Natural Killer Cell Lymphoma Research Articles

The expression and clinical significance of PLK1/p-PLK1 protein in NK/T cell Lymphoma

AimsTo investigate the expression of polo-like kinase 1 protein (PLK1) and its phosphorylation level (p-PLK1) in extranodal NK/T cell lymphoma (NKTCL) and their correlation with clinical characteristics and prognosis.MethodsWe collected 40 cases of NKTCL (referred to as the experimental group), which received diagnoses at the First Affiliated Hospital of Zhengzhou University between January 2018 and October 2022. Concurrently, we assembled a control group, including 20 cases afflicted with nasopharyngeal mucosal lymphoid hyperplasia diseases during the same timeframe. We utilized immunohistochemical techniques to evaluate the levels of PLK1 and p-PLK1 expression in both the experimental and control groups. Subsequently, we conducted an analysis to identify disparities in their expression and explore their relationships with clinical characteristics and patient prognosis.ResultsAmong the 40 NKTCL patients, there were 27 males and 11 females, with a median age of 51 years (range 12–80 years). Compared to the control group, the tissue samples of NKTCL patients exhibited significantly elevated expression levels and active phosphorylation levels of PLK1 (P < 0.05). Correlation analysis of the immunohistochemical H score and Ki-67 positive rate of PLK1 and p-PLK1, revealed a significant positive correlation for both (P < 0.0001, each). No statistically significant differences were observed in the distribution of PLK1 and p-PLK1 expression in NKTCL patients with respect to gender, age, Ann Arbor stage, PINK-E score, B-symptoms, lactate dehydrogenase, β2-microglobulin, blood EBV-DNA, bone marrow invasion, and lymph node metastasis (p > 0.05). Grouping based on PLK1 and p-PLK1 immunohistochemical H-scores revealed that the high expression of PLK1 and p-PLK1 was associated with poor prognosis.ConclusionsThe expression levels and active phosphorylation levels of PLK1 were significantly increased in NK/T cell lymphoma, and patients with overexpression of PLK1 and p-PLK1 had a poorer prognosis.

EZH2 K63-polyubiquitination affecting migration in extranodal natural killer/T-cell lymphoma

BackgroundOverexpressed EZH2 is oncogenically involved in the pathogenesis of different cancerous contexts including extranodal natural killer/T cell lymphoma (ENKTL). However, the underlying mechanisms of EZH2 upregulation have not been fully clarified and it is still difficult to target EZH2 in ENKTL.ResultsCurrent study identifies an E3 ligase TRIP12 that triggers K63-linked polyubiquitination of EZH2 in ENKTL and unexpectedly, stabilizes EZH2. As determined by gene expression profiling (GEP), TRIP12 and EZH2 levels correlate with each other in ENKTL patient samples. Aided by quantitative mass spectrometry (MS) and follow-up analysis, we identify K634 as the ubiquitination site of EZH2. Further study confirms that TRIP12-mediated EZH2 K634 ubiquitination enhances the interaction between EZH2 and SUZ12 or CDK1 and increases the level of EZH2 T487 phosphorylation. This study further demonstrates the TRIP12-EZH2 signaling might be regulated by cytoplasmic HSP60. Importantly, the TRIP12-EZH2 axis mediates ENKTL cell migration via accelerating epithelial-mesenchymal transition (EMT). Moreover, our study finds out dexamethasone treatment manipulates TRIP12-EZH2 signaling and may represent a novel therapeutic strategy against ENKTL metastasis.ConclusionsAltogether, TRIP12 induces K63-linked site-specific polyubiquitination of EZH2 for stabilization, which promotes ENKTL cell migration and could be targeted by dexamethasone treatment.

Diverse Microbiome Characteristics in Treatment Naïve Extranodal NK/T-Cell Lymphoma Patients

Background: It is still unclear why extranodal NK/T cell lymphoma (ENKTL) emerged in certain regions, such as Asia and South America, and initiated ENKTL lymphomagenesis in restricted upper airway or aerodigestive tract regions. Recently, reports have highlighted the profound relationship between the gut microbiome and immune system modulation. These associations can potentially influence cancer development and impact the effectiveness of chemotherapy. Therefore, we conducted a stool analysis to determine the newly diagnosed ENKTL (ND-ENKTL) microbiome characteristics. Additionally, a comparison analysis of microbiome composition among healthy individuals, newly diagnosed diffuse large B-cell lymphoma (ND-DLBCL), and ND-ENKTL was performed to identify potential clues that could influence ENKTL development and outcomes. Methods: We have gathered and analyzed microbial data from 40 feces samples obtained from 40 ND-ENKTL patients from 2020 to 2022. Fecal samples from ENKTL patients were collected using a stool sampling kit (CJ Bioscience. Inc, Seoul, Korea) designed explicitly for microbial community analysis using shotgun sequencing. We used pre-analyzed gut microbiome data from the healthy control (HC) or newly diagnosed DLBCL (ND-DLBCL) group to compare the gut microbiome specificities. Results : 32 (80.0%) presented stage I/II, and 7 (17.5%) or 6 (15.0%) had a high PINK (≥2) or high PINK-E score (≥3). In the alpha diversity analysis, ENKTL exhibited significantly lower diversity than HC. Additionally, we observed significant differences in microbial composition between these two groups (PERMANOVA, P-value=0.001). Species such as Escherichia coli, Citrobacter portucalensis and Citrobacter freundii were identified as significant biomarkers associated with dysbiosis in ENKTL. All these species belong to the Enterobacteriaceae family, a facultative aerobic opportunistic bacteria group. In the functional analysis, we observed an increase in genes related to aerobic bacterial metabolism in the ENKTL group. Conversely, a trend of decreased short-chain fatty acid (SCFA) metabolism and folate biosynthesis was identified. The noteworthy observation in ENKTL is the dominant Enterobacteriaceae family presence, which bears similarity to the microbiome associated with gut microbial dysbiosis in DLBCL, previously reported. While no significant differences were observed at the family level, a notable distinction was found between ENKTL and DLBCL at the genera. Specifically, a significantly higher proportion of Escherichia was dominant in ENKTL compared to DLBCL, which was dominant in Enterobacter and Citrobacter. In the chi-square analysis, a high abundance of Enterobacteriaceae presented statistically correlated with advanced stage (p=0.001) and PINK-high (≥2, p=0.003). Moreover, statistically significant associations were observed between the high proportion of the Escherichia genus and advanced stage (p=0.037), PINK-High (≥2, p=0.020), and a high PD-L1 CPS (≥10, p=0.029). During the median 21.1 months (95% CI 16.2-25.9) follow-up duration, the patients with high Enterobacteriaceae abundance exhibited significantly poorer progression-free survival (PFS) compared to those without such abundance (5.6 months vs. NR, p=0.001). Furthermore, patients with high Escherichia abundance demonstrated inferior PFS compared to those without (5.2 months vs. NR, p=0.001). Conclusion: By comparing the intestinal microbes of ND-ENKTL with those of healthy individuals and ND-DLBCL, we could determine the distinct distribution of intestinal microbiome specific to ENKTL. Although the finding of the Enterobacteriaceae family was similar result with previously reported microbiome composition in DLBCL, we determined a high abundance of Escherichia genea in ENKTL but high Enterobacter and Citrobacter in DLBCL through subgroup analysis. Interestingly, Escherichia genea in ENKTL showed a statistical correlation with a high disease burden, PD-L1 expression, and inferior PFS. Based on the results, we expected that it would be possible to predict the disease characteristics and treatment susceptibility in ENKTL by intestinal microbes' composition and diversity in the future.

