Association Between Polymorphism Of Diabetes Susceptibility Gene IGF2BP2 And The Risk Of Diffuse Large B-cell Lymphoma

Diffuse large B cell lymphoma(DLBCL)is one of the most common non-Hodgkin’s lymphomas and represents a group of malignant tumors with significant heterogeneity in clinical presentation and prognosis.A multi-center study in China showed that DLBCL in China accounted for 45.8%of the total non-Hodgkin’s lymphoma population and 40.1%of all lymphomas.More importantly,viral infection,environmental pollution,aging population and other possible factors are the high incidence of lymphoma.Diabetes mellitus is a metabolic syndrome caused by the combined effects of environmental and genetic factors,the main manifestation of which is chronic hyperglycemia.The number of population with diabetes is about 366 million worldwide,and the World Health Organization evaluates that the amount of population combine with diabetes will reach 552 million by 2030.The study data indicated the prevalence of diabetes in women and men over 20 years old was8.8%and 10.6%,respectively.The prevalence of pre-diabetes in the same period was 15.5%.According to the results of the survey,the number of diabetics in China will reach more than 92 million,and the number of pre-diabetes is more than 148 million.With the rapid development of society and economy,people’s living standards are also improving,and the population begins to enter the age of aging.With these changes,the incidence of lymphoma and diabetes is gradually increasing.In the world,China has become one of the fastest increasing rates of diabetes prevalence.Lymphoma rates have nearly doubled worldwide over the past two decades.Numerous studies had shown that patients with diabetes,especially type 2 diabetes,were prone to multiple malignant tumors,which affected the prognosis of patients and increased the risk of death in patients with malignant tumors.Metformin is the first-line drug for patients with type 2 diabetes.The anti-hyperglycemic effect of metformin is mediated by reducing gluconeo-genesis,glucose absorption and hepatic glucose production.Metformin can reduce insulin resistance by increasing peripheral glucose uptake.With the advent of aging society,the incidence of DLBCL and diabetes is increasing day by day,and more DLBCL patients with diabetes can be seen in clinical work.Many studies had shown that the use of metformin in T2DM patients could reduce cancer incidence and cancer-related mortality.However,there were few studies on whether metformin can improve prognosis in DLBCL population.The insulin-like growth factor 2 m RNA-binding protein 2(IGF2BP2)gene is located on chromosome 3 and is a member of the insulin-like growth factor 2 m RNA-binding protein family.IGF2BP2 can bind to insulin-like growth factor 2(IGF2)m RNA,promote its translation,and affect the function of insulin-like growth factor family members.At the same time,IGF2BP2 can also affect cell differentiation,proliferation through various ways,and play an important role in the occurrence and development of cancer.Multiple studies had shown that IGF2BP2 SNPs rs4402960,rs1470579,rs11705701 and rs9826022 were susceptibility genes for type 2 diabetes.IGF2BP2 was also associated with insulin resistance,lipid metabolism,and tumor development.Studies had shown that diabetes was an important risk factor affecting the survival of DLBCL.Type 2 diabetes significantly reduced the overall survival and progression-free survival of DLBCL patients,and patients had a higher risk of death.In this study,DLBCL population was taken as the research object to study whether hyperglycemia affected the prognosis of DLBCL population,and whether metformin,a hypoglycemic drug,had a protective effect on DLBCL population.Furthermore,the correlation between the polymorphism of IGF2BP2 rs4402960,rs1470579,rs9826022 and rs11705701 and the risk of DLBCL was further explored.Part One Clinical features and prognosis of diffuse large B-cell lymphoma patients complicated with hyperglycemiaObjective:To discuss clinicopathological features and prognosis of patients with DLBCL and hyperglycemia.Method:1767 patients with definite pathological diagnosis of DLBCL in the Department of Hematology,the Fourth Hospital of Hebei Medical University were gathered from January 1,2010 to June 1,2020.Age,sex,height,body weight,family history,stage,presence or absence of B symptoms,lactate dehydrogenase(LDH),international prognostic index(IPI)score,clinical diagnosis,β2 microglobulin,immunohistochemistry and compli-cations were collected.The follow-up was conducted by telephone of the follow-up center of our hospital,outpatient