 level in tumor tissue of ENKTL patients was significantly lower than that of DLBCL patients (P < 0.05), while the PD-L1 level in tumor tissues of ENKTL patients was not different from DLBCL (P < 0.05). In addition, the ENKTL patients with B symptoms, elevated lactate dehydrogenase (LDH) levels and decreased hemoglobin (HGB) concentrations had lower level of PD-1 in tumor tissue. PD-L1 level in tumor tissues, the LDH level, Epstein-Barr genome (EBV-DNA) copy and Ki-67 index may affect the outcomes of ENKTL patients (P < 0.05), but they were not independent factors. PD-L1 levels in tumor tissues has clinical significance in ENKTL patients, which suggested that the PD-1/PD-L1 signal pathway may be involved in the immune escape of ENKTL and play different roles in different lymphoma subtypes.

The Acetyltransferase KAT5 Inhibitor NU 9056 Promotes Apoptosis and Inhibits JAK2/STAT3 Pathway in Extranodal NK/T Cell Lymphoma.

BACKGROUND: Extranodal natural killer/T cell lymphoma (ENKTL) is an aggressive malignant non- Hodgkin's lymphoma (NHL) with a poor prognosis. Therefore, novel therapeutic biomarkers and agents must be identified for the same. KAT5 inhibitor, NU 9056, is a small molecule that can inhibit cellular proliferation; however, its role in ENKTL has not been studied. OBJECTIVE: The present study investigated the effect of NU 9056 in ENKTL cells and explored the possible molecular mechanism for its antitumour effect. METHODS: The role of NU 9056 in ENKTL cells was investigated through the Cell Counting Kit-8 assay, flow cytometry, Western blot, and real-time quantitative polymerase chain reaction assay. RESULTS: NU 9056 inhibited ENKTL cell proliferation and induced G2/M phase arrest. NU 9056 also induced apoptosis by upregulating DR4, DR5, and caspase 8 expressions. Additionally, NU 9056 increased the expression of Bax, Bid, and cytochrome C and decreased the expression of Bcl-2, Mcl-1, and XIAP. Furthermore, NU 9056 activated endoplasmic reticulum (ER) stress and inhibited the JAK2/STAT3 signalling pathway. The p38 mitogen-activated protein kinase (MAPK) signalling pathway was also activated by NU 9056, and the ERK signalling pathway was suppressed in natural killer/T cell lymphoma cells. CONCLUSION: NU 9056 inhibited cell proliferation, arrested cell cycle in the G2/M phase, and induced apoptosis through the stimulation of ER stress, thus inhibiting the JAK2/STAT3 signalling pathway and regulating MAPK pathways in ENKTL cells.

Primary breast CD20-positive extranodal NK/T cell lymphoma with stomach involvement: a case report and literature review.

BACKGROUND: We present a unique case of primary breast CD20-positive extranodal NK/T cell lymphoma with stomach involvement in a young Chinese female patient. CASE PRESENTATION: The patient presented with a mass in her right breast that rapidly increased in size over approximately 2 months. Upper gastrointestinal endoscopy showed a giant serpentine ulcer in the stomach. Biopsy was performed, and microscopic inspection revealed that the fibrous tissue was diffusely involved by medium to large abnormal lymphocytes. The cytoplasm was low to moderate. The tumor cells had irregular nuclei and inconspicuous nucleoli. The lymphoid cells were strongly immunoreactive to CD20, CD3, CD4, CD56, TIA-1, EBER, and Ki-67 (90%). Epstein-Barr virus genomes were also found in tumor cells by in situ hybridization. A whole-body positron emission tomography (PET)-CT scan revealed intense FDG uptake in the right breast and greater curvature of the stomach. Monoclonal rearrangements of the T cell receptor (TCR-γ) and immunoglobulin heavy chain (IgH) were identified by genetic analysis. Whole-genome next-generation sequencing was performed, and up to 12 gene mutations, including a frameshift mutation in exon 4 of the BCOR (G97Rfs*87; 44.3%) gene and a base substitution mutation (Q61H 17.6%) in exon 3 of the KRAS gene, were detected. Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed using the database for annotation, visualization, and integrated discovery, which showed that rare primary breast CD20-positive extranodal NK/T cell lymphoma had a unique genetic background compared with diffuse large B cell lymphoma and extranodal NK/T cell lymphoma without CD20 expression. The patient received four cycles of the modified SMILE regimen. The second whole-body PET-CT scan revealed that the right breast mass was significantly smaller than before; additionally, FDG uptake in the stomach wall disappeared. CONCLUSIONS: Systemic examination, extensive immunohistochemistry, and molecular profiling are essential for an accurate diagnosis. More similar cases are required to clarify the biological pathways and even the potential molecular mechanisms of rare lymphomas, which may help direct further treatment.