Treatments and Outcomes of Lymphoma-Associated Hyperinflammation- a Report from the HLH in Adults Working Group for the Histiocyte Society

Purpose: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening inflammatory syndrome. Lymphoma is the most common cause of HLH in adults. Previous studies have indicated poor patient outcomes in lymphoma-associated HLH. The recently introduced optimized HLH inflammatory (OHI) index has improved prognostication and identification of the hyper-inflammatory phenotype of lymphoma; however, more comprehensive information on treatments and outcomes in a large cohort in the contemporary era is necessary. Our study aimed to investigate the clinical features, treatments, and outcomes of lymphoma-associated HLH through a multi-national effort by the HLH in Adults Working Group of the Histiocyte Society (NCT05898477). Methods: We conducted a multicenter, retrospective study evaluating patients diagnosed with lymphoma and hyperinflammation, defined by fulfilling 5/8 HLH diagnostic criteria or the OHI index from 2003-2022. Clinical characteristics were summarized using median and interquartile ranges (IQR). Survival analysis employed Kaplan-Meier curves and the log-rank test, considering HLH diagnosis as the zero time-point. Results: The analysis encompassed 181 patients with lymphoma: 55% had B-cell non-Hodgkin lymphoma (NHL), 35% had natural killer (NK) cell or T-cell lymphoma, and 10% had classical Hodgkin lymphoma. 35% of patients were from China, 21% from the USA, 17% from Israel, 16% from Europe, and 11% from Japan. Of the entire cohort, 157 (87%) patients met the HLH-2004 criteria, and 153 (83%) met the OHI index; The median age at HLH diagnosis was 60 years (IQR, 45-68), and 59% were males. 50% of patients presented with HLH before/at the time of lymphoma diagnosis (median time to lymphoma diagnosis 9 days), while 50% developed HLH after the initial lymphoma diagnosis (median time to HLH diagnosis 181 days). The median values of hemoglobin, absolute neutrophil count (ANC), and platelet count at HLH diagnosis were 8.6 g/dL, 2.1 K/microL, and 54.7 K/microL, respectively. The median values of triglycerides and fibrinogen were 241 mg/dL and 250 mg/dL, respectively. Interestingly, the median values for ANC, triglycerides, and fibrinogen fell outside the HLH-2004 diagnostic threshold. The median and interquartile ranges for ferritin and sCD25 (sIL-2r) were 3017 ng/mL (1337-8958) and 14098 U/mL (7894-39916), respectively. 48% of physicians altered their initial treatment approach due to the HLH diagnosis. Of these instances, 48% were treated with dexamethasone and etoposide outside of a formal treatment protocol, 11% with the HLH-94/2004 protocol, 24% with DEP regimen (liposomal doxorubicin, etoposide, and methylprednisolone), and 4% with DEP and ruxolitinib. Dexamethasone alone was used in 15% of the patients, while another 4% were treated with anakinra +/- steroids. After a median follow-up duration of 2.1y (range, 0.005-6.1), the median overall survival was 0.58 years. 46% of the patients died from multi-organ dysfunction/HLH, 17% from single-organ dysfunction, 16% from infection or septic shock, and 8% from bleeding. The risk of mortality among T-cell or NK cell lymphoma was higher than B-cell lymphoma (HR 1.7, 95% CI 1.16-2.61, Figure 1A). Finally, patients positive for the OHI index alone at diagnosis had a higher mortality risk (Figure 1B) compared to those positive for both the OHI index and HLH-2004 (HR 2.5; CI 1.26 - 4.8) or those positive for HLH-2004 alone (HR 3.72; CI- 1.62-8.58). Conclusions: Preliminary findings from this large multi-institutional study show that the overall survival for patients with lymphoma and HLH remains poor despite the institution of HLH-specific therapies. In nearly half of the cases, the physicians altered lymphoma treatment to control HLH, primarily through dexamethasone and etoposide-based or DEP regimens. Although B-cell lymphoma was our cohort's most common cause of HLH, overall survival was worse among those with T-cell or NK cell lymphoma, suggesting that the underlying lymphoma type might drive the prognosis even in cases with hyperinflammation. The most common causes of death included multi-organ dysfunction and infection. The OHI index-positive patients experienced higher mortality risk than those meeting the HLH-2004 criteria. The study is ongoing, and we plan to present data on the differential effects of treatment regimens at the meeting.

Novel Protac-Based EZH2 Degraders Effectively Target EZH2-Driven Lymphoid Malignancies By Abolishing Both Methylation-Dependent and -Independent Functions

Across various cancers, overexpression or mutation of the epigenetic silencer EZH2 correlates with invasive cancer growth, resistance and poor survival outcomes. While the tumorigenic role of EZH2 is largely attributed to the H3K27me3-mediated repression of tumor suppressor genes, reports have also established a methylation-independent role for EZH2 in the transcriptional activation of oncogenes, particularly in aggressive cancers like Natural Killer T-cell Lymphoma (NKTL). Here, traditional EZH2 inhibitors have been ineffective. We hypothesized that targeted depletion of the EZH2 protein will more effectively disrupt oncogenic signals arising from enzyme-independent activities of EZH2. Thus, we embarked on the development of EZH2 Proteolysis Targeting Chimeras (PROTACs) to trigger targeted ubiquitination and proteasome-mediated degradation of EZH2. No clinical EZH2 PROTAC has been reported yet. Our initial efforts focused on the iterative synthesis of a series of PROTAC candidates considering factors such as permeability, solubility and stable ternary complex formation. We developed a library of 53 compounds with various permutations of EZH2- and E3 ligase-binding ligands as well as linker lengths. Through extensive screening and testing of the EZH2 degradation activity via the Nano-Glo HiBiT lytic detection system and immunoblotting, we identified three lead Protac candidates. Compared to the clinical EZH2 inhibitor Tazemetostat (Taz), EZH2 PROTACs, C2911 and C1311 demonstrated higher cytotoxic activity in diffuse large B-cell lymphoma (DLBCL), a cancer in which Taz was evaluated during clinical trial. Importantly, C2911 and C1311 also successfully triggered EZH2 degradation in a dose-dependent manner thereby qualifying for further evaluation in NKTL. Previous work has highlighted a critical role for JAK3-mediated phosphorylation of EZH2 in promoting NKTL proliferation. Treatment of NKTL with C2911 and C1311 leads to a dose and time-dependent EZH2 degradation, loss of cell viability and increased apoptosis. These effects were absent with Taz and Pomalidomide treatments. The concomitant loss of H3K27me3 levels as well as PRC2 complex subunits, SUZ12 and EED suggest effective degradation of the EZH2 protein. We also observed that induction of NKTL cell death required a longer treatment period as compared to DLBCL. This may be potentially ascribed to the longer half-life of the EZH2 protein in NKTL as determined through cycloheximide treatment. In order to validate the mechanism of action of the EZH2 PROTACs, cells were either pretreated with pomalidomide or siRNA targeting cereblon. Indeed, this mitigated PROTAC-induced degradation of the EZH2 protein and rescued cancer cell death. Similarly, inhibiting the activity of the 26S proteasome through bortezomib also prevented EZH2 degradation. Characterization of EZH2 PROTAC-induced protein expression changes through immunoblotting highlighted that there was specific loss of H3K27me3 and H3K27me2 but not H3K27me1. Of note, we detected a concomitant decrease of Jak3 protein expression and STAT5 phosphorylation levels, more pronounced than PRC2 complex subunits. This suggests that the PROTAC may act by disrupting the non-canonical binding of EZH2 to Jak3 in NKTL. Furthermore, gene expression analyses revealed that EZH2 PROTAC treatment more effectively reactivated canonical EZH2 target genes such as MYT1 and ANPEP as compared to Taz. As the JAK-STAT-MYC axis is dysregulated in NK leukemia, we also evaluated the gene expression of c-Myc and c-Myc target gene, E2F1. EZH2 PROTAC treatment reduces the transcription levels of these oncogenes corroborating with loss of c-Myc protein expression. Studying the pharmacokinetics of the lead PROTACs in a mouse model revealed good plasma stability, half-life (&amp;gt; 4 h), and low clearance. Collectively, our data supports a critical role for EZH2 PROTACs in abolishing both the enzyme-dependent and independent activities of EZH2 through protein degradation, resulting in enhanced anti-cancer activity against DLBCL and NKTL. This may potentially benefit other EZH2-driven cancers. Further evaluation of the EZH2 PROTACs for clinical application is in progress. EZH2 PROTACs will next be tested in vivo. An in-depth investigation of the mechanisms stabilizing EZH2 protein levels and underlying PROTAC induced-cell death will be made to identify potential mechanisms of resistance.