review and inpatient review,and the deadline of follow-up was August 15,2021.The main observed endpoints were progression-free survival(PFS)and overall survival(OS).SPSS 26.0software was used for statistical analysis.Cox regression risk models were used in variable analysis to evaluate independent risk predictors of DLBCL population.Kaplan-Meier method was used to draw the survival curve,P<0.05 considered statistically significant.Results:1.General clinical data of the patients:A total of 1767 DLBCL patients were included in this study.The patients were divided into hyperglycemia group and non-hyperglycemia group according to whether they were complicated with hyperglycemia,and 732 people were in hyperglycemia combine with DLBCL group.The study found that DLBCL patients aged>60years had hyperglycemia than those aged≤60 years(P<0.001).Patients with hyperglycemia had higher BMI,stage,IPI score,LDH level,and comorbidities(all P<0.05).2.Baseline clinical characteristics of DLBCL patients in primary hyperg-lycemia group,secondary hyperglycemia group and non-hyperglycemia group:There were 732 patients in DLBCL combined with hyperglycemia(including466 patients in DLBCL combined with primary hyperglycemia,266 patients in DLBCL combined with secondary hyperglycemia),and 1035 patients in DLBCL combined with non-hyperglycemia.Compared with secondary hyperglycemia,the primary hyperglycemia group had higher age,higher expression rates ofβ2 microglobulin and C-MYC(all P<0.05).Compared with non-hyperglycemia group,patients with secondary hyperglycemia were more likely to have higher age,BMI,stage,IPI score,LDH level,and comorbidities(all P<0.05).3.Cox univariate and multivariate analysis evaluated the influencing factors of progression-free survival in DLBCL population:Cox univariate analysis showed that hyperglycemia,age,sex,BMI,stage,IPI score,B symptoms,pathological source,LDH level,β2 microglobulin,BCL-2 and C-MYC high expression were correlated with PFS in DLBCL population(all P<0.05).Cox multivariate analysis showed that hyperglycemia,age>60 years,BMI≥24kg/m~2,stage III-IV,IPI score 3-5,B symptoms,high LDH level,highβ2 microglobulin level,extranodal involvement,and BCL-2 positive were all associated with PFS in DLBCL population(all P<0.05).4.Cox univariate and multivariate analysis evaluated the influencing factors of overall survival in DLBCL population:Cox univariate analysis showed that hyperglycemia,age,BMI,stage,IPI score,B symptoms,pathological source,LDH level,β2 microglobulin level,BCL-2 and C-MYC high expression were correlated with the OS of DLBCL population(all P<0.05).Cox multivariate analysis showed that hyperglycemia,age>60 years,BMI≥24kg/m~2,stage III-IV,IPI score 3-5,B symptoms,high LDH level,highβ2 microglobulin level,extranodal involvement,and BCL-2 positive were all associated with poor prognosis of OS in DLBCL population(all P<0.05).5.Survival analysis:The follow-up time ended on August 5,2021.The shortest follow-up time was 1 month,the longest was 281 months,and the mean follow-up time was 35.8±33.4 months.Compared with non-hypergly-cemia group,the hyperglycemia group had a poor prognosis,and the differences in PFS and OS were statistically significant(all P<0.001).Compared with secondary hyperglycemia,the prognosis was worse in the primary hyperglycemia(all P<0.001),and the secondary hyperglycemia also affected the PFS and OS of patients compared with the non-hyperglycemia group(all P<0.001).Summary:1.DLBCL patients with hyperglycemia had higher age,higher BMI,IPI score 3-5,stage III-IV,higher LDH level and comorbidities.2.Hyperglycemia,age>60 years,BMI≥24kg/m~2,stage III-IV,IPI score3-5,B symptoms,high LDH level,highβ2 microglobulin level,extranodal involvement and BCL-2 positive associated with PFS and OS in DLBCL population.3.Hyperglycemia affected the survival time of DLBCL population(PFS:HR=1.41,95%CI,1.16-1.70,P<0.001,OS:HR=1.33,95%CI,1.09-1.61,P=0.004).4.This study suggested that secondary hyperglycemia also affected the prognosis of DLBCL population.Compared with the secondary hypergly-cemia group,the prognosis was worse in the primary hyperglycemia group combined with DLBCL.Part Two Metformin can improve the prognosis of diffuse large B-cell lymphoma with hyperglycemiaObjective:To investigate whether metformin can improve the prognosis of DLBCL population.Method:There were 732 patients diagnosed as DLBCL combined with hyperglycemia in the department of hematology,the Fourth Hospital of Hebei Medical University from January 1,2010 to June 1,2020,all of whom were pathologically confirmed to meet the diagnostic criteria of DLBCL.Data collected included