Clinical Significance Of The Circulating Cell-free EBV DNA Copies For Patients With Extranodal Nasal-type NK/T-cell Lymphoma

Part I:Establishment and optimization of real-time polymerase chain reaction for detection of the circulating cell-free EBV DNA copies for patients with extranodal nasal-type NK/T-cell lymphomaObjective Real-time polymerase chain reaction(RT-PCR) is the most common method to detect the circulating cell-free EBV DNA copies for patients with extranodal nasal-type NK/T-cell lymphoma. The aim of this study is to establish and optimize a stable and standard RT-PCR for detection of the circulating cell-free EBV DNA copies for patients with extranodal nasal-type NK/T-cell lymphoma, and to ascertain the detected target gene and sample origin for further large-scale experiment.Methods The primers and probes for BamHI-W and Pol-1 genes were biochemically synthesized. Considering the plasm samples from 6 patients with extranodal nasal-type NK/T-cell lymphoma along with EBV-positive in the tumor tissue as the PCR (polymerase chain reaction) template, BamHI-W and Pol-1 gene fragment were expanded by PCR, purified and cloned to the plasmid vector of pGM-T. A standard plasmid was constructed and comfirmed by gene sequencing, then the standard curves spanned 5-logs with serial 10-fold dilutions containing 100 up to 104 and 103 up to 107 plasmid targets for RT-PCR were performed. The blood samples of patients with extranodal nasal-type NK/T-cell lymphomas and healthy control were collected, respectively. Using RT-PCR with taget gene BamHI-W and Pol 1, the cell-free EBV DNA copies in the plasm for 6 patients with extranodal nasal-type NK/T-cell lymphom were preliminarily detected and the efficacy comparison was analyzed. Healthy persons as negative control and blank control were done as well. Then, the cell-free EBV DNA copies in the plasm and serum for 30 patients with extranodal nasal-type NK/T-cell lymphoma (of these,25 with stage IE disease) were detected using BamHI-W gene. Finnally, the cell-free EBV DNA copies in the plasm and serum were ascertained and difference comparison was analyzed.Results BamHI-W and Pol 1 gene fragments were synthesized and comfirmed by gene sequencing. The BamHI-W gene was easier expanded, higher positivity than Pol 1 gene. The standard curves were constructed containing two different concentrations of plasmid targets. Linear correlation was excellent and the curve slope and expanded efficacy were under the standard area. Using RT-PCR with taget gene BamHI-W, preliminarily results showed the cell-free EBV DNA copies for all 6 patients can be detected, while undetectable for negative and blank control. Using the taget gene BamHI-W, the median cell-free EBV DNA copies in the plasm and serum for 30 patients with extranodal nasal-type NK/T-cell lymphom before radiotherapy were 1497 copies/mL and 1639 copies/mL (p=0.200), respectively, and the spearman correlation coefficient (rp) was 0.729 (p=0.000). The corresponding cell-free EBV DNA copies after radiotherapy were 115 copies/mL and 12 copies/mL (p=0.093), respectively, and rp was 0.497 (p= 0.005). For 25 patients with stage IE extranodal nasal-type NK/T-cell lymphom before radiotherapy were 1023 copies/mL and 1088 copies/mL(p=0.244), respectively, and rp was 0.771 (p=0.000). The corresponding cell-free EBV DNA copies after radiotherapy were both 0 copies/mL (p=0.109), and rp was 0.547 (p=0.005). The positive rate for 30 patients with extranodal nasal-type NK/T-cell lymphom before radiotherapy was a little higher in the plasm than that in the serum (83.3% vs 66.7%, p=0.136). The corresponding positive rate after radiotherapy were 43.3% and 46.7%, respectively (p= 0.795).Conclusions A stable and standard RT-PCR for detection of the circulating cell-free EBV DNA copies for patients with extranodal nasal-type NK/T-cell lymphoma had been established. BamHI-W gene was considered as the primary detected target gene. The cell-free EBV DNA copies in the plasm and serum for total and stage IE patients with extranodal nasal-type NK/T-cell lymphom pre- and post-radiotherapy had no significant difference with positive correlation and similar positive rate. The absolute positive rate of cell-free EBV DNA copies before radiotherapy in the plasm was higher than that in the serum. Plasm was considered as the primary selection for sample origin. All these data clearly indicated further large-scale samples experiment and invastigated the correlation between cell-free EBV DNA copies and clinical characteristics and prognosis for patients with extranodal nasal-type NK/T-cell lymphom. PARTâ…¡:Clinical significance of the circulating cell-free EBV DNA copies for patients with extranodal nasal-type NK/T-cell lymphomaObjective To explore the positivity, distribution, change patterns between pre- and post-radiotherapy of the circulating cell-free EBV DNA copies for patients with extranodal nasal-type NK/T-cell lymphoma and its correlation with clinical stage and prognosis.Methods Thirty patients newly diagnosed with extranodal nasal-type NK/T-cell lymphoma were prospectively reviewed. According to the Ann Arbor stage system, there were 25 patients with stage IE disease,4 with stageâ…¡E, and 1 with stage IVE. Radiotherapy was the primary treatment for patients with early stage disease. Among 30 patients,15 patients were treated with curative