SLC1A1 mediated glutamine addiction and contributed to natural killer T-cell lymphoma progression with immunotherapeutic potential.

BACKGROUND: Metabolic reprogramming plays an essential role on lymphoma progression. Dysregulation of glutamine metabolism is implicated in natural-killer T-cell lymphoma (NKTCL) and tumor cell response to asparaginase-based anti-metabolic treatment. METHODS: To understand the metabolomic alterations and determine the potential therapeutic target of asparaginase, we assessed metabolomic profile using liquid chromatography-mass spectrometry in serum samples of 36 NKTCL patients, and integrated targeted metabolic analysis and RNA sequencing in tumor samples of 102 NKTCL patients. The biological function of solute carrier family 1 member 1 (SLC1A1) on metabolic flux, lymphoma cell growth, and drug sensitivity was further examined in vitro in NK-lymphoma cell line NK-92 and SNK-6, and in vivo in zebrafish xenograft models. FINDINGS: In NKTCL patients, serum metabolomic profile was characterized by aberrant glutamine metabolism and SLC1A1 was identified as a central regulator of altered glutaminolysis. Both in vitro and in vivo, ectopic expression of SLC1A1 increased cellular glutamine uptake, enhanced glutathione metabolic flux, and induced glutamine addiction, leading to acceleration of cell proliferation and tumor growth. Of note, SLC1A1 overexpression was significantly associated with PD-L1 downregulation and reduced cytotoxic CD3+/CD8+ T cell activity when co-cultured with peripheral blood mononuclear cells. Asparaginase treatment counteracted SLC1A1-mediated glutamine addiction, restored SLC1A1-induced impaired T-cell immunity. Clinically, high EAAT3 (SLC1A1-encoded protein) expression independently predicted superior progression-free and overall survival in 90 NKTCL patients treated with asparaginase-based regimens. INTERPRETATION: SLC1A1 functioned as an extracellular glutamine transporter, promoted tumor growth through reprogramming glutamine metabolism of NKTCL, while rendered tumor cells sensitive to asparaginase treatment. Moreover, SLC1A1-mediated modulation of PD-L1 expression might provide clinical rationale of co-targeting metabolic vulnerability and immunosuppressive microenvironment in NKTCL. FUNDING: This study was supported, in part, by research funding from the National Natural Science Foundation of China (82130004, 81830007 and 81900192), Chang Jiang Scholars Program, Shanghai Municipal Education Commission Gaofeng Clinical Medicine Grant Support (20152206 and 20152208), Clinical Research Plan of SHDC (2020CR1032B), Multicenter Clinical Research Project by Shanghai Jiao Tong University School of Medicine (DLY201601), Shanghai Chenguang Program (19CG15), Shanghai Sailing Program (19YF1430800), Medical-Engineering Cross Foundation of Shanghai Jiao Tong University (ZH2018QNA46), and Shanghai Yi Yuan Xin Xing Program.

TP73-AS1 promotes malignant progression of NK/T cell lymphoma by regulating DKK1 methylation.

PURPOSE: Long non-coding RNA (lncRNA) TP73-AS1 is abnormally expressed in multiple types of tumors, which is able to mediate tumor cell signals. This study aims to explore the role of TP73-AS1 in affecting biological functions of NK/T-cell lymphoma (NKTCL) and DKK1 methylation. METHODS: TP73-AS1 levels in peripheral blood of NKTCL patients and healthy volunteers was detected by quantitative real-time polymerase chain reaction (qRT-PCR). After knockdown of TP73-AS1, proliferative and migratory abilities in SNK-6 and HANK-1 cells were assessed by cell counting kit-8 (CCK-8) and Transwell assay, respectively. Regulatory effect of TP73-AS1 on DKK1 methylation in NKTCL cells was evaluated through methylation-specific PCR (MSP), dual-luciferase reporter assay and RNA Binding Protein Immunoprecipitation (RIP). Rescue experiments were conducted to further validate the interaction between TP73-AS1 and DKK1. RESULTS: TP73-AS1 level was higher in peripheral blood of NKTCL patients than that of healthy volunteers. Knockdown of TP73-AS1 in vitro weakened proliferative and migratory functions of NKTCL cells. TP73-AS1 induced methylation of DKK1 promoter through DNMT1/DNMT3, thus regulating NKTCL cell functions. CONCLUSIONS: TP73-AS1 level was higher in peripheral blood of NKTCL patients. Through inducing methylation of DKK1 promoter, TP73-AS1 aggravates the malignant progression of NKTCL.