age,sex,height,weight,pathological diagnosis,Ann Arbor stage,presence or absence of B symptoms,international prognostic index(IPI),lactate dehydrogenase(LDH),β2 microglobulin,blood glucose levels and hypoglycemic drugs.The metformin group refers to the hypoglycemic drug metformin used for at least 6 months,and the non-metformin group refers to the group that never used metformin or used metformin for less than 6 months.Follow-up was conducted by telephone of our hospital’s follow-up center,outpatient review and inpatient review.The deadline for follow-up is August15,2021.The primary study endpoints were progression-free survival(PFS)and overall survival(OS).Data were statistically analyzed by SPSS 26.0software,baseline clinical features of patients were analyzed by Chi-square test,and Cox regression risk model was used for variable analysis to assess independent risk factors affecting the prognosis of DLBCL.Kaplan-Meier method was used to draw the survival curve.P<0.05 considered statistically significant.Results:1.Comparison of clinical data of 732 DLBCL patients:A total of 732patients were included in this study,all of whom met the diagnostic criteria of DLBCL and hyperglycemia.The study was divided into two groups:146patients(19.9%)in the metformin group and 586 patients(80.1%)in the non-metformin group.Compared with the non-metformin group,the IPI score of the metformin group was lower and the LDH value was mostly normal(P<0.05).2.Comparison of living status of 732 DLBCL patients combine with hyperglycemia:Age>60 years,BMI≥24kg/m~2,stage III-IV,IPI score 3-5,LDH value higher than the upper limit of the normal range,β2 microglobulin value higher than the upper limit of the normal range,B symptoms,primary hyperglycemia had worse prognosis(P<0.05).3.Cox univariate and multivariate analysis assessed the influencing factors of PFS in DLBCL patients with hyperglycemia:Cox univariate analysis indicated that non-metformin application,secondary hyperglycemia,age,BMI,stage,IPI score,B symptoms,LDH,β2 microglobulin were correlated with poor PFS in DLBCL patients(all P<0.05).Cox multivariate analysis showed that the no application of metformin,secondary hypergly-cemia and IPI 3-5 score were associated with poor prognosis of PFS in DLBCL population(all P<0.05).4.Cox univariate and multivariate analysis assessed the influencing factors of OS in DLBCL patients with hyperglycemia:Cox univariate analysis indicated that non-metformin application,secondary hyperglycemia,age,BMI,stage,IPI score,B symptoms,LDH,β2 microglobulin and hyperglycemia were correlated with poor OS of the DLBCL patients(all P<0.05).Cox multivariate analysis showed that non-metformin application,secondary hyperglycemia,BMI≥24kg/m~2,and IPI 3-5 score were associated with poor OS in DLBCL population(all P<0.05).5.Kaplan-Meier curve of DLBCL patients with hyperglycemia:The5-year OS in the metformin or non-metformin group was 66%,55%,respectively.The 5-year PFS in the metformin or non-metformin group was65%,53%,respectively.The median survival time was 77.83 months in the non-metformin group and 118.13 months in the metformin group.Kaplan-Meier curve showed that no application of metformin could reduce PFS and OS in DLBCL patients combine with hyperglycemia(all P<0.05).Summary:For DLBCL patients combine with hyperglycemia,the prognosis of those treated with metformin was significantly better than that non-metformin(PFS:HR=0.69,95%CI=0.49-0.96,P=0.028,OS:HR=0.68,95%CI=0.49-0.95,P=0.024).Therefore,in clinical work,metformin is preferred for hypogly-cemic treatment in patients with DLBCL complicated with hyperglycemia as long as there is no obvious contraindication.As for whether metformin can improve the prognosis of DLBCL,further studies are needed to confirm.Part Three Association between polymorphism of diabetes susceptibility gene IGF2BP2 and risk of diffuse large B-cell lymphomaObjective:To investigate the association between the polymorphism of IGF2BP2 rs4402960,rs1470579,rs9826022 and rs11705701 and the risk of DLBCL.Method:In this study,295 patients diagnosed with DLBCL in the Department of Hematology,the Fourth Hospital of Hebei Medical University from January 2018 to December 2021 were collected as the research objects,and 331 healthy people from the physical examination center were selected as the control group.All subjects had an empty stomach overnight,and about5m L of peripheral venous blood was extracted in the test tube containing EDTA anticoagulant in the morning of the next day.After the red blood cells were lysed,the whole blood genomic DNA was stored in the refrigerator at-80℃for extraction.Genomic DNA was extracted in strict