Analysis of Efficacy and Prognosis of Extranodal Natural Killer/T Cell Lymphoma

Objective: Extranodal natural killer/T cell lymphoma (ENKTCL) is one type of non-Hodgkin's lymphomas. It's highly malignant and aggressive. The survival rate of patients with ENKTCL is low and the prognosis is extremely pessimistic. With regard to the common therapeutic regimen, there is no absolute answer. Thus, this research aims to retrospectively analyze clinical features, efficacy and prognosis based on the data of ENKTCL patients from two different clinical centers, so as to provide real-world evidence for the clinical treatment of the disease. Methods : Clinical data of 18 ENKTCL patients treated in Hematological Department of Zhongda hospital Southeast University from January 2013 to December 2022 and 104 ENKTCL patients from Jiangsu Cancer Hospital from January 2017 to December 2022 were retrospectively collected. Clinical data included age at initial diagnosis, gender, stage, clinical classification, B symptom, number of extranodal involvement sites, the situation of regional lymph nodes involvement, the situation of bone marrow involvement, KPS, ECOG, PINK, NRI, immunohistochemistry, the clinical symptom, therapy regimen, laboratory and imaging test. Then analysis of efficacy and prognosis was done. Follow-up was conducted by telephone contact with the patient and/or family until April 2023. Results: 1. Among the different treatment options, the efficacy of a combination of chemotherapy with radiotherapy group was statistically significant compared with either single chemotherapy group (P=0.000) or single radiotherapy group (P= 0.009) for patients with limited-stage disease (stage I/II), but there was no significant difference in the efficacy between chemotherapy group and radiotherapy group (P&amp;gt;0.05).Similarly, the efficacy of chemotherapy with radiotherapy group was statistically higher than that of single chemotherapy group (P=0.012), and that of a combination of chemotherapy with targeted therapy group was statistically higher compared with single chemotherapy group (P= 0.041) for patients with advanced-stage disease (stage III/IV), but there was no statistical difference between the single chemotherapy group and its combination with radiotherapy and targeted therapy group, the combination of chemotherapy with radiotherapy group and their combinations with targeted therapy group, the combination of chemotherapy with targeted therapy group and their combinations with radiotherapy group (All P &amp;gt;0.05). For all patients receiving chemoradiotherapy, there was no statistical significance in the efficacy of the sequential chemoradiotherapy group compared with the sandwich chemoradiotherapy group (P=0.547). 2. Univariate analysis showed that age at initial diagnosis, stage, presence of B symptoms, lactate dehydrogenase (LDH), ECOG score, extranodal involved site and number, and therapy regimen were factors related to the prognosis of patients (All P&amp;lt;0.05). Multivariate analysis further showed that patients' age at initial diagnosis, LDH, ECOG score and therapy regimen were the main factors affecting the prognosis of ENKTCL patients (P&amp;lt;0.05). Conclusion: 1. In the treatment scheme of ENKTCL, the combination of radiotherapy and chemotherapy has a better effect for patients at stage I and II, whereas chemotherapy should be combined with radiotherapy or targeted therapy to improve the efficacy of patients at advanced stage III and IV. For all patients with chemoradiotherapy, the efficacy of sequential chemoradiotherapy is similar to sandwich chemoradiotherapy. 2. The age at initial diagnosis, LDH, ECOG score and therapy regimen are the main factors affecting the prognosis of ENKTCL patients.

Anti-PD-1 Antibody Sintilimab in Combination with Anlotinib and Pegaspargase As a Highly Effective Salvage Regimen for the Treatment of Relapsed or Refractory Natural Killer /T-Cell Lymphoma

Natural killer/T-cell lymphoma (NKTCL) is a rare and aggressive neoplasm associated with Epstein-Barr virus (EBV) infection. When patients experience relapse or disease progression after initial treatment, options for salvage therapy are limited. Targeting the PD-1/PD-L1 pathway has emerged as a highly potent treatment approach for NKTCL. However, the overall response rate (ORR) and complete response (CR) rate were limited with a short median progression-free survival (PFS) of 2.7 months. Therefore, improving the efficacy of immune checkpoint inhibitors is a critical clinical concern. In this study, we reported the efficacy and safety of a novel triplet regimen called LEAP, which combines PD-1 blockade (sintilimab 200mg on day 1), pegaspargase (2500U/m2 capped at 3750U on day 1), and angiogenesis inhibitor anlotinib (8mg on day 1-14), as a salvage treatment for relapsed/refractory (R/R) NKTCL. From March 2019 to May 2020, a total of 33 patients with R/R NKTCL were included in this study. The median age of patients was 48 years (range: 18-76), with 75.8% being male. At study entry, 88% of patients had stage IV disease, while 4 patients had stage II disease, with a history of primary refractory response to radiotherapy and L-asparaginase-based combination therapy. Moreover, 69.7% of patients had refractory disease, including those who were progressive or non-responsive to L-asparaginase-based regimens. Additionally, 10 patients experienced relapse or disease progression within 6 months after prior L-asparaginase-based therapy, and 10 patients had a history of PD-1/PD-L1 blockade monotherapy. A total of 221 cycles of the LEAP regimen were administered to these patients. The median number of cycles was 8 (range: 2-8). All patients reported at least one or more adverse events (AE). Hematological toxicities were mild, with no grade 3/4 neutropenia, anemia, or thrombocytopenia observed. Non-hematological AEs were common, and the most frequent (&amp;gt;20%) included hypofibrinogenemia (66.7%), hyperbilirubinemia (57.5%), APTT prolongation (54.5%), ALT elevation (54.5%), AST elevation (51.5%), hypoalbuminemia (48.5%), appetite loss (60.6%), nausea (57.5%), fatigue (57.5%), weight loss (42.4%), and edema (27.3%). These adverse events were typically attributed to pegaspargase. Other adverse events such as hyponatremia (24.2%), hypertriglyceridemia (21.2%), hyperglycemia (21.2%), hypothyroidism (30.3%), hypertension (24.2%), fever (39.4%), and dysphonia (21.2%) were most likely due to sintilimab or anlotinib. Most adverse events were manageable with supportive care. Among the 33 patients, the ORR was 81.8%, and CR was 45.5. The progression-free survival (PFS) rate at 1-year was 54.6% (95% CI, 36.3% to 70.0%), and at 2 years, it was 44.2% (95% CI, 26.6% to 60.4%). The median PFS time was 21.7 months. The overall survival (OS) rate at 1-year was 66.7% (95% CI, 47.9% to 80.0%), and at 2 years, it was 51.5% (95% CI, 33.5% to 66.9%). The median OS time was not reached during the study period. In conclusion, our study demonstrates that the LEAP regimen has a favorable safety profile, and is highly effective as a salvage treatment for patients with relapsed and refractory NKTCL.

Targeting Aberrantly Activated Glycolysis in Natural Killer T Cell Lymphomas

Background and aims: With advances in the diagnosis and treatment of malignant lymphoma, improvements in long-term survival have been achieved for most common lymphoma types. However, natural killer/T-cell lymphoma (NKTL), which is rare in Europe and North America (0.3%) but relatively common in Asia and Latin America, has been left behind as a subtype of lymphoma with a high relapse rate and a poor prognosis. In particular, the outcome of patients with advanced or recurrent NKTL is dismal, with an overall survival of only a few months. This is mainly attributed to the intrinsic resistance of NKTL cells to anthracycline due to the high expression of P-glycoprotein. Molecular-targeted drugs used for other hematological malignancies, such as anti-CD30 and anti-CD52 monoclonal antibodies, have only limited effects on NKTL, and conversely, these drugs can cause severe immunodeficiency and severe autoimmune diseases, often with a fatal course. The development of NKTL-specific and effective molecular-target therapies is expected to improve the prognosis of NKTL. Methods: We performed a gene expression array targeting genes involved in glucose cell metabolism to compare the gene signatures of NK cell lines (NKL, NK92, YT, and KYHG1) and freshly isolated NK cells from three healthy donors (FNK). Results: The expression of various glucose metabolism genes was higher in NK cell lines than in FNK cells, and in particular, the PKM gene was highly enriched in NK cell lines. The PKM gene encodes the pyruvate kinase muscle isozyme 2 (PKM2), which is responsible for ATP production within the glycolytic sequence and plays an essential role in aerobic glycolysis (the Warburg effect), a dominant metabolic pathway used by cancer cells. Immunohistochemistry studies revealed high PKM2 protein expression in 17 out of 20 tumor tissues derived from patients with extranodal NK/T cell lymphoma. Treatment of NKL, NK92, YT, and KYHG1 cells with Benserazide or Shikonin, two known PKM2 inhibitors, robustly induced cell apoptosis in a time- and dose-dependent manner and inhibited colony formation in in vitro clonogenic assays. Western blotting studies showed a dose-dependent inhibition of STAT1 and STAT3 in NK cell lines exposed to PKM2 inhibitors. A colorimetric assay showed that PKM2 glycolytic activity correlated with the PKM2 gene expression in NK cells, with significantly higher glycolytic activity in malignant NK cell lines than in normal FNK cells. Further mechanistic studies showed that in normal FNK cells, PKM2 is mainly in the tetrameric form, where glucose is converted to pyruvate to generate ATP. In contrast, in malignant NK cell lines, PKM2 is mainly expressed in its dimeric form, with a low affinity for pyruvate and a higher affinity for glycolytic intermediates that promote the synthetic processes and thereby lead to persistent cell proliferation. Conclusions: These results substantiate the importance of aerobic glycolysis in malignat NK cells and indicate that targeting this pathway by blocking PKM2 activity has therapeutic potential for NKTL.

