Clinical Features And Prognosis Of Diffuse Large B-cell Lymphoma And The Significance Of NEK2 Expression

Background:DLBCL is the most common subtype of invasive non-Hodgkin’s lymphoma(NHL)in adults,its clinical classification is quite complicated,due to its high heterogeneity in histomorphology,molecular immunology,cytogenetics,and invasive sites.With the emergence of new targeted drugs in recent years,the prognosis of some patients has greatly improved.There are still some patients with disordered cellular regulatory mechanisms,including chromosomal instability(CIN),gene rearrangement and abnormal signal transduction pathways,they progressed to relapsed-refractory DLBCL,it seriously affects the long-term quality of life.Although the current IPI scoring system and the Hans classification,to some extent,can distinguish the different prognosis of disease,which make it possible for some patients to achieve a hierarchical diagnosis and treatment plan,but it is limited to relying on IPI scores and Hans classification,which simply considered from clinical features and the hierarchical subtype of pathology.it can no longer effectively evaluate the prognosis of patients with various genetic abnormalities.Therefore,in the future,it is great of necessary and urgency to explore new prognostic factors to perfect the pre-existing prognostic stratification system.NEK2 is one of the member of the NEKs family,which belong to the centrosome-associated kinase family-NIMA protein kinase.A large number of studies have confirmed that it is abnormally expressed in a variety of malignancies,and closely associated with the prognosis of the disease.Further studies found that NEK2 can participate in the formation and localization of spindle when they moving to two poles,by acting on the centrosome,play an important role in chromosome movements and regulate the chromosome separation during the process of cell mitosis.The abnormal expression of NEK2 can lead to mitotic disruption,which in turn causes CIN and the formation of aneuploidy,and then promotes genetics to expressing abnormal in cellular,consequently activated proto-oncogenes or inactivated the tumor suppressor genes and eventually lead to the occurrence and development of tumors.Therefore,the centrosome regulatory factors have become a hot spot topic for researchers in the field of cancer research in recent years.Great amount of studies had further shown that abnormal expression of NEK2 may cause changes in biological functions such as tumor cell proliferation and drug resistance,suggesting that NEK2 may become a potential target gene for the treatment of human malignant tumors,which is of great significance for improving the prognosis.Therefore,we retrospectively analyzed the clinical data of patients with DLBCL,and applied the IHC staining to detect the expression of NEK2 in DLBCL and LNRH patients,and explored its relationship with clinicopathological parameters and prognosis,providing experimental basis for the subsequent study on the mechanism of NEK2 acting in DLBCL.Objectives:1.Retrospectively analyze the clinical characteristics,efficacy and survival of DLBCL and related prognostic factors;2.Detect the expression of NEK2 in DLBCL and LNRH patients and analyze its relationship with clinical characteristics and prognosis.Methods:1.A total of 129 newly diagnosed patients with DLBCL treated at the First Affiliated Hospital of Nanchang University from August 2014 to August 2017 were collected.All cases were relatively completed with medical records and treated with CHOP or RCHOP chemotherapy regimens.Recording the basic information and clinical features of all patients before treatment,including age,gender,primary site,number of extra-nodal involvement lesions,B symptoms,ECOG score,serum LDH,IPI score,Ann Arbor clinical stage,pathological subtypes,Ki-67 index,bone marrow involvement,masses,and chemotherapy regimens,summarized the short-term efficacy of patients,assessed the prognostic factors.2.62 cases(newly diagnosed in our hospital from August 2014 to August 2017,confirmed by pathological IHC and archived paraffin blocks,no serious complications)were selected as the experimental group,patients with reactive hyperplasia served as controls.The expression of NEK2 protein in the pathological tissues of DLBCL and LNRH was detected by IHC.The expression of NEK2 protein was assessed according to the Fromowitz.and the correlation between this protein and the prognosis of DLBCL patients was evaluated.3.SPSS 25.0 statistical software was used for statistical analysis,chi-square test or Fisher exact test to analyze the relationship between NEK2 protein expression and clinicopathological data.Kaplan-Meier method was used to draw survival curves,and survival rate was compared by Log-rank test.Firstly,the single factor Cox regression analysis was used to find out the clinicopathologic factors that affected the prognosis of DLBCL patients.Then Cox proportional hazards model was used for multivariate analysis.P<0.05 was considered the difference between the two groups