accordance with the instructions and stored at-20℃.The polymorphism of IGF2BP2 gene was detected by PCR-LDR.The research data was analyzed using SPSS 26.0software.The Hardy-Weinberg equilibrium test was conducted for genotype distribution frequency of the study population by chi-square test.T-test or chi-square test were used to compare demographic characteristics and the differences in the distribution of IGF2BP2 genotypes between the case group and the control group.Logistic regression was used to evaluate the association of IGF2BP2 single nucleotide polymorphisms with risk and clinicopatholo-gical parameters of DLBCL,and age,sex and BMI were adjusted.P<0.05indicated statistical significance.Results:1.Comparative analysis of basic conditions between DLBCL group and the control group:the average age of DLBCL group was 61.58±13.25 years old,and the control group was 54.98±12.53 years old,with significant difference between the two groups(P<0.001).The average BMI of DLBCL group was 24.32±4.30kg/m~2and the control group was 25.18±3.78kg/m~2,there was a significant statistical difference between the two groups(P=0.008).2.Frequency distribution of alleles and genotypes of IGF2BP2 rs440-2960(G/T),rs1470579(A/C),rs9826022(A/G)and rs11705701(G/A)in DLBCL group and the control group:After adjusting for age,sex and BMI,IGF2BP2 rs4402960(G/T)GT(OR=1.54;95%CI,1.08-2.19,P=0.016),TT(OR=2.0;95%CI,1.09-3.68,P=0.026)genotype and T genotype carriers(GT+TT)(OR=1.62;95%CI,1.17-2.25,P=0.004)significantly increased the risk of DLBCL.IGF2BP2 rs1470579(A/C)AC(OR=1.55;95%CI,1.11-2.17,P=0.010),CC(OR=2.18;95%CI,1.17-4.06,P=0.014)genotype and C geno-type carriers(AC+CC)(OR=1.64;95%CI,1.19-2.26,P=0.002)signifi-cantly increased the risk of DLBCL.Neither IGF2BP2 rs9826022(A/G)nor IGF2BP2 rs11705701(G/A)increased the risk of DLBCL.3.Hierarchical analysis of clinical data of IGF2BP2 rs4402960(G/T)in DLBCL group:Patients with IGF2BP2 rs4402960(G/T)GT+TT genotype showed higher age(age>60 years),lower BMI(BMI<24kg/m~2),and higher IPI score(3-5 points),later clinical stage(stage III-IV)(all P<0.05).4.Hierarchical analysis of clinical data of IGF2BP2 rs1470579(A/C)in DLBCL group:Patients with IGF2BP2 rs1470579(A/C)AC+CC genotype showed higher age(age>60 years),lower body weight(<24kg/m~2),later clinical stage(stage III-IV),non-germinal center B cell,and BCL-6 positive(all P<0.05).5.Hierarchical analysis of clinical data of IGF2BP2 rs9826022(A/G)in DLBCL group:Patients with IGF2BP2 rs9826022(A/G)AG+GG genotype showed higher age(age>60 years),later clinical stage(stage III-IV),higher IPI score(3-5 points),B symptoms and BCL-6 positive(all P<0.05).6.Hierarchical analysis of clinical data of IGF2BP2 rs11705701(G/A)in DLBCL group:Patients with the IGF2BP2 rs11705701(G/A)AG+AA genotype usually showed B symptoms(P<0.05).Summary:1.The age of patients in the DLBCL group was significantly higher than that in the control group,suggesting that higher age was a risk factor for DLBCL patients.The BMI in the DLBCL group was significantly lower than that in the control group,which may be related to weight loss after malignant tumors.2.In this study,we found that the polymorphism of IGF2BP2 rs4402960gene was associated with the occurrence and development of DLBCL.After adjusting for age,sex and BMI,GT(OR=1.54;95%CI,1.08-2.19,P=0.016),TT(OR=2.00;95%CI,1.09-3.68,P=0.026)and T genotype carrying(GT+TT)(OR=1.62;95%CI,1.17-2.25,P=0.004)significantly increased the risk of DLBCL.IGF2BP2 rs4402960 GT+TT genotype was associated with age>60years,BMI<24kg/m~2,stage III-IV,and IPI score 3-5 points.3.We also found that the IGF2BP2 rs1470579 polymorphism was associated with the development of DLBCL.After adjusting for age,sex and BMI,AC(OR=1.55;95%CI,1.11-2.17,P=0.010),CC(OR=2.18;95%CI,1.17-4.06,P=0.014)and C genotype carrying(AC+CC)(OR=1.64;95%CI,1.19-2.26,P=0.002)significantly increased the risk of DLBCL.The IGF2BP2rs1470579 AC+CC genotype was associated with age>60 years,BMI<24kg/m~2,stage III-IV,non-germinal center B cell,and BCL-6 positive.Conclusions:1.Hyperglycemia affected survival time in DLBCL patients(PFS:HR=1.41,95%CI,1.16-1.70,P<0.001,OS:HR=1.33,95%CI,1.09-1.61,P=0.004).Secondary hyperglycemia also affected the prognosis of DLBCL population,Compared with them,the prognosis of the group of DLBCL combined with primary hyperglycemia was worse.2.For DLBCL patients with hyperglycemia,the prognosis of those who took metformin was significantly better than those who did not take metformin.3.This study also found that polymorphisms of the diabetes suscepti-bility gene IGF2BP2 rs4402960 and rs1470579 were associated with an increased risk of DLBCL.