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BACKGROUND: Extranodal natural killer/T-cell lymphoma, nasal type (ENKTL) is an aggressive form of non-Hodgkin lymphoma. up to now, optimal therapeutic strategies for ENKTL have not been fully defined yet. However, approximately 25-50% patients experience local relapse or systemic failure who receive RT alone. The addition of chemotherapy is emphasized to reduce the risk of recurrence. Therefore, we evaluated efficacy and safety of P-GEMOX regimen in patients with newly diagnosed stage I/II ENKTL. METHODS: We conducted this pilot study to evaluate the efficacy and safety of pegaspargase combined with gemcitabine and oxaliplatin (P-GEMOX) followed by extensive involved-field radiotherapy(EIFRT) in patients with stage I/II ENKTL. We enrolled 56 newly diagnosed stage I/II patients. All patients received P-G GEMOX chemotherapy. The P-GEMOX dosage was as follows: gemcitabine 1000 mg/m2 intravenous infusion in 30 minutes ondays 1 and 8; oxaliplatin 100 mg/m2 intravenous infusion in 2 hours on day 1; pegaspargase deep intramuscular injection of 2000 U/m2 at two different sites on day 1. The regimen was repeated every 3 weeks. Patients underwent 4 cycles of induction chemotherapy, followed by EIFRT. After achieving complete response (CR), partial response (PR), or stable disease (SD). EIFRT was 56 Gy in 28 fractions over 4 weeks. Primary EIFRT was delivered using 6-MeV linear accelerator using 3-dimensional conformal treatment planning. Clinical target volume (CTV) included gross tumor volume with a margin of at least 20mm and the bilateral nasal cavity, bilateral parasinusess. Planning target volume (PTV) included CTV with a 5mm margin. For stage IIE disease, CTV and PTV also included the involved the cervical lymph node area. RESULTS: The median follow-up was 35.2 months (range: 10.6-51.4 months). The objective response rates(ORR) of P-G GEMOX regimen was 89.3% (50/56), 35(62.5%) patients achieved CR and 15 (26.8%) patients achieved PR, respectively. After EIFRT, ORR increased to 94.6% (53/56), CR rate increased to 89.3% (50/56). The 4-year overall survival(OS) and progression-free survival(PFS) rate was 90.7¡À4.0% and 89.1¡À4.2% for the whole cohort. The OS and PFS of stage I patients were superior to patients with stage II (Figure1A,B). No treatment-related death was observed. No allergic reactions occurred. Common toxicities (>50%) were neutropenia (80.3%), thrombocytopenia (55.3%), hypoproteinemia (75.0%). Fibrinogen decrease rate was 44.6%. The most common grade III/IV toxicities (>10%) were granulocytosis (23.2%), thrombocytopenia (19.6%) and hypoproteinemia (10.7%). CONCLUSION: The P-GEMOX regimen followed by radical radiotherapy yielded very promising longterm survival for patients with stage I/II ENKTL with good tolerance. Further investigation of P-GEMOX in a larger series of patients, is required. Figure 1. Survival for different stage. (A) Overall survival(P =0.056). (B) Progression free survival (P =0.023). A. B. Figure 1. Survival for different stage. (A) Overall survival(P =0.056). (B) Progression free survival (P =0.023). / A. B. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

Purpose Extranodal natural killer/T-cell lymphoma (ENKTL) is an aggressive form of non-Hodgkin's lymphoma. The prognosis for patients with advanced stages or relapsed/ refractory ENKTL is extremely poor. Optimal combined chemotherapy remain to be defined. Therefore, the purpose of this study is to evaluate efficacy and safety of P-GEMOX (Pegaspargase, Gemcitabine and Oxaliplatin) in patients with newly diagnosed stage III/IV or relapsed/refractory ENKTL. Patients and methods We retrospectively analyzed the effectiveness and toxicity of P-GEMOX in 60 patients with newly diagnosed stage III/IV and relapsed/refractory ENKTL between February 2008 and August 2014. The P-GEMOX dosage was as follows: Gemcitabine 1000 mg/m2 iv d1,d8; Oxaliplatin 100 mg/m2; d 1, Pegaspargase 2000 U/m2 im, two different sites. The regimen was repeated every three weeks for a maximum six cycles. Patients underwent autologous hematopoietic stem cell transplantation (ASCT) as consolidation if they achieved CR. Results 57 patients were available for evaluation of response. The objective response, complete remission(CR), of whole cohort were 73.7% (42/57), 36.8% (21/57), respectively. It can be easily administered in out-patients clinic. The median follow-up was 29.1 (range, 2.4¨C54.2 months). Median OS and PFS was 23.0 months (95% confidence interval [CI], 16.441-29.559) and 12.8 months(95% confidence interval [CI], 8.109-17.491), respectively. The 4-year OS and PFS rate was 43.0¡À7.3% and 36.5¡À6.9%, respectively (Figure1). There was no difference between newly diagnosed stage III/IV and relapsed/refractory