CD94 expression patterns in reactive and neoplastic T-cell and NK-cell proliferations.

Lymphomas and leukemias of T-cell and NK-cell lineages are highly heterogeneous disorders and lack effective therapeutic strategies. Targeted therapies including anti-CD94 agents are currently under clinical investigation, but studies of CD94 expression on mature T/NK-cell neoplasms are limited. In this study, we investigated the landscape of CD94 protein expression in 15 patients with reactive T/NK-cell proliferations and 124 patients with various T/NK cell neoplasms. CD94 expression was detected at a high level in reactive NK-cells, with a lower level of expression in a subset of reactive CD8 + T-cells; reactive CD4 + T-cells were negative for CD94 expression. All NK-cell neoplasms surveyed had high-level CD94 expression, which was significantly higher than that in T cell neoplasms (p = 0.0174). In neoplastic T-cell proliferations, CD94 expression was positive in all 10 hepatosplenic T-cell lymphoma cases tested, with a high mean fluorescence intensity. Fifty-six percent of T-cell large granular lymphocytic leukemia cases were positive for CD94 expression in a subset of neoplastic cells. All T-cell prolymphocytic leukemia and 97 % of peripheral T-cell lymphoma cases showed no CD94 expression. Our findings demonstrate a broad range of CD94 expression among T/NK-cell neoplasms, in some at levels that suggest therapeutic vulnerability to CD94-targeted therapies.

Overexpression of S100A9 in tumor stroma contribute to immune evasion of NK/T cell lymphoma and predict poor response rate.

NK/T cell lymphoma (NKTCL) represents an aggressive lymphoid malignancy characterized by dismal prognosis. Immune-checkpoint blockade has shown promising efficacy in NKTCL. However, the molecular mechanisms underlying immune evasion in NKTCL have never been explored. Here, proteomic analysis was used to identify the differentially expressed proteins between NKTCL patients and healthy individuals. We found that S100A9, an immunosuppressive molecule, was much higher in NKTCL patients both in serum and tumor stroma. Elevated level of S100A9 was associated with advanced stage, poor overall response and early recurrence. Moreover, percentage of myeloid-derived suppressor cells (MDSCs) in peripheral blood was positively correlated with levels of S100A9. Low concentration of S100A9 promoted proliferation of NKTCL cells, while did not affect cell apoptosis and cell cycles. Furthermore, programmed death ligand 1 (PD-L1) expression on NKTCL cells was up-regulated by S100A9 through activation of ERK1/2 signaling. Inhibition of ERK1/2 signaling significantly decreased tumor growth and PD-L1 expression induced by S100A9. In conclusion, our research firstly identified S100A9 as an immune suppressor in the tumorigenesis of NKTCL via accumulation of MDSCs and upregulation of PD-L1 expression. S100A9 may serve as a potential target to increase the efficacy of immunotherapy in NKTCL.

Interleukin-6 reverses Adriamycin resistance in nasal NK/T-cell lymphoma via downregulation of ABCC4 and inactivation of the JAK2/STAT3/NF-κB/P65 pathway.