to be statistically significant.Results:1.Clinical characteristics and short-term efficacy analysis1.1 Clinical data: age≤ 60 in 93 patients,>60 in 36 cases,median age was 59 years;73 males and 56 females;primary intra-nodal in 26 cases,103 cases were primary extra-nodal patients;32 cases with B symptoms;76 cases of ECOG 0~1;53 cases of ECOG 2~4;70 cases of elevated serum LDH;64 cases of IPI scores 0~2,65 cases of IPI 3~5;Ann Arbor stage III~IV in 62 cases;54 cases of GCB type;86 cases with high-expression of Ki-67;30 cases with extra-nodal involvement lesions≥2;26 cases with bone marrow involvement;31 cases with huge tumors;71 patients treated with rituximab,58 patients were not treated with rituximab.1.2 The short-term efficacy of 129 patients with DLBCL: 79 patients achieved effective chemotherapy(CR + CRu + PR),the effective rate was 61.2%.Age,IPI score,Ann Arbor stage,bone marrow involvement,B symptoms,combination of rituximab chemotherapy or not,pathologic subtypes were statistically significant,P < 0.05.other clinical characteristics show no significant correlation with the short-term efficacy.1.3 Clinical data in 62 patients with DLBCL: 32 males and 30 females,;19 patients of aged ≤60 years,43 patients of aged >60 years;49 cases with primary extra-nodal site;17 cases with B symptoms;35 cases with ECOG 0~1;37 cases with elevated serum LDH;29 patients in IPI 0~2 and 33 patients with IPI 3~5;33 patients at Ann Arbor stage I~II,29 patients at stage III to IV;28 patients with of GCB type;48 cases with high-expression of Ki-67;13 cases with extra-nodal lesions ≥ 2;bone marrow involvement in 12 cases;huge tumors in 16 cases;41 cases were treated rituximab,21 patients were not treated with rituximab.2.Survival analysis of 129 patients with DLBCLThe overall median follow-up time was 23 months,82 cases survived,and 47 cases died.The 3-year OS rate was 63.6%.The MS was not reached.2.1 Univariate survival analysis showed that age ≤ 60 years,serum LDH,IPI score 0~2,GCB type,Ki-67≤70%,and treated with rituximab have longer Survival time,P <0.05.Other clinical parameters were not significantly associated with the patient’s survival.2.2 Multivariate survival analysis suggested that age,serum LDH and IPI scores are independent prognostic factors of DLBCL,age >60 years,elevated serum LDH and IPI score 3~5 show more worse prognosis,the difference was statistically significant,P <0.05.3.Comparison of expression of NEK2 in DLBCL and LNRH3.1 There were 49 cases with positive NEK2 protein expression(+~+++)in 62 cases,13 cases with no expression(-).There were 28 cases with low expression and 34 cases with high expression.For LNRH,NEK2 was weakly positive(+)in 9 cases and negative in 11 cases.The total positive rate of NEK2 protein expression was significantly higher in the DLBCL group than LNRH group,P<0.05.4.Correlation analysis of NEK2 expression in DLBCL with clinical characteristics and short-term efficacy of patients4.1 Correlation with clinical characteristics: the expression of NEK2 protein was correlated with age >60 years,with B symptoms,elevated serum LDH,IPI score 3~5 points,non-GCB type,huge masses,the difference between the two groups was statistically significant,P<0.05.The expression of NEK2 protein was not associated with other clinical parameters.4.2 Correlation with short-term efficacy: There were 41 patients with effective(CR+CRu+PR)in 62 patients with DLBCL and 21 patients with ineffective(SD+PD)patients,and the overall effective rate was 66.1%.The patients of positive NEK2 group(+~+++)was worse than negative(-)group,the difference between two groups was statistically significant,P=0.003.The short-term efficacy of the NEK2 protein high expression(++~+++)group was lower than the low-expression(-~+)group,and the difference was statistically significant,P=0.016.5.Survival Analysis of 62 Patients with DLBCLThe median follow-up time was 24 months,45 patients survived,17 patients died,the 3-year OS rate was 65.6%,MS was not reached.5.1 Univariate survival analysis showed that patients with elevated serum LDH,IPI score 3~5,not treated with rituximab and high expression of NEK2 protein had a lower survival rate,with a statistically significant difference,P<0.05.There was no significant difference in correlation with other clinical parameters.5.2 Multivariate survival analysis suggests that both high expression of NEK2 and IPI score were independent prognostic factors for DLBCL patients,P<0.05.Conclusion:1.Newly dignosed DLBCL with age ≤ 60 years,IPI score 0-2,Ann Arbor stage I-II,no bone marrow invasion,no B symptoms,low expression of Ki-67,treated with rituximab and GCB type,the short-term efficacy is better.Age,IPI score,and serum LDH were independent prognostic factors for DLBCL.2.The high expression of NEK2 was associated with age >60 years,with B symptoms,elevated serum LDH,IPI score 3~5,non-GCB type and with huge masses,and patients with high expression are less effective than those with low expression.3.High expression of NEK2 and IPI score can identified DLBCL patients with poor prognosis.