in OS and PFS. The long term survival CR responders were superior to patients with other response, and there was significant difference between the three group(Figure 2, P50%): neutropenia (85.0%), thrombocytopenia (72.0%), hypoproteinemia (86.7%), and anorexia (63.3%). In addition, hypofibrinogenemia was 46.7%. The most common grade III/IV toxicities (>10%) were granulocytosis (31.6%), thrombocytopenia (26.67%) and hypoproteinemia (13.3%)(Table 1). Intracranial bleeding occurred in one patient during the first cycle with discontinuation of pegaspargase in the consecutive cycles. No treatment related death confirmed. Conclusion The P-GEMOX regimen is a safe and effective combination for newly diagnosed advanced and relapsed/refractory ENKTL. Promising long term outcome can be expected by addition of ASCT consolidation after response to induction chemotherapy. In comparison to other combined regimen in literatures, P-GEMOX is effective with less toxic, simplified and high cost-effective. Further clinical trials urgently needed. Table 1. Toxicities of whole cohort All cases (%) Grade 1/2 (%) Grade 3/4 (%) Toxicities Neutropenia 51(85.0) 32(53.3) 19(31.6) Thrombocytopenia 22(36.7) 15(25.0) 7(11.7) Anemia 43(71.6) 18(66.7) 3(5.0) lymphocytoponia 14(23.3) 12(20.0) 2(3.3) AST/ALT elevated 26(43.3) 22(36.7) 4(6.7) Hypoproteinemia 53(88.3) 48(84.9) 8(13.3) Fbg decrease 41(68.3) 39(65.0) 2(3.3) APTT prolong 16(26.7) 16(26.7) 0 Hyperglycemia 7(11.6) 7(11.6) 0 Total bilirubin elevated 9(15.0) 9(15.0) 0 Nausea 21(35.0) 21(35.0) 0 Anorexia 32 (53.3) 32 (53.3) 0 Vomiting 19(31.6) 19(31.6) 0 Allergic reactions 1(1.7) 1(1.7) 0 herpes zoster 3(5.0) 3(5.0) 0 Figure 1. 4-year OS and PFS of whole patients Figure 1. 4-year OS and PFS of whole patients Figure 2. Survival of whole patients, based on response Figure 2. Survival of whole patients, based on response Figure 3. OS: patients with ASCT vs. Non-ASCT Figure 3. OS: patients with ASCT vs. Non-ASCT Disclosures No relevant conflicts of interest to declare.

Backgroud It has been demonstrated that Natural/killer T-cell lymphoma (NK/TCL) derives from chronic EBV infection of nasopharynx. In recent years, IL-6 has been proved to be associated with a variety of inflammation-related malignancies in terms of carcinogenesis, tumor proliferation and metastasis. However, there hasn’t been any report about IL-6 on the pathogenesis and development of NK/TCL. Patients and Methods From January 2005 to December 2012, the paraffin sections of 91 patients with NK/TCL in Sun Yat-sen University Cancer Center were collected. The activation/expression of NF-ΚB(P50, P52, P65), IL-6,P-STAT3(Tyr705),BCL-2 in tumor microenviroment were investigated by immunohistochemistry. The NK/TCL cell lines were treated by IL-6(80ng/ml) and neutralizing IL-6 antibody, then the viability and apoptosis of lymphoma cell lines (NK-YS, SNK-6 and SNT-8) were assessed by CCK-8 and flow cytometry; and the expression of p-STAT3, BCL-2, BCL-XL, MCL-1, Survivin、p-ERK and p-AKT were examined by Western blot . Results Overall, ninety-one patients with NK/TCL were analyzed. There were 68 male patients. The median age was 43 years (range 10-68 years). There were 52, 14 and 25 patients diagnosed as stage I, II and IV disease, respectively. Sixty-one patients had B symptoms. The P52 activation was observed in the microenviroment of most patients, while P65 and P50 activation were observed in minority of patients. In patients with P52 activation, P52 activation was observed both in macrophages and lymphoma cells, and vice versa. IL-6 was primarily secreted by macrophages and tumor cells, and 69.2% (63/91) patients were positive (IL-6 secreting cells ≥ 10 / HP). The STAT3 activation was observed in 67.0% (61/91) of patients, most of which were combined with expression of BCL-2. The expression of P52、IL-6、P-STAT3、bcl-2 in the microenvironment of NK/TCL were in uniform (all positive or all negative). Patients with positive expression of IL-6 were associated with fever, advanced stage, high serum level of C-reactive protein (CRP), chemotherapy resistance and poor survival (P < 0.05). Comparing with blank control group and the neutralizing IL-6 antibody group, the cell viability and antiapoptosis ratio of NK-YS、 SNK-6、SNT-8 cells were higher in the IL-6 group (P Conclusions The NK-κB(P52)/IL-6/STAT3 pathway is activated in tumor microenvironment, and is associated with fever, advanced stage, high serum level of CRP in patients with NK/TCL. IL-6 could activate the JAK/STAT3 pathway in NK/T lymphoma cell lines, and promote lymphoma proliferation and anti-apoptosis. The activation of NK-κB(P52)/IL-6/STAT3 pathway may lead to chemotherapy resistance and poor survival in patients with NK/TCL. Disclosures: No relevant conflicts of interest to declare.