Chemotherapy is generally effective for extranodal natural killer (NK)/T-cell lymphoma (ENKTCL), nasal type. Nevertheless, multidrug resistance (MDR) remains a key challenge in treating nasal NK/T-cell lymphoma. Interleukin-6 (IL-6) is reportedly an important regulator of MDR in many cancers, implicating a role of IL-6 in the chemotherapy response. However, the effects and mechanism of IL-6 in nasal NK/T-cell lymphoma remain unclear. Herein, we demonstrated that the IL-6 serum level was decreased in nasal NK/T-cell lymphoma patients compared to chronic rhinitis patients. Lower serum levels of IL-6 were closely correlated with Ki67 expression and patient survival. ATP-binding cassette (ABC) drug transporter ABCC4 in patients was abnormally upregulated. IL-6 significantly inhibited resistance to Adriamycin (ADM) in ADM-resistant SNK-6 cells (SNK-6/ADM). Moreover, IL-6 resulted in cell cycle arrest and led to apoptosis in SNK-6/ADM cells. Furthermore, IL-6 decreased ABCC4, p-JAK2, p-STAT3, and phospho-NF-κB p65 expression in SNK-6/ADM cells. IL-6 in combination with ADM inhibited tumor growth and increased the survival of SNK-6/ADM xenograft mice. In conclusion, our findings suggest that IL-6 can inhibit the upregulation of ABCC4 and inactivate the JAK2/STAT3/NF-κB/P65 pathway to sensitize NK/T-cell lymphoma to ADM, indicating that combination therapy with IL-6 and other chemotherapeutic drugs may be effective in reversing acquired resistance in nasal NK/T-cell lymphoma.

Metabolic activity of extranodal NK/T cell lymphoma on 18F-FDG PET/CT according to immune subtyping.

Disseminated extranodal NK/T cell lymphoma (ENKTL) is associated with dismal prognosis. Hence, distinct tumor immune microenvironment (TIME) subtypes were proposed to explain their influence on ENKTL progression and help predict treatment response. In this study, we investigated the capacity of FDG PET/CT to discern ENKTL TIME subtypes. A total of 108 pretreatment FDG PET/CT scans of 103 patients with newly diagnosed or relapsed ENKTL were retrospectively analyzed. TIME subtype was determined using three key immunohistochemical markers. SUVmax, MTV and TLG were measured, and metabolic features associated with TIME subtype were statistically extracted. TIME subtype was immune tolerance (IT) in 13.9%, immune evasion A (IE-A) in 56.5%, immune evasion B (IE-B) in 21.3%, and immune silenced (IS) in 8%. The IS group showed the highest SUVmax (15.9 ± 6.4, P = 0.037), followed by IE-A (14.1 ± 7.8), IE-B (10.9 ± 5.6), and IT groups (9.6 ± 5.1). Among 53 with only nasal FDG lesions, 52 had non-IS subtype. Among 55 with extra-nasal FDG lesions, those with IS subtype more often had adrenal (P = 0.001) or testis involvement (P = 0.043), greater MTV (P = 0.005), greater TLG (P = 0.005), and SUVmax located at extra-nasal sites. The presence of 0-2 and 3-4 of these four findings was associated with low probability (2/46) and high probability (6/9) of IS subtype, respectively. Furthermore, patients showing IS subtype-favoring PET/CT pattern had worse overall survival compared to their counterparts. These results demonstrate that FDG PET/CT can help predict immune subtype in ENKTL patients. The different patterns between glycolytic activity and involved site according to TIME subtype might be related to the interplay between tumor cells and immune cells in the tumor microenvironment.

Primary Adrenal Lymphoma: Two Case Series From China.

Objective: Primary adrenal lymphoma (PAL) is a rare form of adrenal mass. We summarize our experience in its clinical presentation, biochemical indexes, radiological features, pathological information, therapy regimens, and outcomes. Methods: This was an institutional review board-approved retrospective review of medical records and surgical pathology specimens of patients with a diagnosis of PAL at the Chinese People's Liberation Army General Hospital and the First Affiliate Hospital of Xiamen University between July 2007 and July 2017. Results: Twenty-six patients were identified. The mean age at presentation was 60.84 ± 13.14 years with a male-to-female ratio of 2.25:1 (18:8). The most common presenting symptoms were loss of appetite (65%, 17/26), weight loss (62%, 16/26), abdominal pain (58%, 15/26), and fatigue (58%, 15/26). The levels of lactate dehydrogenase (75%, 15/20), ß2-microglobulin (100%, 10/10), C-reactive protein (82%, 14/17), and ferritin (88%, 7/8) and the erythrocyte sedimentation rate (83%, 10/12) were elevated. Bilateral involvement was seen in 21 of 26 patients (81%); 12 of 19 evaluated patients with bilateral lesions (63%) were confirmed to have adrenal insufficiency. On computed tomography (CT), the mean tumor diameter was 7.31 ± 3.35 cm and the median Hounsfield density was 37.0 HU (range: 31.0-45.0 HU); 67% (10/15) and 27% (4/15) of lesions presented with mild and moderate enhancement after injection of contrast medium. 18F-fluorodeoxyglucose positron emission tomography (FDG PET)-CT revealed not only an adrenal tumor but also extra-adrenal lesions. Diffuse large B-cell lymphoma (DLBCL) was the most common phenotype (92%, 24/26). Ninety-two percent (24/26) of patients received chemotherapy while 8% (2/26) received unilateral adrenalectomy plus chemotherapy. The prognosis of PAL was poor, with a general survival time of 7.20 ± 5.18 months. Conclusion: PAL is a rare disease. The clinical characteristics of PAL include loss of appetite and weight loss. Endocrine evaluation should be performed to determine whether patients have adrenal insufficiency, especially patients with bilateral lesions. FDG-PET appears to be more accurate than other imaging modalities in revealing extra-adrenal sites. Better therapy is required to improve the poor prognosis of PAL.

