| Objective: Real-time fluorescence PCR was used to quantify circulating cell-free DNA(cf-DNA)and to observe the changes in cf-DNA concentration and integrity before and after chemotherapy in patients with primary diffuse large B-cell lymphoma(DLBCL),and to analyze The relationship between cf-DNA levels and patient outcomes.The correlation between clinical characteristics such as age,gender,IPI score,NCCN-IPI score,Ann Arbor stage,presence of systemic symptoms,ECOG physical status score,lactate dehydrogenase level,Hans immunophenotyping and number of extra-nodal involvement and cf-DNA concentration and integrity was also investigated.Methods: Plasma samples collected from 40 patients with primary DLBCL were analyzed.Plasma cf-DNA was determined by quantitative PCR(q PCR)by amplifying ALU sequences,and cf-DNA integrity was calculated as the ratio of q PCR results(ALU247/115).The correlation between treatment efficacy and plasma cf-DNA concentration and integrity was monitored in 29 paired patients out of 40 patients at the midterm efficacy assessment and then in 9 paired patients at the final efficacy assessment.Results: 1.Changes in cf-DNA concentration and integrity before and after treatment in the remission group: 29 DLBCL patients were grouped and analyzed according to the results of the midterm efficacy assessment(3-4 courses of treatment).17 cases had a median ALU247 fragment concentration of 61.44 ng/ml after treatment,which was lower than 76.76(44.66,116.04)before treatment,and the difference was not statistically significant The median value of ALU115 fragment concentration after treatment was 95.49ng/ml,which was higher than 91.21ng/ml before treatment,and the difference was not statistically significant;the median value of cf-DNA integrity after treatment was 0.62,which was lower than 0.77 before treatment,and the difference was statistically significant(P=0.015<0.05).2.Changes in cf-DNA concentration and integrity before and after treatment in the refractory group: 29 DLBCL patients were grouped and analyzed according to the results of the mid-term efficacy assessment(3-4 courses of treatment).12 patients in the refractory group,the median value of ALU247 fragment concentration after treatment was 96.10 ng/ml,which was higher than that before treatment 78.98 ng/ml,and the difference was not statistically significant;after treatment The median value of ALU115 fragment concentration was 116.00ng/ml after treatment,which was lower than141.86ng/ml before treatment,and the difference was not statistically significant;the median value of cf-DNA integrity was 0.71 after treatment,which was higher than 0.56 before treatment,and the difference was statistically significant(P=0.009<0.05).3.Changes in cf-DNA concentration and integrity in the remission group at midterm and final chemotherapy assessments: 3 patients with PR and 2 patients with CR at midterm assessment,all 5 patients had achieved CR by the final assessment(6-8courses).median ALU247 fragment concentration increased from 55.42ng/ml to177.52ng/ml,with no statistically significant difference;median ALU115 fragment concentration increased from 70.57ng/ml to 206.43ng/ml,with no statistically significant difference.The median value of ALU115 fragment concentration increased from 70.57ng/ml to 206.43ng/ml,the difference was not statistically significant;the median value of cf-DNA integrity decreased from 0.79ng/ml to 0.74ng/ml,the difference was statistically significant(P=0.043<0.05).4.In the refractory group,the changes in cf-DNA concentration and integrity were compared with the last chemotherapy assessment: after 3 to 4 courses of treatment,3patients with PD and 1 patient with CR whose efficacy was evaluated for refractory treatment still had disease progression or recurrence when the efficacy was evaluated again after 6 to 8 courses of treatment.The median concentration of ALU247 fragment increased from 97.81ng/ml to 104.29ng/ml,and the difference was not statistically significant.The median concentration of ALU115 fragment increased from 92.26ng/ml to 77.19ng/ml without statistical significance.5.No matter in remission group or refractory group,ALU247 and ALU115 concentrations did not change regularly throughout treatment.cf DNA integrity was reduced in patients with disease remission throughout treatment,but the reduction degree of the last chemotherapy was less than that of the middle stage,and the residual level was basically maintained.cf-DNA integrity is consistently elevated in patients who present disease progression throughout treatment.6.Correlation between patients’ cf-DNA concentration and integrity and clinical characteristics: Analysis of DLBCL patients grouped by clinical characteristics showed that there was no significant correlation between ALU247 fragment concentration and other clinical characteristics of DLBCL patients.ALU115 fragment concentration correlated with IPI scoring system and NCCN-IPI scoring system(both P < 0.05).The cf-DNA integrity was correlated with gender,disease stage,IPI scoring system,and NCCN-IPI scoring system in DLBCL patients(all P < 0.05).7.The correlation between the concentration and integrity of cf-DNA of patients and immunohistochemistry: The immunohistochemistry of patients included BCL-2,BCL-6,Ki-67 and MUM-1,and the results were not significantly correlated with the concentration and integrity of ALU247 and ALU115 of patients(all P > 0.05).Conclusions: 1.The changes of cf-DNA integrity in DLBCL patients were consistent with the results of interim efficacy evaluation.The cf-DNA integrity was decreased in patients with remission(including CR/PR)and increased in patients with refractory disease(including SD/PD).This suggests that cf-DNA integrity can be used as an indicator for patient treatment evaluation.2.In DLBCL patients who had achieved disease remission(including CR/PR),the integrity of cf-DNA was reduced but the residual level was basically maintained at the last efficacy evaluation for further remission;DLBCL patients with refractory disease(including SD/PD)were still progressive at the last evaluation of efficacy,and cf-DNA integrity was again significantly elevated.The results showed that the integrity of cf-DNA was consistent with that of traditional imaging examinations such as PET-CT in the evaluation of clinical efficacy.3.There was no significant correlation between ALU247 concentration and clinical characteristics of DLBCL patients,while ALU115(actual concentration of cf-DNA)was correlated with IPI scoring system and NCCN-IPI scoring system.ALU247/115(cf-DNA integrity)was associated with gender,disease stage,IPI scoring system,and NCCN-IPI scoring system of DLBCL patients.4.The change of cf-DNA integrity level was consistent with the evaluation of efficacy,and the actual concentration of cf-DNA was strongly correlated with cf-DNA integrity in prognostic scoring systems such as IPI and NCCN-IPI.The results indicated that the combined use of cf-DNA concentration and integrity could improve the prediction accuracy of disease prognosis. |
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