Background and Objective Natural killer/T-cell lymphoma (NK/TCL) is featured with inflammatory symptoms and poor prognosis. Currently, new treatment targets and modalities are urgently needed for patients with high risk NK/TCL. As one of the most important factors in the microenvironment of lymphomas, tumor-associated macrophages (TAM) may display either positive or negative effect on tumor proliferation and metastases, depending on the polarization of macrophages (M2 or M1 type). It is well established that the activation and differentiation of TAM are determined by different stimuli from the microenvironment. The characteristic cytokine spectrum of M1 type macrophages is high expression of IL-6 and IL-12, and low expression of IL-10. In contrast, the characteristic cytokine spectrum of M2 type macrophages is high expression of IL-10 and low expression of IL-12. Prior studies have revealed that IL-4, IL-10 and IL-13 are key cytokines which stimulate the differentiation from monocytes to M2 polarized TAM. Our study was designed to explore the cytokine spectrum secreted by NK/TCL and TAM in the microenvironment of NK/TCL. We aimed to prove that NK/TCL could induce the differentiation from monocytes to M2 polarized TAM, by secreting special cytokines. Material and Methods The cell lines involved in the study were as follows: the human NK/TCL cell lines (NK-YS, SNK-6 and SNT-8); the human diffuse large B-cell lymphoma cell line (OCI-LY8); and the human acute monocytic leukemia cell line (THP-1). We analyzed the cytokine spectrums in the supernatant of the cell lines mentioned above by using a multiplex suspension array (Bio-Plex Pro Human Cytokine 27-Plex Panel, Bio-rad). Then, we examined the cytokine spectrums in the supernatant of THP-1 cell lines which were cultured in the following groups of medium: A. Roswell Park Memorial Institute (RPMI) 1640 medium; B. RPMI 1640 medium with phorbol-12-myristate- 13-acetate (PMA, PMA can stimulate the activation of M1 polarized macrophages from THP-1 cell line); C. RPMI 1640 medium mixed with supernatants from NK/TCL cell lines; D. RPMI 1640 medium mixed with supernatant from the OCI-LY8 cell line. To balance the existing cytokines secreted by lymphoma cells, a same sample of medium mixed with the corresponding supernatants from lymphoma cell lines were used for baseline concentration as controls, respectively, in group C and D. Results There were a variety of elevated cytokines detected in NK/TCL cell lines, including: IL-1ra, IL-4, IL-6, IL-8, IL-10, IL-9, IL-13, IP-10, MCP-1 and IFN-¦A. There were no IL-4, IL-10 or IL-13 detected in the supernatant of OCI-LY8 cell line. The morphology and cytokine concentration of THP-1 cell lines in different groups were showed in Figure 1 and Table 1, respectively. In group B, the concentration of IL-1b was significantly higher than group A; In group C, the concentration of IL-1ra, IL-10 and IL-6 were significantly higher than group A, while the concentration of IL-12 was significantly lower, and the concentrations in group C gradually increased (IL-12 decreased) with time prolonged, which indicated a M2 polarization. There was no significant difference in terms of cytokine concentration between group A and group D (Table 1). Table 1. The cytokine spectrum of THP-1 cell lines in different groups. Group A Group B Group C Group D p-value Control PMA NK-YS SNK-6 SNT-8 OCI-LY8 IL-1b 1.97 129.02 25.36 27.94 23.58 1.82 0.01 IL-1ra 20.42 154.24 162.19 130.58 158.22 19.24 IL-6 2.58 426.40 240.14 168.25 299.33 2.18 0.001 IL-10 1.35 2.80 20.58 5.85 6.37 1.32 IL-12 16.58 40.19 13.24 12.01 13.87 15.9 Download : Download high-res image (57KB) Download : Download full-size image Figure 1. The morphology of THP-1 cell lines in different polarization (200X). A. undifferentiated monocytes (Group A); B. M1 type macrophages (Group B) there were pseudopodia and phagocytosed particles in macrophages; C. M2 type macrophages (Group C). Conclusions Our study suggests that the NK/TCL cell lines can secrete various cytokines. Some of these cytokines could induce the differentiation from monocytes to M2 polarized TAM, and the key cytokines might be IL-4, IL-10 and IL-13. The interactions between NK/TCL and TAM may provide novel targets for treatment of NK/TCL. Disclosures: No relevant conflicts of interest to declare.

