Unusual presentation of an Epstein barr virus-negative extranodal natural killer/T cell lymphoma: A diagnostic dilemma

Extra-nodal Natural killer/T cell lymphoma (ENKTL) is a well-defined and highly aggressive form of NonHodgkin’s lymphoma with a scarcity of cases reported in literature. The most common primary site of involvement is the nasal cavity followed by skin and the gastrointestinal tract (GIT). Cutaneous involvement is a rarity. More than 95% of cases are usually in association with Epstein Barr Virus (EBV) infection. EBV negative ENKTL can be similar in clinical, pathological, and prognostic characteristics with EBV positive ENKTL. This malignancy is usually characterized by its poor prognosis irrespective of clinical stage and therapy. We describe here, a 58-year-old man presenting with multiple nodular lesions over legs and trunk, had an ileal perforation later, and was diagnosed as ENKTL on the ileal biopsy specimen. This case is being reported in view of the fulminant clinical course of the disease, simultaneous involvement of the GIT and skin without nasal or midline involvement, the usefulness of immunohistochemistry in arriving at a diagnosis, and EBV negativity which is quite rare in the Asian population.

Extranodal natural killer/T-cell lymphoma nasal type with extensive ocular tissue involvement: a case report

Background To report a rare case of extranodal natural killer/T-cell lymphoma (ENKTL), nasal type related to extensive ocular tissue, including conjunctiva, ciliary body, vitreous and retina. Case presentation A 52-year-old woman who had been treated by radiotherapy for ENKTL, nasal type in the right nasal cavity presented with a dramatic deterioration of vision in right eye. Physical and accessory examination showed extensive ocular tissue related, including conjunctiva, ciliary body, vitreous and retina. Vitreous specimen and conjunctiva biopsy revealed the presence of ENKTL, nasal type in the right eye. She was treated with systemic and ophthalmic chemotherapy, her ocular symptoms significantly improved, and systemic condition remained stable 7 months after the diagnosis. Conclusions Extranodal natural killer/T-cell lymphoma, nasal type is an aggressive disease and may relate extensive ocular tissue and course dramatic vision deterioration. It is important to observe ocular related and begin aggressive combined therapy as early as possible after diagnosis.

Daratumumab Resistant Natural Killer/T-Cell Lymphoma Exhibit an Addiction to the Exosome Biogenesis Pathway for Survival

A phase 2 clinical trial has demonstrated that daratumumab monotherapy was safe and well tolerated in relapsed/refractory Natural Killer/T-Cell Lymphoma (NKTL). However, no patients achieved complete response, and duration of response was short. Similarly, in Multiple Myeloma (MM), while daratumumab based combinations are approved for front line treatment, responses are heterogeneous and development of treatment resistance inevitable. Therefore, elucidation of mechanisms which can overcome daratumumab resistance is essential for the optimization of therapeutic response in patients. To this end, 2 pairs of isogenic daratumumab resistant and sensitive models of NKTL were developed in vitro via sequential exposure of cell lines to increasing concentrations of daratumumab in the presence of complement serum. A 3rd model was also studied in vivo whereby long-term administration of daratumumab in mice identified a sub-group of tumors which stopped responding to treatment and began to rapidly enlarge ('Resistant') as compared to others which remained similar to or smaller ('Sensitive') than the tumour volume at the initiation of treatment. RNA sequencing was performed on these models and genes commonly upregulated or downregulated analyzed. Differential gene expression analysis highlighted an enrichment for the upregulation of genes involved in exosome biogenesis and secretion in both cell line and mouse-derived daratumumab resistant NKTL models. An additional daratumumab resistant model was developed in an MM cell line to further validate these findings and extend the study in an MM model. Immunoblotting of the 3 pairs of isogenic sensitive and resistant cell line models demonstrate that there is indeed an upregulation of proteins regulating exosomal biogenesis and secretion; Alix, TSG101 and Rab27b in the daratumumab resistant phenotype. This is associated with a concomitant increase in secreted exosomes levels in the tumour microenvironment. The size and quantification of extracellular vesicles (EV) secreted in the media were studied by nanoparticle tracking analysis. Extracellular vesicles ranged in the size of 70-150nM corroborating with the size of exosomes and nanoparticle quantification revealed a higher concentration of exosomes present in the tumour microenvironment of resistant cells as compared to sensitive cells. Subsequently, exosomes were purified via ultracentrifugation and protein expression analysis confirmed elevation of exosome markers CD63 and CD81. To study the role of exosomal-mediated mechanisms in the survival of daratumumab resistant cells, we treated isogenic models with neticonazole and ketoconazole (azoles) which have been identified as selective inhibitors of exosomal biogenesis in a drug repurposing study for advanced cancers. Interestingly, azole treatment demonstrated a selective and more effective suppression of tumour cell viability in daratumumab resistant than sensitive cell lines. Immunoblot analysis showed that azole treatment at identical concentrations resulted in a more extensive downregulation of Alix, Rab27b and CD81 protein expression in resistant than sensitive cells. Additionally, depletion of Alix and Rab27b protein expression via siRNA knockdown induced cell death confirming that daratumumab-resistant cell lines are dependent on exosomal-mediated pathways for survival. Current research is focused mainly on intrinsic or immune cell-mediated mechanisms of daratumumab resistance, but little is known regarding the effect of extrinsic components in the tumour microenvironment. We demonstrate that daratumumab resistant models exhibit a distinct upregulation of proteins mediating exosome biogenesis resulting in enhanced exosome secretion. Daratumumab resistant cells are targeted more efficaciously with exosome biogenesis inhibitors than sensitive counterparts thereby suggesting an addiction to exosome-mediated mechanisms of survival. This is further supported by gene silencing studies. In future, we aim to perform miRNA profiling of exosomes purified from the tumour microenvironment of isogenic daratumumab-resistant and sensitive cell line models as well as from bone marrow plasma of daratumumab treated patients. miRNA which are enriched in the exosomes of resistant phenotypes will be characterized, unique biomarkers of response identified and in depth mechanisms of resistance studied. Disclosures No relevant conflicts of interest to declare.