Addition of Chidamide to a Sandwich Protocol, Pegaspargase, Gemcitabine and Oxaliplatin Chemotherapy Combined with Radiotherapy, As First-Line Treatment in Early Stage NK/T-Cell Lymphoma: Real World Outcomes in a Retrospective Study

Background: Extranodal NK/T-cell lymphoma, nasal type (ENKTL-NT) is a highly invasive tumor with a short doubling time and poor prognosis. Clinically, it usually involves the upper aerodigestive tract and mainly presents with early-stage. Initially, intensity modulated radiation therapy (IMRT) has been considered the preferred first-line therapy for early-stage ENKTL due to its high response rate. In recent years, more and more studies show that adding chemotherapy, especially asparaginase (ASP)-based regimens, provide survival benefit in early-stage patients. However, the optimal chemotherapy regimen has not been fully defined. Epigenetic regulatory mechanisms related to histone deacetylase (HDAC) have been found to play a role in the evolution and progression of ENKTL. Chidamide, a new HDAC inhibitor, has been confirmed to be effective and tolerable to treat lymphoma. Recently, Xue et al showed that chidamide in combination with cisplatin, etoposide or gemcitabine showed synergistic effect and reversed the chemotherapy resistance in the relapsed/refractory B-cell lymphoma. Here, we investigate the safety and efficacy of adding chidamide to a sandwich protocol, P-GEMOX (pegaspargase/gemcitabine/oxaliplatin) regimen (CP-GEMOX) combined with IMRT as first-line treatment in early-stage ENKTL-NT patients. Methods: We retrospectively reviewed data of early-stage ENKTL-NT patients in the first affiliated hospital of Soochow University from March 2020 to April 2023. A total of 21 patients diagnosed with early-stage (stage Ⅰ or Ⅱ) ENKTL-NT were enrolled and treated with 2 cycles of CP-GEMOX followed by IMRT and 1 or 2 cycles of CP-GEMOX sequentially as consolidation. After consolidation, chidamide was administrated as a maintenance therapy for 1 year. In the induction therapy phase, chidamide was administered orally at a dose of 20 mg twice a week for two weeks, with a one-week discontinuation period; During the maintenance treatment phase, the original dose of chidamide was still maintained and used without pause. The baseline clinical features for the 21 patients were summarized in Table 1. Results: Among 21 patients, 14 were males and 7 were females, and their median age was 57 (range, 41-75) years. Elevated lactate dehydrogenase, elevated β2-microglobulin, stage II disease and primary tumor invasion were present in 4.8% (1/21), 19.0% (4/21), 61.9% (13/21) and 61.9% (13/21) patients. According to the early stage-adjusted nomogram-revised risk index, the majority of patients (76.2%, 16/21) were classified into intermediate- (1-2) and high (≥3) risk groups. The baseline clinical features for the 21 patients were summarized in Table 1. During treatment, all patients were well-tolerated and the most common adverse events were hematological toxicities with neutropenia, thrombocytopenia and anemia. Among non-hematological adverse events, nausea, vomiting and reduced appetite were most common. Other side effects included coagulation dysfunction, skin reaction, diarrhea, serum amylase and alanine aminotransferas /aspartate aminotransferase /bilirubin elevation. No anaphylactic reaction to pegaspargase, hemophagocytic syndrome, pancreatitis or sepsis were observed during chemotherapy. Some patients had mucositis and xerostomia when receiving local radiotherapy. All side effects could be reversed with proper treatment, and there were no therapy related mortality. All patients completed 2 cycles of CP-GEMOX, 19 patients achieved complete remission (CR) and 2 patients achieved partial remission (PR) with a CR rate of 90.5% (19/21) and overall response rate (ORR) of 100.0% (21/21) after 2 cycles of CP-GEMOX. Among the 21 patients, 18 patients completed treatment and CR rate was improved to 100.0% (18/18), giving an ORR of 100% (18/18). After a median follow-up of 25.6(range, 2.6-39) months , all patients were alive except one patient relapsed 20 months after treatment and died of COVID-19 related pneumonia 1 month later. The 2-year progression-free survival and overall survival were all 91.5% (Figure 1). Conclusions: This study demonstrates that sandwich CP-GEMOX chemoradiotherapy is a safe and promising approach to managing patients with early-stage ENKTL-NT. Keywords: Extranodal NK/T-cell lymphoma; chidamide; pegaspargase; gemcitabine; radiation therapy.

Upfront Autologous Stem Cell Transplant at First Complete Remission in Patients with Advanced-Stage Extra-Nodal NK/T Cell Lymphoma: A Multicenter Retrospective Study

Objective: Treatment outcome of patients with advanced-stage extranodal NK/T cell lymphoma (NKTCL) remains unsatisfactory. The role of consolidation therapy with autologous stem cell transplantation (ASCT) at first complete remission (CR1) in these patients is controversial. This study aimed to investigate the effect of upfront ASCT on the prognosis of patients with advanced-stage NKTCL achieving CR1 to frontline therapy. Methods:This is a multicenter retrospective study. Data were collected from patients diagnosed with NKTCL between 2006 and 2021 in the China Lymphoma Collaborative Group (CLCG) database. The inclusion criteria were as follows: pathologically confirmed diagnosis of NKTCL, age≤65 years at diagnosis, stage III or IV disease, and achievement of CR1 at the completion of frontline induction therapy. Clinical characteristics, treatment including the use of ASCT, and follow-up information were reviewed. The primary endpoints were progression-free survival (PFS) and overall survival (OS). Results: A total of 99 patients from 14 medical centers in China were included in this study. The median age was 38 years (range: 6-64 years). Sixteen patients (16%) had an ECOG-PS≥2 at diagnosis, and 26 patients (26%) presented with primary extra-nasal disease. The baseline Prognostic Index for NK-cell lymphoma (PINK) was≥2 in 63 patients (64%). Regarding first-line treatment, 85 patients (86%) received non-anthracycline-based chemotherapy, and the remaining patients received anthracyline-based chemotherapy. At the time of CR1, 32 patients (32%) received upfront ASCT consolidation and 67 patients did not. Baseline clinical characteristics were comparable between the ASCT group and non-ASCT group. With a median follow-up time of 33 months, median PFS was not reached in the ASCT group versus 46 months in the non-ASCT group (3-year PFS: 76.6% versus 54.9%, P=0.012). OS was not significantly different between the ASCT group and the non-ASCT group (3-year OS: 85.2% versus 69.4%, P=0.098). In multivariate analyses, upfront ASCT was independently associated with better PFS (HR=0.40, 95%CI 0.17-0.94, P=0.037) after adjusting for baseline PINK score and types of first-line chemotherapy (anthracyline-based versus non-anthracyline-based). Only non-anthracyline-based first-line chemotherapy (HR=0.32, 95%CI 0.11-0.88, P=0.028) was associated with better OS in multivariate analysis. Conclusions: In this multicenter real-world study, upfront ASCT improves PFS of patients with advanced stage NKTCL achieving CR1 to front-line chemotherapy, but the effect of upfront ASCT on OS in the era of non-anthracycline-based chemotherapy remains uncertain. The specific subgroups of patients most likely to gain a survival benefit from upfront ASCT need to be further investigated.

Survival Outcome of Autologous or Allogeneic Stem Cell Transplantation in 49 Patients with Extranodal NK/T-Cell Lymphoma