Backgroud Natural/killer T-cell lymphoma (NK/TCL) was previously named as lethal midline granuloma. Most patients presented with inflammatory symptoms such as fever and necrosis, which was associated with poor survival. It is still unclear what exactly the role of severe inflammation on the pathogenesis and development of NK/TCL, and there has been few report about the serum cytokines profile and its clinical significance in patients with NK/TCL. Patients and Methods From January 2005 to December 2012, the serums and paraffin embed tissues of 59 patients with NK/TCL in Sun Yat-sen University Cancer Center were collected. The Human Cytokine 25-Plex Panel kits (Invitrogen) were used to detect the serum level of the cytokines. And the real-time quantitative polymerase chain reaction (RT-PCR) was employed for detection of the plasma EBV-DNA copies. Results Overall, fifty-nine patients with NK/TCL were analyzed. There were 44 male patients. The median age was 39 years (range 10-69 years). There were 37, 5 and 17 patients diagnosed as stage I, II and IV disease, respectively. Forty-one patients had B symptoms and ten patients were diagnosed hemophagocytic syndrome. Fifty-eight patients were followed up, the median follow-up time was 25 (0.2¨C70) months. Comparing with the 12 normal control cases, there were high proinflammatory cytokines and chemokines detected in most patients with NK/TCL(without hemophagocytic syndrome, HPS), including, interleukin-6 (IL-6)(2.56 (0.00-22.29) pg/ml), IL-1ra(88.98 (0.00-1210.05) pg/ml),interferon-¦Ã(IFN-¦Ã)(2.78 (0.00-61.60) pg/ml), IP-10 (41.85 (0.00-1057.41) pg/ml),MIG(49.90 (0.00-799.00) pg/ml), IL-10(0.43 (0.00-13.48)pg/ml), sIL-2R(168.82 (0.00-1916.61) pg/ml),IL-15(0.00(0.00-443.76) pg/ml)(datum express the median serum concentration of cytokines and their range). The serum levels of these cytokines and chemokines mentioned above dramatically increased in 10 patients with hemophagocytic syndrome. The serum concentration of these proinflammatory cytokines and chemokines was notably higher in patients with fever, advanced stage, and microenvironment NF-kB alternative pathway (P52) activation. In patients with P52 activation, the concentration of these cytokine were higher in those plasma EBV-DNA copies ≥103copy/ml (P≤¼0.05). Multivariate analysis showed that the Korean Prognostic Index (KPI)(P=0.003), the serum concentration of IL-6 (P=0.035) and IL-2R(P=0.044) were independent prognostic factors. High serum IL-6 and IL-2R correlates with poor survival. Twenty-nine patients with serum IL-6 level≤3.97pg/ml, had better survival, with 68.0±8.8% of 3-year overall survival (OS); while 3-year OS of the higher serum IL-6 group was 21.3±8.1% (P=0.001). Conclusion ≤°Our study indicate that patients with NK/TCL had special serum proinflammatory cytokines and chemokines profile, and these cytokines were notably higher in those with fever, advanced stage, the NF-kB alternative pathway activation in microenvironment, and EBV-DNA copies ≥103copy/ml. Moreover, high serum concentration of IL-6 and IL-2R were correlated with poor survival. Disclosures: No relevant conflicts of interest to declare.