Anti-PD-1-Antibody (Tislelizumab) Combined with Deacetylase Inhibitor (Chidamide), Lenalidomide and Etoposide for the Treatment of Refractory/Relapsed Extranodal Natural Killer/T Cell Lymphoma, Nasal Type (r/r-ENKTL): Preliminary Results from a Prospective, Multicenter, Single -Arm, Phase II Trial

B ackground: Extranodal Natural Killer/T Cell Lymphoma(ENKTL) is a highly aggressive non-Hodgkin lymphoma (NHL), with higher incidence in Asia, which is related to Epstein-Barr virus (EBV) infection. Patients with r/r-ENKTL have a poor prognosis, and is lack of effective treatment. Anti-PD-1-Antibody plus Chidamide was effective and well tolerated on r/r-ENKTL (SCENT Trial, 2020 ASH). Lenalidomide is an immunomodulatory agent, previous studies showed activity in r/r-lymphoma with an acceptable safety. Etoposide is an important chemical agent in the treatment of ENKTL. Here we present preliminary results of Anti-PD-1 antibody combined with Chidamide, Lenalidomide and Etoposide for the r/r-ENKTL. Methods: This was a prospective, single-arm, open-label, multi-center, phase II clinical trial. This trial enrolled eligible patients with histologically confirmed r/r-ENKTL failing from at least 1-line treatment; ECOG performance status ≤ 2; adequate organ function and bone marrow function; and at least one measurable or evaluable lesion. Patients received 6 cycles of Tislelizumab (200mg), Chidamide (20mg q3d), Lenalidomide (25mg d1-10) and Etoposide (100mg/m 2 d1-3), every 21 days cycle. Then received 10 cycles of Tislelizumab (200mg) maintenance treatment, every 21 days cycle. The primary endpoints were objective response rate (ORR) based on Lugano 2014 criteria, duration of response (DOR), progression-free survival (PFS), overall survival (OS) and disease control rate (DCR). Secondary endpoints were median survival time (MST) and safety. Adverse events (AE) were rated according to the NCI Common Terminology Criteria for Adverse Events (CTCAE) 5.0. This trial is registered at ClinicalTrials.gov (NCT04038411). R esults: From July 2020 to July 2021, 12 patients were screened and 10 eligible patients were enrolled, median age was 36.5 years (range 18-58), 8 (90.0%) male, 8(80.0%) patients with stage Ⅲ-Ⅳ of disease at screening, 4 (40.0%) patients received ≥2 lines of prior systemic therapy. Of 8 response evaluable patients, 7 (87.5%) achieved response including 5 (62.5%) patients with CR. The median time to initial response was 4.0 weeks (2-6 weeks). 10 Patients reported treatment-related AEs (TRAEs). The most frequently observed TRAEs were neutropenia (10 patients), thrombocytopenia (4 patients), transaminase increased (3 patients), nausea (4 patients). The most frequent Grade (G) ≥3 TRAEs were neutropenia (6 patients). Immune-related AEs were reported in 2 patients with G1 hypothyroidism. No death was related to the study. C onclusion: Tislelizumab combined with Chidamide, Lenalidomide and Etoposide regimen have a very high response rate in r/r-ENKTL for the first time, and the safety is under control. It is a promising therapeutic option for this population, further investigation is warranted. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Antitumor Activity of Dual BCL-2/BCL-Xl Inhibitor Pelcitoclax (APG-1252) in Natural Killer/T-Cell Lymphoma (NK/TCL)