Objective:Extranodal NK/T-cell lymphoma (ENKTL) is an uncommon tumor featured by high invasiveness, the ease of developing drug resistance, low curative rate, and short survival time. Hematopoietic stem cell transplantation (HSCT) is now recommended for patients with advanced or relapsed/refractory (r/r) ENKTL, but which HSCT is the most suitable remains to be conclusively determined. Here, we performed a retrospective single-center study to compare the prognostic role of autologous and allogeneic HSCT after induction chemotherapy in ENKTL patients. Methods: Clinical data on 49 ENKTL patients who underwent HSCT in the First Affiliated Hospital of Soochow University from 8/2007 to 9/2022 was reviewed. We divided all patients into two subgroups: cohort A) auto-HSCT and cohort B) allo-HSCT. Observation period started from the day of stem cell infusion in both cohorts. The primary endpoint was overall survival (OS). The secondary endpoints were progression-free survival (PFS) and non-relapse mortality (NRM). NRM was defined as death from any cause related to transplantation without evidence of lymphoma progression. Results: Of 49 patients included, 28 (57%) patients received auto-HSCT (cohort A), while 21 (43%) patients received allo-HSCT (cohort B). The two cohorts of patients were different in some terms of baseline characteristics (Figure A). A total of 8 patients with stageⅡdisease received transplantation due to r/r status or primary lesions beyond the nasal area. The patients in cohort B were younger at the time of transplant (median age: 33 years vs. 37 years, p=0.0031) and had a higher proportion of bone marrow involvement (7% vs. 43%, p=0.005). After a median follow-up of 35 months, patients who received allo-HSCT had a worse prognosis compared to patients who underwent auto-HSCT (2-year OS:61% vs 96%, p=0.0015). There was no transplantation-related mortality in the auto-HSCT group, while the NRM was 33.3% in the allo-HSCT group. Limiting the analysis to only patients with complete remission (CR) (allo-HSCT=13, auto-HSCT=25) before HSCT, no differences in 2-year OS and PFS were reported despite 61% vs 4% bone marrow involvement, respectively (76% vs 96% OS and 76.9% vs 84.1% PFS at +24 months, p&amp;gt;0.05). In the allo-HSCT group, patients who achieved first CR(CR1) before HSCT showed higher OS and lower NRM rates compared to those without CR1, with 2-year OS of 88.9% and NRM of 11.1% (p=0.028, p=0.06, respectively). In terms of donor sources, no significant difference in PFS and OS was observed in patients who received haploid conjugate (n = 14) vs. HLA-matched related and unrelated donors(p&amp;gt;0.05). Conclusions: Overall, consolidation therapy with HSCT improved OS in patients with advanced or relapsed/refractory ENKTL who achieved CR after induction therapy. Given the high NRM in the allo-HSCT, auto-HSCT is probably the preferred clinical option in most patients and allo-HSCT may be more appropriate for younger patients with bone marrow involvement and CR1 acquired before transplantation. Larger studies are needed to validate our results.

The Lymphocyte Subsets Activation Profiles in Peripheral Blood May Contribute to the Diagnosis of Natural Killer/T-Cell Lymphoma-Associated Hemophagocytic Lymphohistiocytosis

The lymphocyte subsets activation profiles in peripheral blood may contribute to the diagnosis of natural killer/T-cell lymphoma-associated hemophagocytic lymphohistiocytosis Background: Hemophagocytic lymphohistiocytosis (HLH) is a rampant and potentially fatal systemic hyperinflammation characterized by dysfunction of macrophages and cytotoxic T cells. Natural killer/T-cell lymphoma (NKTCL) has been reported to be one of the most common etiologies of HLH among hematologic neoplasms. Although, the diagnosis of natural killer/T-cell lymphoma-associated hemophagocytic lymphohistiocytosis (NK/T-LAHS) refers to the HLH-2004 diagnostic guidelines for primary HLH, most of them are based on non-specific clinical features. Identification of the immunophenotype characteristics is important for the diagnosis of NK/T-LAHS. In this study, we investigated the lymphocyte subsets activation profiles of peripheral blood mononuclear cells (PBMC) from NKTCL patients, and preliminarily screened molecular marker with potential diagnostic value for NK/T-LAHS. Methods: This study retrospectively collected peripheral blood samples from 58 patients diagnosed with NKTCL in Sun Yat-sen University Cancer Center from September 2020 to December 2022. Among them, 29 patients had HLH at the time of enrollment, and peripheral blood samples from 10 healthy people were collected as the control group. NKTCL was diagnosed according to the 2016 WHO classification of malignant lymphoma. All HLH patients met HLH-2004 diagnostic criteria and primary HLH was excluded. Flow cytometric staining of PBMC was performed on BD Fortessa according to the manufacturer's instructions. All samples were analyzed by Flowjo（V10）software. The ROC curve was drawn according to the percentage of lymphocyte subsets to determine the optimal cutoff threshold. RStudio 4.2.1 was used to calculate the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and likelihood ratio (LR) of the related indicators. Graphpad prism 8.0.1 and SPSS 26.0 (IBM) was used for drawing and statistical analysis. All statistical tests were two-sided, and p＜0.05 were considered statistically significant. Results:The median age of 58 patients was 41 (range, 18-83) years old, and 10 patients (17.2%) had poor performance status (ECOG PS≥2). Forty-two patients (72.4%) were stage III-IV, and 26 patients (44.8%) had PINK-E score≥3. Firstly, we compared the immune cell activation profiles between healthy donors, NKTCL patients with or without HLH. We observed that the proportion of functionally activated T cells, NK cells, NKT cells that expressed CD86, CD69, or HLADR was significantly higher than healthy donors ( p＜0.05), except for CD86+NK cell levels was no significance between the three groups (Figure1). In addition, the percentage of CD86+NK/T cells, CD69+NK cells, HLADR+NK cells and HLADR+NKT cells were significantly higher than NKTCL patients without HLH and healthy controls( p＜0.05), and no significant difference of these immune cells subsets between those non-HLH patients and healthy controls was observed (Figure1). Based on the significance of these lymphocyte subsets in NKTCL patients with and without HLH, we evaluated the diagnostic value of CD86+NKT cells, CD69+NK cells, HLADR+NK cells, and HLADR+NKT cells in NK/T-LAHS patients by ROC analysis to determine the optimal diagnostic threshold. We observed that HLADR+NK &amp;gt; 15.9% could better distinguish NK/T-LAHS from non-HLH patients, with a sensitivity of 0.759 (95%CI: 0.603-0.914) and specificity of 0.828(95%CI:0.690-0.965), and the AUC was 0.775 (95%CI:0.647-0.904) (Table 1). HLADR+NK cells may have potential application value for the diagnosis of NK/T-LAHS. Conclusions: These activated T cells, NK cells, and NKT cells that express functional activation markers was increased in NK/T-LAHS patients. Among them, HLADR+NK &amp;gt; 15.9% could better distinguish NK/T-LAHS from NKTCL patients without HLH and healthy donors, which may be a potential biomarker for the diagnosis of NK/T-LAHS patients and contribute to better management.

Pembrolizumab in Relapsed/Refractory Extranodal NK/T Cell Lymphoma and Mature T Cell Lymphoma: A Prospective Phase II Study

Background Pembrolizumab, a monoclonal antibody against programmed death 1 receptor (PD-1) has been shown to be efficacious in relapsed/refractory (R/R) extranodal NK/T cell lymphoma (ENKTL), with an overall response rate (ORR) up to 100% in retrospective case series 1. In R/R mature T cell lymphoma, an overall response rate of 33% has been demonstrated in a phase II study 2. In this prospective phase II study, we aim to evaluate the efficacy of pembrolizumab in patients with R/R mature T- and NK-cell lymphomas. Methods Patients with R/R mature T- and NK-cell lymphomas who failed at least one line of prior therapies were recruited. Pembrolizumab was administered at 200mg intravenously every three weeks for up to two years (35 cycles) unless there was intolerable toxicity or disease progression. PET/CT was performed after at least three cycles of treatment for response evaluation. The primary endpoints were the efficacy and safety of pembrolizumab treatment. Secondary endpoints included progression-free survival (PFS) and overall survival (OS). Response assessment was made according to published criteria 3. Survival was analyzed using Kaplan-Meier method. Statistical calculations were performed with SPSS Statistics version 28. Results ENKTL cohort: Ten women and six men at a median age of 57 (range: 41-83 years) were included. Fourteen patients had been treated with L-asparaginase-containing regimens. Ten patients (62.5%) had relapsed disease while six patients (37.5%) had refractory disease. Other relevant features included staging (I, N=3, 18.8%; II, N=3, 18.8%; IV, N=10, 62.5%). Response assessments were performed in all patients, and their best responses were as below: complete response (CR), N=7, 44%; partial response (PR), N=1, 6%; indeterminate response (IR), N=2, 13%; stable or progressive disease (SD/PD), N=6, 38%. At a median follow-up of 24 months (range 1-51 months), the median PFS was 10 months and the OS was 25 months (Figure 1). Adverse events included immunotherapy-related adverse events (IRAE) in five patients (31%), including two endocrine (one grade 1 and one grade 2 toxicity), two hepatic (both grade 2) and one gastrointestinal (grade 2). Haematological toxicity was mild with one patient having grade 1 anaemia. Mature T-cell lymphomas cohort: Four men and four women at a median age of 60 (range: 22-80 years) were included. Underlying diseases were peripheral T cell lymphoma, not otherwise specified (PTCL-NOS) (N=4, 50%), angioimmunoblastic T-cell lymphoma/PTCL with T-follicular helper phenotype (AITL/PTCL-TFH) (N=2, 25%), hepatosplenic T-cell lymphoma (HSTCL) (N=1, 12.5%) and systemic Epstein-Barr Virus (EBV)-positive T cell lymphoma of childhood (N=1, 12.5%). All patients had advanced-stage diseases (stage 3, N=1, 12.5%; stage 4, N=7, 87.5%). The median line of the prior regimens was 2 (range: 1-3). Response assessment was performed in seven patients, and their best responses were as follows: CR, N=1, 14.2%; IR, N=2, 28.4%; SD/PD, N=4, 57.1%. At a median follow-up period of nine months, the progression-free survival was 4 months and overall survival was 7 months. The only patient with a durable response was the one with systemic EBV-positive T-cell lymphoma of childhood. Toxicity included IRAE in two patients (endocrine, N=1, grade 1; pulmonary, N=1, grade 4) and haematological toxicity in three patients (37.5%) (anaemia, N=1, grade 2; neutropenia, N=1, grade 1; thrombocytopenia, N=1, grade 1). Conclusion Pembrolizumab is a safe and efficacious therapy for R/R ENKTL with potentially durable remission. In R/R mature T-cell lymphomas, the benefit of pembrolizumab treatment was limited. With the durable response observed in EBV-positive T-cell lymphoma of childhood, the use of pembrolizumab in EBV-positive T-cell lymphoma warrants further investigation. Reference: 1. Kwong YL, Chan TSY, Tan D, et al. PD1 blockade with pembrolizumab is highly effective in relapsed or refractory NK/T-cell lymphoma failing l-asparaginase. Blood 2017; 129 (17): 2437-2442 2. Barta SK, Zain J, MacFarlane AW 4 th, et al. Phase II Study of the PD-1 Inhibitor Pembrolizumab for the Treatment of Relapsed or Refractory Mature T-cell Lymphoma. Clin Lymphoma Myeloma Leuk 2019; 19(6): 356-364.e3 3. Cheson BD, Ansell S, Schwartz L, et al. Refinement of the Lugano Classification lymphoma response criteria in the era of immunomodulatory therapy. Blood 2016 Nov 24;128(21):2489-2496