radiotherapy alone,8 with radiotherapy followed by chemotherapy and 7 with chemotherapy followed by radiotherapy with or without chemotherapy. Totally, tweenty-three patients treated with initial radiotherapy and 7 with initial chemotherapy. Real-time polymerase chain reaction (RT-PCR) using target BamHI-W gene was performed to detect the circulating cell-free EBV DNA copies for these patients. The positivity, distribution, change patterns between pre- and post-radiotherapy of the circulating cell-free EBV DNA copies and its correlation with clinical stage and prognosis were ananlyzed.Results The median follow-up was 21 months (range:4-35 months). The 2-year overall survival (OS) and progression-free survival (PFS) rates for all patients were 78.6% and 55.1%, respectively. The corresponding OS and PFS for 25 patients with stage IE were 83.8% and 63.0%, respectively. The median cell-free EBV DNA copies in the plasm and serum for all patients before radiotherapy were 1497 copies/mL and 1639 copies/mL, respectively. The median cell-free EBV DNA copies for patients with stage IIE-IVE was higher than those for patients with stage IE (2062 copies/mL vs 1023 copies/mL for plasm, p=0.043,2673 copies/mL vs 1088 copies/mL, p=0.068 for serum). Of patients with stage IE disease, the median cell-free EBV DNA copies for a subgroup of patients with T3/T4 tumor stage (n=15) was higher than that with T1/T2 (n=10) (2852 copies/mL vs 206 copies/mL for plasm, p=0.047; 4654 copies/mL vs 131 copies/mL for serum, p=0.066). The 2-year OS for patients with primary radiotherapy was superior to those with primary chemotherapy (87.5% vs 44.4%, p=0.009). The 2-year PFS for patients achieved complete remission after treatment was superior to those who did not achieved (59.0% vs 0%, p=0.002). The 2-year OS for all patients with equal to or less than 500 copies/mL of the median cell-free EBV DNA copies before radiatherapy was superior to those with more than 500 copies/mL (100% vs 66.3% for plasm, p=0.015; 100% vs 63.2%, p=0.064 for serum). The 2-year OS for patients with 0 copies/mL of the median cell-free EBV DNA copies in the plasm after radiatherapy was superior to those with more than 0 copies/mL (93.8% vs 58.9%, p=0.058). The corresponding 2-year PFS in the serum was 73.8% and 35.0%, respectively (p=0.009). For patients with stage IE disease, patients with equal to or less than 500 copies/mL of the median cell-free EBV DNA copies before radiatherapy showed a trends toward better OS compared with those with more than 500 copies/mL (2-year OS,100% vs 73.3% for plasm, p=0.089; 100% vs 71.4%, p=0.063 for serum). The 2-year OS for patinets with 0 copies/mL of the median cell-free EBV DNA copies in the plasm after radiatherapy was superior to those with more than 0 copies/mL (100% vs 62.3%, p=0.013). The corresponding 2-year PFS in the serum was 76.9% and 50.0%, respectively (p=0.044).Conclusions The cell-free EBV DNA copies for patients with extranodal nasal-type NK/T-cell lymphoma in the plasm and serum before radiotherapy are strongly correlated with clinical Ann Arbor stage and T stage. The cell-free EBV DNA copies both pre- and post-radiotherapy in the plasm and serum are very important prognostic factors for patients with extranodal nasal-type NK/T-cell lymphoma, even for patients with stage IE disease. PART III:Clinical features and treatment outcome of extranodal nasal-type NK/T-Cell lymphoma in children and adolescentsObjective Extranodal nasal-type NK/T-cell lymphoma in children and adolescents is very rare. The aim of this study is to investigate the clinical features, prognosis and treatment outcome in these patients.Methods Thirty-seven patients were retrospectively reviewed. There were 19 patients with stage IE,14 with stageâ…¡E,2 with stageâ…¢E, and 2 with stage IVE. Of 37 patients, 19 patients were treated with initial radiotherapy with or without chemotherapy, and 18 patients with chemotherapy followed by radiotherapy with or without chemotherapy. Among the patients with stage IE and HE disease,19 patients were treated with initial radiotherapy with or without chemotherapy, and 14 patients with chemotherapy followed by radiotherapy. Four patients with stageâ…¢E and IVE disease received primary chemotherapy and additional radiation of the primary tumor.Results Children and adolescents with extranodal nasal-type NK/T-cell lymphoma usually presented with early stage, low frequency of B symptoms, good performance, low-risk age-adjusted international prognostic index (aalPI), chemoresistance, and radiosensitivity. The complete remission (CR) rate after initial radiotherapy was 73.7%, which was significantly higher than 16.7% after initial chemotherapy (p=0.002). The 5-year overall survival (OS) and progression-free survival (PFS) rates for all patients were 77.0% and 68.5%, respectively. The corresponding OS and PFS for stage IE and HE were 77.6% and 72.3%, respectively. The prognosis of the patients with complete remission (CR) was significantly better than those without CR. The 5-year OS and PFS rates of the patients with CR and those without CR were 84.8% and 30%(p=0.006), 77.3% and 0% (p=0.000), respectively.Conclusions Children and adolescents with early stage extranodal nasal-type NK/T-cell lymphoma treated with primary radiotherapy had a favorable prognosis. CR rate after treatment was an important prognostic factor.