Study Of Clinical Prognosis Of Primary Gastric Diffuse Large B-cell Lymphoma Or Thyroid Lymphoma And Bioinformatics Analysis Of The Pathogenesis

Objective:Primary extranodal non-Hodgkin's lymphoma(EN-NHL)is a group of heterogeneous malignant tumors involving many organs and there are significant differences in tumor location,natural history,biological expression,prognostic factors and treatment options.Gastrointestinal tract is the most common site of EN-NHL,most commonly in the stomach,followed by the small intestine and ileocecum.Primary gastric diffuse large B-cell lymphoma(PG-DLBCL)is the most common type of lymphoma affecting the gastrointestinal tract,accounting for 39-58%.Although the early prognosis of PG-DLBCL is good,there are still a small number of patients who are at risk of recurrence and have a poor prognosis,which requires aggressive therapy,including chemotherapy,stem cell transplantation.In the immuno-oncology therapeutics era,the prognosis and predictive indicators of PG-DLBCL have attracted much attention.It has been reported that Helicobacter pylori negative,advanced Lugano stage and high serum lactate dehydrogenase(LDH)level are adverse prognostic factors of PG-DLBCL.The overall survival(OS)of PG-DLBCL depends on tumor characteristics and host-related factors.So,it is necessary to extensively explore other prognostic factors,such as host immune status and molecular markers.Thyroid is the most common endocrine organ in primary EN-NHL,but primary thyroid lymphoma(PTL)accounts for only 1-2%of EN-NHL.Autoimmune thyroiditis is considered as a risk factor for PTL and among patients with Hashimoto's thyroiditis(HT),the risk of developing PTL is 40-80 times higher than that of patients without thyroiditis.Early diagnosis contributes to better treatment of PTL and ensure that the success rate of treatment is improved without the radical surgery.The treatment strategy is based on histopathological classification,grading and disease staging.At present,the effective treatments of PTL are systemic chemotherapy(including biological agents)and local radiotherapy.In this study,the clinical characteristics of primary EN-NHL originated from gastric and thyroid were statistically analyzed to explore the prognostic factors of PG-DLBCL and PTL.Then,bioinformatics analysis was used to excavate biomarkers in the pathogenic process of PTL,so as to provide basis for early diagnosis and target treatment of PTL.Method:1.The detailed clinical data of 72 patients with newly diagnosed PG-DLBCL,who were admitted to the Department of Hematology of Shanxi Tumor Hospital from January2012 to December 2017,were collected and followed up by telephone.2.Immunohistochemical methods were used to detect the expression of CD10,Bcl-2,Bcl-6,MUM1 and Ki67 in 72 patients with PG-DLBCL.3.Lymphocyte subsets in 72 patients with PG-DLBCL were detected by flow cytometry.4.A variety of statistical methods were used to identify the independent prognostic factors affecting the survival of PG-DLBCL.5.The clinical informations of 39 patients with PTL treated in Shanxi Tumor Hospital from January 2010 to December 2018 were analyzed retrospectively,and the clinical indicators of PTL patients were analyzed for survival.6.The microarray dataset was downloaded from the GEO database,and the differentially expressed genes(DEGs)of DLBCL and HT were screened by GEO2R tool.7.GO annotation and KEGG pathway enrichment analyses of DEGs by the Web Gestalt database.8.PPI network construction and hub genes identification.9.The validation of the expression and survival assessment of the hub genes by using the online tool gene expression profiling interactive analysis(GEPIA).Result:1.The clinical characteristics of PG-DLBCLAmong the 72 patients with PG-DLBCL,38 cases were male(53%),34 cases were female(47%),the age of onset was 18-83 years old,and the median age was 54 years old.At the time of diagnosis,there were 31 cases(43%)of gastric antrum,38 cases(53%)of corpus and 3 cases(4%)of gastric fundus.Peripheral blood lymphocytes were less than 10×10⁹/L in 18 cases(26%),low albumin in 33 cases(46%),and CD4/CD8 ratio<1 in 38 cases(58%),?₂-macroglobulin(?