Efficacy and Safety of Avatrombopag in the Treatment of Chemotherapy-Induced Thrombocytopenia Refractory to Rhtpo in Patients with Lymphoma

Background: Chemotherapy-induced thrombocytopenia (CIT) is a common cause of chemotherapy dose reductions and treatment delays. With applications of recombinant human interleukin 11 (rhIL-11) and recombinant human thrombopoietin (rhTPO), outcomes of patients with CIT have improved significantly. however，some patients with severe thrombocytopenia still have poor responses to the above therapies. Avatrombopag is an orally administered thrombopoietin receptor agonist and can stimulate thrombopoiesis in haematopoietic stem cells. Avatrombopag has been approved in the USA, Europe, and China for thrombocytopenia in patients with chronic liver disease who are scheduled to undergo a surgery. It also has been approved for patients with chronic immune thrombocytopenia who are unresponsive to previous treatments in the USA and Europe. The aim of this study is to evaluate the efficacy and safety of avatrombopag in the treatment of refractory CIT. Methods: The clinical data of 23 lymphoma patients with grade 3/4 CIT unresponsive to rhTPO treatment for 7-10 days were collected from April 2022 to May 2023. All patients received avatrombopag orally at a dose of 40 to 60 mg / day combined with subcutaneous injection of rhTPO (300 U/kg/day). Response (R) was defined as a platelet count≥ 50 × /L, or doubling of the baseline platelet count, and absence of bleeding. Platelet transfusion was allowed when the platelet count was less than 10×109/L or with bleeding events. When the platelet count recovered to ≥100 ×10 9/L or increased 50 ×10 9/L from baseline, treatments of avatrombopag and rhTPO were stopped. Results: The median age of the 23 patients with refractory CIT was 57 (range 17‒83) years. The pathological subtypes were diffuse large B cell lymphoma (DLBCL) (n=10), Burkitt lymphoma (n=4), angioimmunoblastic T-cell lymphoma (AITL) (n=3), follicular lymphoma (FL) (n=2), extranodal NK/T cell lymphoma (ENKTL) (n=1), anaplastic large cell lymphoma (ALCL) (n=1), B-lymphoblastic lymphoma (n=1), and mantle cell lymphoma (MCL) (n=1). The treatment consisted of CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) (n=11), Hyper-CVAD (hyperfractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone), high-dose methotrexate with cytarabine (n=6), liposomal mitoxantrone-containing regimen (n=4), bendamustine-containing regimen (n=1), and peg-asparaginase-based regimens (n=1). At baseline, 19 patients experienced grade 3 and 4 patients experienced grade 4 thrombocytopenia after chemotherapy. The median lowest platelet level was 12×10 9/L (range 2‒44×10 9/L). Two patients developed grade 1 bleeding events and one patient occurred grade 2 bleeding events. Nineteen patients received platelet transfusion. After receiving the combination therapy of avatrombopag, 73.9% (17/23)of patients achieved the primary end point of corrected platelet counts (≥50×10 9/L )and were free from platelet transfusion. The median time to response was 11 (range 5‒25) days. No treatment-related adverse events such as elevated aminotransferases, thrombosis, fatigue, fever and dizziness were noticed during administration. Two patients received avatrombopag as secondary prophylactic treatment, and there were no grade 4 thrombocytopenia or bleeding events during subsequent cycles of chemotherapy. Conclusion: Avatrombopag is effective and safe in the management of CIT for lymphoma and it can be used as an alternative / combination treatment for the patients who refractory to rhTPO. The further clinical research is still needed to confirm this conclusion.

Genomic Landscape of Epstein-Barr Virus in Endemic Burkitt Lymphoma

Introduction: Endemic Burkitt lymphoma (eBL) is a fast-growing B-cell malignancy strongly linked to Epstein-Barr virus (EBV) infection, a member of the herpes virus family. The virus is widespread, infecting 95% of the global population, and has also been linked to several other cancers, including nasopharyngeal carcinoma (NPC), diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma (HL), natural killer T-cell lymphoma (NKTCL), gastric carcinoma (GC) and leiomyosarcomas. The distribution of these cancers is inconsistent in different regions, raising the hypothesis of whether different strains are linked to specific cancers. Using high throughput sequencing, we obtained EBV whole genome sequences from the plasma of children with eBL diagnosis from a large cohort study in East Africa. Methods: Twenty-one EBV-positive patients (Sixteen BL, one diffuse large B-cell lymphoma, one lymphoblastoid lymphoma, one clear cell sarcoma, one small blue cell tumour and one alveolar rhabdomyosarcoma) were recruited from St Mary's Hospital Lacor, in Uganda, between 2020 and 2023. Venous blood samples were collected in appropriate DNA tubes, and ~ 5 ml of plasma was used for cfDNA extraction using QIAamp circulating Nucleic acid kit (Qiagen, USA). The cfDNA libraries were prepared using the Thruplex Tag- Seq kit (Takara Bio Inc. Japan), target capture and enrichment was performed using an EBV-custom panel (IDT Inc. USA) before sequencing. The Illumina fastq files were processed using an in-house pipeline, and paired-end reads were aligned to a bespoke hybrid EBV reference genome (NC_007605.1 and EBNA genes from NC_009334) using BWA with an alt-ware method. The average sequencing depth and EBV genome coverage were 5164 and 97.8%, respectively. Following the GATK best practice workflows (version 4.0), 15,318 variants were called across all samples using Varscan and Vardict after base and variant recalibration. Functional annotation was performed using the SNPEff package (v5.1d) according to the reference genome (NC_007605.1, NCBI annotation, Nov 2013). For the phylogenetic analysis, 322 public EBV genomes were downloaded from the GenBank and aligned with 21 EBV genomes from the current study using a multiple sequence alignment program (MAFFT v7.52), and the maximum likelihood of the phylogenetic relationship was inferred using RAxML (v8.0), assuming a general time reversible (GTR) model. The inferred phylogeny was subsequently rooted using the evolutionary placement (EPA) algorithm from RAxML using a Macacine herpesvirus 4 genome sequence (NC_006146) as an outer group. The tree was annotated using FigTree software (v1.4.4). The study was approved by the Oxford Tropical Research Ethics Committee (OxTREC Ref: 15-19), National Institute of Medical Research (NIMR/HQ/R.8a/Vol.IX/3408), Uganda National Council of Science and Technology (Ref: HS529ES), and St Mary's Hospital Lacor Institutional Research Ethics Committee. Participants provided written informed consent and assent for minors. Results: We examined the genomes of 21 EBV samples, including 18 type-1 and 3 type-2 strains. Our analysis revealed an average of 729 mutations per sample, comprised of 707 SNVs and 26 indels (&amp;lt;50bp) per sample (Figure 1). Within 16 eBL tumours, we observed the 30-bp deletion in LMP-1 in 43.8% (7/16) cases. This deletion has been previously reported in NPC in Southeast Asia and is associated with high oncogenic potential and weak immunogenicity. We also identified a novel frameshift variant (15-bp deletion) in LMP-1 in 66.7% (14/21) of tumour samples. The LMP-1 gene encodes a 356-amino acid polypeptide with a cytoplasmic amino terminus, a transmembrane domain, and a cytoplasmic carboxy terminus. The C-terminus interacts with cellular proteins, activating the transcriptional nuclear factor-kB (NF-kB) pathway associated with antiapoptotic cell signalling. Additionally, we observed a high degree of polymorphism in EBNA-1, EBNA3s, LMP-2A, BALF1, and BOLF-1 viral genes. Our phylogenetic analysis revealed distinct clustering of African EBV genomes compared to Asian strains and NPC versus eBL (Figure 2). Conclusions: Overall, our study provides the first-ever comprehensive mutational profile of EBV in eBL clinical samples, offering valuable insights into the somatic events of EBV in tumour biology.