Natural killer/T-cell lymphoma (NK/TCL) is one of the most common subtypes (10.4%) of peripheral T-cell lymphoma, which in turn accounts for 10% to 15% of all cases of non-Hodgkin lymphoma. NK/TCL occurs more frequently in Asia and Latin America than other regions, and although associated with Epstein-Barr virus (EBV) infection, NK/TCL has an unclear pathogenesis of genetic and molecular alterations. Asparaginase-based chemotherapy regimens are frequently used, typically with unsatisfactory outcomes. Novel targeted therapies, such as histone deacetylase (HDAC) inhibitors and programmed death-1 antibodies, are reported effective either as single agents or in combination with other agents. Studies have also shown that EBV-positive NK/TCL cell lines are B-cell lymphoma-extra large (BCL-xL) dependent. Other preclinical experiments have shown that BH3-mimetic drugs targeting BCL-xL-induced cell death in NK/TCL cell lines were effective in NK/TCL xenograft models. BCL-xL inhibitors have shown narrow therapeutic windows in clinical trials because of dose-limiting on-target thrombocytopenia. Pelcitoclax (APG-1252) is a novel dual BCL-2/BCL-xL inhibitor under clinical development for solid tumors. As a result of its unique prodrug design, APG-1252 can overcome the undesired platelet toxicity. This study evaluated the potential antitumor effect of APT-1252 in preclinical models of NK/TCL. Cell-based antiproliferation studies showed activity of APG-1252 and its more potent metabolite APG-1252-M1 toward NK/TCL cell lines that overexpressed BCL-xL. Half-maximal inhibitory concentrations (IC 50) for APG-1252 in SNK-1, SNK-6, and SNK-8 (EBV-positive NK/TCL) cell lines were 2.652 ± 2.606 µM, 1.568 ± 1.109 µM, and 0.557 ± 0.383 µM, respectively. Corresponding values for APG-1252-M1 were 0.133 ± 0.056 µM, 0.064 ± 0.014 µM, and 0.020 ± 0.008 µM, respectively. Mechanistic studies demonstrated that APG-1252 and APG-1252-M1 disrupted the complex of BCL-xL/BCL-2-associated X protein (Bax) and BCL-xL/BCL-2 homologous antagonist killer protein (Bak) in SNK-6 cells, thereby liberating these proapoptotic proteins and further activating downstream apoptosis pathways by cleaving poly-ADP ribose polymerase-1 (PARP-1) and caspase-3. In an SNK-6 xenograft model, administration of APG-1252 at 65 mg/kg and 100 mg/kg either twice or once weekly resulted in significant antitumor effects, with tumor growth rate (T/C%) values ranging from 13.7% to 30.7%. Furthermore, the combination of APG-1252 with HDAC inhibitor chidamide or DDGP (dexamethasone, cisplatin, gemcitabine, and pegaspargase) chemotherapy demonstrated synergistic effects. Pharmacokinetic assessment in mice showed that APG-1252 had a long half-life in plasma (127 hours) and tumor tissues (25.2 hours), justifying intermittent dosing schedules used in vivo. Importantly, the transformation of APG-1252 to APG-1252-M1 was 16 times higher in tumor tissues compared to plasma (22% vs. 1.3%) after administration of APG-1252, thereby suggesting that APG-1252 can reduce platelet toxicity caused by APG-1252-M1 in plasma. In conclusion, APG-1252 has promising antitumor effects in NK/TCL, either as a single agent or in combination with an HDAC inhibitor or chemotherapy. These findings provide evidence to further evaluate APG-1252 as a potential treatment for NK/TCL. Disclosures Wang: Ascentage Pharma Co., Ltd., Shanghai: Current Employment, Current equity holder in publicly-traded company. Liang: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company. Ming: Ascentage Pharma Co., Ltd., Shanghai: Current Employment, Current equity holder in publicly-traded company. Rui: Ascentage Pharma Co., Ltd., Taizhou: Current Employment, Current equity holder in publicly-traded company. Tang: Ascentage Pharma Co., Ltd., Taizhou: Current Employment, Current equity holder in publicly-traded company. LV: Ascentage Pharma Co., Ltd., Shanghai: Current Employment, Current equity holder in publicly-traded company. Ge: Ascentage Pharma Co., Ltd., Shanghai: Current Employment, Current equity holder in publicly-traded company. Zhang: Ascentage Pharma Co., Ltd., Taizhou: Current Employment, Current equity holder in publicly-traded company. Wang: Ascentage Pharma Co., Ltd., Taizhou: Current Employment, Current equity holder in publicly-traded company. Shang: Ascentage Pharma Co., Ltd., Shanghai: Current Employment, Current equity holder in publicly-traded company. Yang: Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Zhai: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding; Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding.