₂-MG)elevated in 19 cases(26%),and LDH elevated in 20 cases(28%),Lugano stage II2?IV in 39 cases(54%),and International Prognostic Index(IPI)high-intermediate/high groups in 20 cases(28%).2.Pathomorphological and immunophenotypic characteristics:According to Hans classification,there were 22 cases(30%)of germinal center B cell(GCB)type and 35 cases(49%)of non-germinal center B cell(non-GCB)type.Immunohistochemistry showed that CD20 was expressed in all cases of lymphoma while the positive expression rates of CD10,Bcl-6,Bcl-2 and MUM1 were 32%(18/56),74%(34/46),65%(17/26),66%(35/53),respectively.The proliferation index(Ki67)>70%was in 57 cases(80%).Immunophenotypic survival analysis showed that the expression of CD10,Bcl-6 and MUM1 was not statistically significant in OS.3.Lymphocyte immunophenotype5 ml of venous blood was collected from PG-DLBCL patients at newly diagnosis.Using Multi TEST IMK kit to detect the number of CD4⁺,CD8⁺T lymphocytes in peripheral blood by flow cytometry and CD4/CD8<1 was in 38 cases(58%).4.Univariate and multivariate survival analysis of prognosis in patients with PG-DLBCL.Univariate analysis showed that the prognostic factors associated with poor survival were elevated LDH level,IPI score?3,high?2-MG level,and CD4/CD8<1.Multivariate analysis showed that CD4/CD8<1 was associated with poor prognosis.5.Clinical characteristics and survival analysis of patients with PTL.39 patients with PTL were mainly female,accounting for 82%.The age of onset was31-82 years old,and the median age was 59 years old.The median follow-up period was 49months(7?134 months).The pathological classification of all PTL patients was DLBCL not mucosa-associated lymphoid tissue(MALT)lymphoma.Patients with Lugano stage III?IV were in 11 cases(28%),and patients with IPI score?3 were in 20 cases(28%).According to Hans classification,patients with non-GCB type were in 16 cases(53%),ESR increased in18 cases(67%),LDH elevated in 25 cases(64%),?2-MG increased in 13 cases(33%),and hypothyroidism in 10 cases(31%).Survival analysis confirmed the prognostic factors of PTL.High?2-MG level patients have decreased OS compared with the normal groups(P=0.021);Moreover,IPI?3 scores significantly affected survival(P<0.001).Cell-of-origin(COO)classification,elevated erythrocyte sedimentation rate(ESR)level,elevated LDH,and HT status had no significant effect on poor prognosis.6.Two groups of gene expression profiles(GSE74266 and GSE29315)were downloaded from GEO database and 15 both upregulated DEGs of DLBCL and HT were screened by GEO2R.7.Employing the Web Gestalt database to conduct GO annotation and KEGG pathway enrichment analyses of the 15 both upregulated DEGs.GO function analysis included three parts:biological processes(BP),molecular functions(MF)and cellular components(CC).GO BP analysis revealed that the 15 upregulated DEGs were markedly enriched in the immune effector process,the response to other organisms,the external biotic stimulus-response and biotic stimulus-response.For GO MF analysis,the top significantly enriched term was the non-membrane spanning protein tyrosine kinase activity.The top four significantly enriched CC terms included lysosome,lytic vacuole,nuclear nucleosome,and the integral component of the nuclear inner membrane.KEGG pathway for both upregulated DEGs were graft-versus-host disease,transcriptional misregulation in cancer,cytosolic DNA-sensing pathway,and acute myeloid leukemia.8.The PPI network of DEGs was constructed by the STRING tool,and a total of 10Hub genes were identified,including IL10RA,IL6,STAT3,IL10,IL4,CXCL10,CXCR3,CCL5,GZMB,and PRF1 genes.9.The expression of 10 Hub genes was analyzed by GEPIA online tool and then survival analysis was carried out.The expression of CXCL10,CXCR3,IL6 and IL10 were significantly higher than those in normal tissues(P<0.05),but had no significant effect on the prognosis of DLBCL.Conclusion:1.The low CD4/CD8 ratio at newly diagnosis was associated with the poor prognosis of PG-DLBCL patients.Moreover,the CD4/CD8 ratio,which represented the host's immunity,was superior compared to other prognostic factors and may be useful in selecting patients for clinical immune-oncology therapeutics.2.Clinical prognostic analysis of PTL showed that IPI score?3 and high?₂-MG level were associated with worse prognosis of PTL,and systematic bioinformatics assessment showed that IL6,IL10,CXCL10 and CXCR3 genes might be new diagnostic targets during the process of HT to PTL.In addition,oxidative stress may participate in the process of lymphomagenesis via chronic inflammation.