Extracellular Vesicles Secreted from Daratumumab Resistant Cells Promote Resistance and Proliferation of Daratumumab Sensitive Cells, Possibly through the Transfer of miRNA Cargo

Multiple Myeloma (MM) is the second most common hematologic malignancy and remains incurable. Daratumumab (dara) is a potent anti-CD38 monoclonal antibody used for MM treatment, but responses are heterogeneous, and resistance is inevitable. There is hence an urgent need for identification of biomarkers to predict responses and in-depth characterisation of mechanisms of resistance. We have previously demonstrated that the exosome biogenesis pathway plays a significant role in the survival of dara resistant (dara R) cell lines. Here, we highlight that the enriched expression of exosome pathway proteins reported in dara R cell lines is not detected in Lenalidomide or Bortezomib-resistant cell lines, or in other monoclonal antibody resistant cell lines such as anti-CD20 (Rituximab)-resistant cell lines, thus indicating that this mechanism is unique to daratumumab resistance. Coculture of dara sensitive (dara S) cell lines with dara R cell lines separated by a cell-impermeable transwell insert promoted cell proliferation and reduced response to dara treatment in dara S cell lines thereby suggesting a potential role for extracellular vesicles (EVs) secreted by dara R cells in mediating these responses. In this study, we seek to investigate the role of EVs secreted by dara R cells, in particular, that of EV-derived miRNAs in the tumour microenvironment which can help predict and mediate dara resistance. We have originally described the establishment of three dara R cell lines in Natural Killer T-cell Lymphoma (NKTL), T-cell acute lymphoblastic leukaemia (T-ALL) and MM via long-term exposure to increasing concentrations of dara. To assess the specific role of EVs in mediating resistance, we isolated and purified EVs from the media of dara R cell lines and added it to the culture of dara S cells. To confirm uptake of dara R EVs by sensitive cell lines, EVs isolated from resistant cell lines were stained with PKH26 and characterised by binding to CD81 and CD63 beads via flow cytometry. Uptake of dara R EVs by dara S cell lines was confirmed through flow cytometry and confocal microscopy. We observed that uptake of dara R EVs directly promote proliferation and reduced cytotoxic response to dara treatment in dara S cells. Next, we assessed the effect of inhibiting dara R EV secretion by applying Neticonazole and Ketoconazole, prior to coculture of resistant and sensitive cell lines. Interestingly, coculturing azole-treated resistant cell lines with sensitive cell lines mitigated the increase in proliferation and dara resistance. Exosomal miRNAs are known to play a pivotal role in regulating cellular pathways driving cancer cell resistance and survival. To evaluate the potential role of EV-derived miRNA cargo, we first extracted miRNAs from the EVs purified from dara R and dara S cell lines and then performed an miRNA profiling screen with the PanoramiR panel (MiRXES) which targets 376 miRNAs expertly curated from miRBase 22, HMDD 3.2 and The Cancer Genome Atlas. We discovered that EVs derived from resistant cell lines expressed higher levels of proliferative miRNAs, miR-155 and miR-181a. These findings were validated through qPCR, confirming the increased expression of these miRNAs in both resistant cell lines and EVs derived from resistant cell lines. We also observed a concomitant downregulation of mir-155 target genes such as BACH1, JARID2 and SMAD5 in the resistant cell lines. This positive observation led us to extend the study into the clinic. Extracellular vesicles were isolated from the plasma fraction of bone marrow samples obtained from 17 patients with MM prior to treatment with dara based combinations either in the upfront or relapsed setting. miRNA extraction was performed and samples evaluated by miRNA sequencing. We found that the EVs from patients with a progression free survival (PFS) of less than 2 years after dara based therapy (dara R patients) had an enrichment of mir-155 and mir-181a as compared to those with a PFS longer than 2 years. These data align with our findings in the cell lines and suggest that these miRNAs may be utilised as potential biomarkers for daratumumab resistance. We are currently elucidating the biological basis for dara resistance mediated by these miRNAs. A deeper understanding of the mechanisms involved may pave the way for clinical evaluation of EV- directed therapeutics to overcome dara resistance.

A Retrospective Study on the Efficacy and Safety of Chidamide As a Maintenance Therapy for Peripheral T-Cell Lymphoma

Background: Peripheral T-cell lymphomas (PTCLs) are a set of rare and highly heterogeneous tumors of non-Hodgkin's lymphoma (NHL) with inferior outcomes. Chidamide is a novel benzamide class of histone deacetylase (HDAC) inhibitor (HDACi) that was approved by the Chinese Food and Drug Administration in 2014 for the treatment of relapsed or refractory (R/R) PTCL patients. This study aimed to assess the efficacy and safety of chidamide as a maintenance therapy after induction or salvage treatment in patients with PTCLs. Methods: The clinical data of 58 patients with different PTCL subtypes who received chidamide as a maintenance therapy after first-line or salvage treatment were collected. Progression-free survival (PFS), overall survival (OS), and safety were analyzed. Results: Fifty-eight patients enrolled in our study between January 2015 and July 2022, of whom 39 patients received chidamide as a maintenance therapy after first-line treatment and 19 patients after salvage treatment. All patients started chidamide maintenance after reaching at least partial response (PR). The median age was 66 (29-83) years. Patients with angioimmunoblastic T-cell lymphoma (AITL) represented the largest subgroup (34, 58.6%), followed by PTCL, not otherwise specified (PTCL-NOS) (10, 17.2%), anaplastic large cell lymphoma (ALCL) (8, 13.8%), NK/T cell lymphoma (NKTCL) (5, 8.6%) and monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL) (1, 1.7%). Thirty-four (58.6%) patients were stage III-IV, and 38 (65.5%) patients had intermediate or high risk (prognostic index for T-cell lymphoma (PIT) 2-4). Among the 39 patients who underwent chidamide maintenance after first-line treatment, 30/39 (76.9%) patients began maintenance after reaching complete response (CR) and 9/39 (23.1%) after achieving PR. Notably, 8/9 (88.9%) patients achieved CR from PR during maintenance treatment, with a median time of 6 (3-12) months. A total of 11/39 (28.2%) patients experienced disease progression during the maintenance treatment, and their median duration of remission was 6 (3-13) months. Among them, 2 patients experienced disease progression 2 to 3 months after drug withdrawal due to COVID-19 infection. A total of 19 patients who received maintenance treatment after salvage therapy, 9 (47.4%) received maintenance therapy after CR and 10 (52.6%) received maintenance therapy after PR. Among them, 9/19 (47.4%)had disease progression and their median duration of remission was 16 (2-25) months. The remaining 10 patients (8 CR and 2 PR) were still undergoing maintenance treatment and follow-up. The median maintenance treatment time for all patients was 16 (1-72) months, and the median maintenance dose was 20 (15-30) mg twice weekly. The median follow-up time was 39 (2-79) months, the median PFS was 33 months, and the median OS had not been reached (Figure 1 a, b) for all patients. The median PFS and OS of the patients who received first-line maintenance therapy were not reached, which were significantly better compared to patients who received chidamide as salvage treatment maintenance (the median PFS and OS were 7 and 67 months, P &amp;lt; 0.001, P = 0.009, respectively) (Figure 1 c, d). There was no statistical difference in PFS and OS among the different pathological subtypes, but AITL seemed to achieve better PFS (Figure 1 e, f). The most common toxicities were hematologic toxicities, including neutropenia (42, 72.4%), anemia (23, 39.7%), and thrombocytopenia (29, 50.0%), followed by fatigue (35, 60.3%), anorexia (31, 53.4%), liver dysfunction (10, 17.2%), and pneumonia (4, 6.9%). Most adverse events were grade 1 or 2, and the grade 3‒4 adverse events were neutropenia (15/42), thrombocytopenia (7/29), fatigue (1/35), and pneumonia (2/4). Twelve (20.7%) patients underwent a dose reduction and three patients discontinued treatment (two patients with severe pulmonary infections, and one patient had intolerable fatigue) due to adverse events. One patient stopped maintenance treatment after taking chidamide for 10 months for financial reasons and he is still in follow-up. Conclusion: The findings of this study showed that patients with chidamide maintenance therapy had a better PFS and OS with a manageable safety profile, especially as the first-line maintenance therapy. We conclude that chidamide can be used as a maintenance treatment to improve the survival of patients with PTCLs.