m6A methyltransferase Wilms' tumor 1-associated protein facilitates cell proliferation and cisplatin resistance in NK/T cell lymphoma by regulating dual-specificity phosphatases 6 expression via m6A RNA methylation.

Nasal-type natural killer/T-cell lymphoma (NKTCL) is an aggressive malignancy with poor survival outcomes that is relatively resistant to chemotherapy. N6-Methyladenosine (m6A) modification, the most prevalent modification of eukaryotic messenger RNA, is involved in the progression of various tumors. However, it is unclear whether it has a physiological role in NKTCL development. To address this question, we probed its function and molecular mechanisms in NKTCL. Initially, we demonstrated that Wilms' tumor 1-associated protein (WTAP), a major RNA N6-adenosine methyltransferase, was obviously upregulated in human NKTCL cell lines (YTS and SNK-6 cells), compared with normal NK cells. Functionally, depletion of WTAP noticeably repressed proliferation and facilitated apoptosis in YTS and SNK-6 cells. Moreover, intervention of WTAP evidently prohibited NKTCL cell chemotherapy resistance to cisplatin, as reflected by a lower inhibition of cell viability and decreased expression of drug resistance-associated protein expression MRP-1 and P-gp in YTS and SNK-6 cells. With regard to the mechanism, we revealed that WTAP enhanced dual-specificity phosphatases 6 (DUSP6) expression by increasing m6A levels of DUSP6 mRNA transcript, leading to oncogenic functions in NKTCL. Interestingly, WTAP contributed to the progression and chemotherapy sensitivity of NKTCL by stabilizing DUSP6 mRNA in an m6A-dependent manner. Taken together, these findings uncovered a critical function for WTAP-guided m6A methylation and identified DUSP6 as an important target of m6A modification in the regulation of chemotherapy resistance in NKTCL oncogenesis. This study highlights WTAP as a potential therapeutic target of NKTCL treatment.

Calreticulin increases growth and progression of natural killer/T-cell lymphoma.

In this study, we investigated the role of calreticulin (CALR) in the pathogenesis of natural killer/T-cell lymphoma (NKTCL). CALR expression was significantly higher in the NKTCL tissues than normal control tissues in the GSE80632 dataset. High CALR expression correlated with poorer overall survival of NKTCL patients (P = 0.0248). CALR mRNA and protein levels were significantly higher in NKTCL cell lines (NK92, SNK6, and SNT8) than normal NK cells. CALR-silenced SNK6 cells generated significantly smaller xenograft tumors in immunodeficient NCG mice than control SNK6 cells. CALR-knockdown NKTCL cells showed significantly less in vitro proliferation and Transwell migration than the controls. CALR knockdown inhibited G1-to-S phase cell cycle progression by increasing the levels of p27 cell cycle inhibitor and reducing the levels of cyclin E2 and cyclin-dependent kinase 2 (CDK2). CALR knockdown inhibited epithelial-to-mesenchymal transition (EMT) by decreasing the levels of ß-catenin and TCF/ZEB1 and upregulating E-cadherin. These data demonstrate that CALR regulates the growth and progression of NKTCL cells by modulating G1-to-S cell cycle progression and EMT.