| Objective:To evaluate the value of serum amyloid protein A protein(SAA)and misfolded transthyretin protein(TTR)as serum marker predicting relapsed/refractory diffuse large B-cell lymphoma(R/R-DLBCL)for early diagnosis of relapsed/refractory DLBCL.The new,specific and sensitive serum marker provide the basis for optimizing diagnosis and treatment strategies and lay the foundation for the discovery of new protein therapeutic targets and the development of new targeted drugs.Methods:1Isolation and identification of specific serum proteins of relapsed/refractory DLBCL and clinical value studies:60 patients included in the study who were hospitalized in the Department of Lymphoma in Shanxi DA YI Hospital from March 2013 to September 2015.Among them 30 R/R-DLBCL patients with complete clinical data were in research group,20remission/stabilization DLBCL patients and 10 chronic lymphadenitis patients as were in control group.10 cases with healthy Physical examination were in negative control group.Before treatment,3 ml of fasting serum was collected from the reaserch object and SELDI technique was used to search for specific protein fingerprints of relapsed/refractory lymphomas;Tris-Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis(Tris-Tricine-SDS-PAGE)was used to isolate the serum protein,determine and compare serum peptides.The shotgun-LTQ-MS method was used to identify the protein of the differential bands.The bioinformatics technique was used to identify the protein and determine the structure and function of the protein.The relationship between the value of high expression of SAA,misfolded TTR protein and the clinical/pathological features in R/R-DLBCL patients were statistically analyzed using SPSS21.0 software.The chi-square test was used to analyze clinical count data,Kaplan-Meier curve was used to survival analysis,and Log-Rank test was used to compare univariate survival differences(P<0.05 was considered significant).2Research in the predictive value of relapsed/refractory DLBCL specific serum proteins SAA and TTR:From September 2016 to December 2017,72 DLBCL patients who were hospitalized in the Department of Lymphoma in Shanxi DA YI Hospital.The patients were received 3 ml of fasting serum before treatment.The serum SAA and TTR levels were determined by enzyme-linked immunosorbent assay.The efficacy of follow-up after R-CHOP/R-EPOCH chemotherapy was evaluated:43(43/72)patients with relapsed/refractory DLBCL and 29(29/72)patients with remission/stabilization DLBCL.Statistical analysis was performed using SPSS 21.0 software.Measured data were expressed as mean±standard deviation((?)±s).Variance analysis was performed for data that matched the normal distribution or Variance.Independent-Sample T Test was used.The data satisfying a normal distribution was analyzed using a non-parametric test with two independent samples(Mann-Whitney U rank-sum test);risk factors were analyzed with binary logistic regression analysis(P<0.05 was considered statistically significant).The ROC curve was plotted and the area under the curve was calculated using the Parabola estimation method.The accuracy of each index was compared,and the index with the highest prediction accuracy was selected.The sensitivity,specificity,and the Youden index were calculated at the points,and the largest possible index was found.Cut points as cutoffs were found.Results:1Isolation and identificationof the specific serum proteins of relapsed/refractory DLBCL.and clinical value studies:1.1 5 different bands were detected in 30 R/R-DLBCL patients by Tris-Tricine-SDS-PAGE compared with remission/stabilization DLBCL patients,chronic lymphadenitis,and healthy controls.2 high expression bands:A band is a molecular weight of about 12000Da;B band is a molecular weight of about 14000Da;3 low expression bands:C band is a molecular weight about 10000 Da;D band is a molecular weight of about 15000-20000Da;E band is a molecular weight of about 15000Da.1.2 A band and B band were weakly or not expressed in the remission/stabilization DLBCL patients,chronic lymphadenitis group and healthy group.A band is identified as serum amyloid A(SAA1/2),B band is identified as misfolded Transthyretin(TTR)by The shotgun-LTQ-MS method and the bioinformatics technique.1.3 Clinical and pathological features of R/R-DLBCL patients with high expression of SAA and misfolded TTR protein were analyzed:High expression of SAA protein(SAA~+)was significantly associated with B-symptoms and high level of LDH in R/R-DLBCL patients(The P values were 0.02,0.011 respectively);SAA~+was associated with non-GCB(Φcorrelation coefficient was0.390),not associated with C-myc,bcl-2,bcl-6,Ki67,double/triple expression.High expression of TTR protein(TTR~+)was significantly associated with high level of LDH and extranodal lesion in R/R-DLBCL patients(The P values were 0.028,P=0.003 respectively);TTR~+was associated with C-MYC expression and non-GCB(Φcorrelation coefficient were 0.305,0.385 respectively),not associated with BCL-2,BCL-6,Ki67,double/triple expression.The high expression of SAA and TTR protein(SAA~+TTR~+)was significantly associated with extranodal lesion,high level of LDH,and NCCN-IPI scores in patients with R/R-DLBCL(The P values are 0.017,P=0.017,P=0.004 respectively);SAA~+TTR~+correlates with the patient’s non-GCB(Φcorrelation coefficient are 0.538),not associated with BCL-2,C-MYC,BCL-6,Ki67,double/triple expression.1.4 Survival analysis:The survival time of SAA~+group,TTR~+group and SAA~+TTR~+group was shorter than that of the negative group.Statistical analysis were showed significant differences,The P values are P=0.001,P=0.034,P=0.003respectively.2Research in the predictive value of relapsed/refractory DLBCL specific serum proteins SAA and TTR:2.1 The SAA and TTR levels of the first diagnosed DLBCL group were20.18±6.70μg/mL and 80.34±33.56μg/mL respectively,which were significantly higher than those of the healthy control group.Statistically significant differences were observed(P<0.01).2.2 The efficacy after 6 cycles of chemotherapy:In 72 patients,43 were relapsed/refractory group(43/72),29 were remission/stabilization group(29/72);in which 15 cases were complete remission CR(15/29),7 cases were partial remission PR(7/29),7 cases were stable disease SD(15/29).After treatment,TTR levels were significantly higher in the relapsed/refractory group than in the remission/stable group,with statistically significant differences(The P values is 0.003);area under the TTR curve(AUC)was 0.707 in Yoden.The index was 0.492 and the cut-off value was66.63μg/m L.The sensitivity was 0.837 and the specificity was 0.655.There was no significant difference in SAA levels between the relapsed/refractory group and the remission/stabilization group(The P values is 0.897),AUC is 0.511;2.3 The level of combining SAA and TTR was significantly different between the relapsed/refractory group and the remission/stabilization group.The AUC was 0.704,and the Yonden index was 0.481.The sensitivity was 0.791.The specificity was 0.690.Conclusion:1Both SAA and misfolded TTR were poor prognosis of R/R-DLBCL.SAA and misfolded TTR were significantly High expression in R/R-DLBCL;SAA and misfolded TTR were significantly associated with high level of LDH and non-GCB types;SAA was significantly associated with B symptoms;Misfolded TTR was significantly associated with C-MYC expression and extranodal lesions.2 Both SAA and TTR are highly expressed in the newly diagnosed DLBCL:Serum TTR in newly diagnosed DLBCL patients is valuable for predicting patient problely progression to relapse/refractory DLBCL at the point of>66.63μg/mL.The sensitivity is 83.7%and the specificity is 65.5%. |
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