Clinical Characteristics and Survival of Non-Nasal Natural Killer/T-Cell Lymphoma from 2 Cancer Centers from Peru

Background Natural killer/T-cell lymphoma (NKTCL) is an aggressive lymphoma with poor prognosis mainly seen in Asian and South American countries, most patients have nasal involvement. Non-nasal NKTCL have a dismal prognosis. However, data about this very specific population is mostly known in Asians but little known in Latin American (LATAM) population. Our aim is to describe the clinical characteristics and survival of newly diagnosed NKTCL patients in Peru Methods: This is a retrospective study, including all patients with a pathological diagnosis of NKTCL patients >13 years at the Instituto Nacional de Enfermedades Neoplasicas (INEN) and Oncosalud between 2002 and 2019. The estimate of the survival curves was performed by the Kaplan-Meier method, and the difference was computed by the log-rank test. Results: 60 patients were included for the analysis. The median age was 37 years (range 15-83). 62% were male, 51% had B-symptoms, 56% had stage III/IV disease, 54% had elevated serum LDH, 29% had nodal involvement. The most frequent non-nasal sites were oropharynx, soft tissue, cutaneous, gastrointestinal and orbit with 24%, 17%, 12%, 7% and 5%, respectively. 79% patients received systemic chemotherapy, 7% received radiotherapy alone and 14% did not received chemotherapy because poor performance status. Of patients who received chemotherapy only 20% received a L-asparaginase-based chemotherapy. The median follow-up time was 8 months (1-103). The median overall survival (OS) was 14 months (interquartile range [IQR] 4-29), 1y OS was 51% (95% CI 33-67), 3y OS 21% (95% CI 7-40), 5y OS 14% (95% CI 3-34), figure 1. The median overall survival according to LDH level was 10 months regardless of the levels (p=0.84), figure 2. The median OS for female and male were 8 and 10 months, respectively p=0.59) Conclusions: Peruvian patients with non-nasal NKTCL are younger, with presence of B-symptoms, advance stage and elevated LDH levels compared to those reported in nasal NKTCL. The most frequent non nasal NKTCL is located on the oropharynx. OS of non-nasal NKTCL is extremely poor. Elevated LDH and sex are not prognostic factors for OS. A larger cohort and follow-up are needed to evaluate prognostic factors in this population. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

CircADARB1 serves as a new biomarker in natural killer T-cell lymphoma and a potential regulator of p-Stat3

Background Natural killer/T-cell lymphoma (NKTCL) is a rare and aggressive subtype of Non-Hodgkin’s Lymphoma. CircRNA has shown great potential to become a biomarker in plasma. In this study, we aimed to determine circRNA for its diagnostic and prognostic value and biological function in NKTCL. Method The circRNA microarray of plasma from NKTCL patients and healthy donors were conducted. The relative expressions of target circRNA were verified by qRT-PCR. We conducted function experiments in vitro and in vivo. Bioinformatics predicted the target miRNA of the target circRNA and the binding site was detected by the dual luciferase report assay. Downstream target protein was predicted and detected by western blot in vitro and immunohistochemistry in vivo. Result By analyzing the plasma circRNA microarrays in NKTCL, 6137 circRNAs were up-regulated and 6190 circRNAs were down-regulated. The relative expressions of circADARB1 were significantly higher in NKTCL patients. The knockdown of circADARB1 inhibited proliferation of NKTCL cells in vitro and in vivo. CircADARB1 could bind to miR-214-3p in the downstream and regulate the expression of p-Stat3. In nude mice tumor tissue, p-Stat3 was under-expressed in the circADARB1 knockdown group. Conclusion CircADARB1 was highly expressed in NKTCL plasma and circADARB1 was a potential biomarker to assist diagnosis and predict the response in NKTCL. CircADARB1 bound up to miR-214-3p and regulated p-Stat3.