Phase II Study of Lenalidomide Maintenance after Salvage Therapy for Relapsed or Refractory Peripheral T-Cell Lymphomas

Introduction Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of non-Hodgkin lymphomas. Their prognoses are still poor especially after they relapse or become refractory to the first-line treatment. Although salvage chemotherapy followed by autologous stem cell transplantation (ASCT) could be used for those relapsed or refractory patients, a substantial number of patients relapse even after ASCT. Furthermore, ASCT is not always possible because elderly or frail patients are not eligible for high-dose chemotherapy followed by ASCT. Thus, there are limited treatment option for patients who failed after ASCT or ineligible for ASCT because there is no established salvage treatment strategy and the impact of novel therapies in previous trials is unknown. Lenalidomide is widely used for the treatment of B-cell lymphomas as well as multiple myeloma, however, there are a few data about its efficacy in patients with PTCLs. Considering the mode of action is related with the immune modulatory effect on tumor microenvironment, lenalidomide might have a role in the management of relapsed or refractory PTCLs. Thus, we hypothesized that the use of lenalidomide as a maintenance therapy for patients who achieve response after salvage therapy might produce survival benefit comparable to that of ASCT after salvage therapy. Methods This is a prospective, open-labeled, single-arm, multi-center phase II study for relapsed or refractory PTCLs. The inclusion criteria are as follows: 1) Patients should have relapsed or refractory disease after at least one-line of prior chemotherapy; 2) Patients should not be eligible for ASCT; 3) Patients should have any subtypes of PTCLs including PTCL-not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), anaplastic lymphoma kinase (ALK)-positive or -negative anaplastic large-cell lymphoma (ALCL), extranodal NK/T-cell lymphoma (ENKTL), monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), hepatosplenic T-cell lymphoma (HSTL), and mycosis fungoides (MF). When patients eligible for the study achieve complete response (CR) or partial response (PR) after four cycles of any kinds of salvage chemotherapy, they can receive lenalidomide as a maintenance therapy. One cycle is 28 days consisting of lenalidomide 25 mg (day 1 - day 21). The treatment can be repeated until progression or the occurrence of intolerable toxicities (up to 2 years). The primary end point is 1-year progression-free survival, and the target rate is 35%. Results A total of 58 patients were enrolled between February 2019 and May 2023. The median age was 62 years (range: 20 - 83 years) and male was dominant (male: 35, female: 23). Patients who failed after ASCT or could not undergo ASCT due to their health status participated in the study. Patients with AITL (n = 23) and PTCL-NOS (15) accounted for 66% of patients whereas other subtypes included ENKTL (n = 4), ALK-negative ALCL (n = 3), MEITL (n = 3), HSTL (n = 2) and MF (n = 7). Prior to the maintenance therapy with lenalidomide, their disease status was CR (n = 22, 38%) and PR (n = 36, 62%). At the median follow-up of 20.7 months (95% CI: 17.7 - 23.7 months), seven patients completed the planned treatment of 2 years, and three patients withdrew their informed consents. Disease progression was documented in 21 patients during the maintenance and the other 20 patients discontinued the treatment due to their adverse events. At the time of analysis, seven patients were maintaining their treatments. Out of 36 patients with PR before maintenance, five patients achieved CR and 13 patients showed PR. However, 18 patients showed disease progression during maintenance. On the other hand, only three patients showed disease progression among 22 patients with CR prior to lenalidomide maintenance. Thus, 1-year PFS after enrollment was 49% and the median OS was (34.1 months, 95% CI: 7.9 - 18.6 months, Figure 1). The most common hematologic toxicities were neutropenia and thrombocytopenia; thus, dose-reduction should be done in patients with grade 4 hematologic toxicity. The most frequent non-hematologic toxicities included skin rash (40%), nausea (35%), and diarrhea (20%) however, they were manageable. Conclusions The lenalidomide maintenance might be a feasible and effective treatment approach for patients with relapsed or refractory PTCLs who are ineligible for ASCT or intensive consolidation chemotherapy.

Molecular and clinicopathological features of granzyme B-negative extranodal NK/T-cell lymphoma

Extranodal NK/T-cell lymphoma (ENKTL) generally expresses cytotoxic molecules, including granzyme B (GZMB), T-cell-restricted intracellular antigen-1 (TIA-1), and perforin; however, the expression of these molecules varies across cases. We performed gene expression profiling and identified unique biological and clinicopathological features of GZMB-negative ENKTL. We reviewed the clinicopathological characteristics of 71 ENKTL samples. Gene expression profiling on nine ENKTLs using multiplexed, direct, and digital mRNA quantification divided ENKTLs into Groups A (n = 7) and B (n = 2) through hierarchical clustering and t-distributed stochastic neighbor embedding. Group B was characterized by downregulation of genes associated with IL6-JAK-STAT3 signaling and inflammatory responses. GZMB mRNA expression was significantly downregulated in Group B. GZMB protein expression was evaluated with immunohistochemistry in all 71 ENKTLs, and expression data of Tyr705-phosphorylated STAT3 (pSTAT3) and MYC from our previous study was utilized. T-cell receptor gamma (TRG) gene rearrangement in the selected samples was also assessed using PCR. GZMB expression was higher in pSTAT3-positive (p = 0.028) and MYC-positive (p = 0.014) ENKTLs. Eighteen percent (13/71) of all ENKTLs were negative for GZMB (defined by positivity <10 %); patients with GZMB-negative ENKTLs were often in a higher clinical stage (p = 0.016). We observed no other correlations with clinical parameters or TRG rearrangement and no significant association between GZMB expression and survival. In conclusion, GZMB expression is highly heterogeneous in ENKTLs and is associated with the activation of the JAK-STAT3 pathway and higher MYC expression. GZMB-negative ENKTLs correlate with an advanced clinical stage, suggesting the potential utility of GZMB immunohistochemistry as a biomarker of ENKTL.

Extranodal NK/T-Cell Lymphoma Predominantly Composed of Anaplastic Cells: A Frequently Misdiagnosed and Highly Aggressive Variant.

Extranodal NK/T-cell lymphoma (ENKTL) is a non-Hodgkin lymphoma associated with the Epstein-Barr virus that primarily affects individuals in East Asia and indigenous populations in Central and South America. Morphologically, ENKTL typically consists of medium-sized cells or a combination of small and large cells. This report presents 10 cases characterized by predominantly anaplastic cells with diffuse expression of CD30, resembling anaplastic lymphoma kinase-negative anaplastic large cell lymphoma (ALK-negative ALCL) and demonstrating highly aggressive behavior. The cohort included 9 males and 1 female, ranging in age from 29 to 65 years (median age: 47y). Eight patients presented with nasal disease, while 2 had non-nasal disease. Five patients had stage I/II disease, and the remaining 5 had stage III/IV disease. Morphologically, necrosis was observed in 9 cases, angiocentric-angiodestructive growth in 3 cases, and pseudoepitheliomatous hyperplasia in 2 cases. Anaplastic cells predominated in all cases, with some displaying eccentric, horseshoe-shaped, or kidney-shaped nuclei (referred to as "Hallmark" cells). The morphology profile was monomorphic in 3 cases and polymorphic in 7 cases. Immunohistochemically, all cases tested positive for cytotoxic granule markers (TIA1 and granzymeB) and Epstein-Barr virus-encoded RNA. Cytoplasmic expression of CD3ε and CD56 was observed in 9 of 10 cases. Interestingly, most cases (7 of 8) exhibited variable expression of MuM1, ranging from 10% to 90%. All cases showed diffuse positivity for CD30 but were negative for ALK, resulting in 3 cases being initially misdiagnosed as ALK-negative ALCL. Compared with nonanaplastic cases, anaplastic cells predominant ENKTL had a significantly higher frequency of "B" symptoms, bone marrow involvement, hemophagocytic lymphohistiocytosis, and higher Ki67 proliferative index. These findings provide valuable information for pathologists, expanding their understanding of the cytologic spectrum of ENKTL. This rare variant of ENKTL, characterized by the predominance of anaplastic cells and diffuse CD30 expression, exhibits high aggressiveness and should be differentiated from ALK-negative ALCL. Awareness of this uncommon variant is crucial in preventing misdiagnosis and ensuring the timely initiation of therapy.