| Research BackgroundDiffuse large B-cell lymphoma(DLBCL)has significant heterogeneity in genetics,histomorphology,immunophenotype and clinical manifestations,as well as in response to treatment and prognosis.The general growth mode of DLBCL is diffuse infiltration.The tumor destroys part or all of the lymph node structure.In rare cases,tumor cells will selectively invade the lymph sinus and be confined to the lymph sinus,which is called Sinusoidal Large B-cell Lymphoma(SLBCL).SLBCL is very rare,and there are only scattered reports at home and abroad.More in-depth molecular characterization research has not been reported yet.The prognosis of SLBCL is very poor,and there is no effective treatment at present.PurposeThis study is intended to first describe the morphological and immunohistochemical expression changes of SLBCL at the histological level,and then detect the characteristic molecular changes of SLBCL at the molecular level by second-generation sequencing,explore its possible pathogenesis and potential therapeutic targets,and provide theoretical basis and guidance for the treatment of SLBCL.The main purposes of this study include:1.To study the morphological and immunohistochemical characteristics of SLBCL;2.To study the molecular characteristics of SLBCL;3.To analyze the factors affecting the prognosis of SLBCL and compare the difference between SLBCL and DLBCL.MethodThe research methods of histopathology,immunohistochemistry(IHC),fluorescence in situ hybridization(FISH),Sanger sequencing and high-throughput sequencing were used in this study,mainly listed as follows:1.A multi-center SLBCL research team was established to collect SLBCL cases from hospitals across the country with strict standards and follow up their clinical information.2.Hematoxylin-Eosin(HE)staining was used to observe the morphological changes of SLBCL,and the expression of CD30,CD15,CD5,CD20,PD-L1,MYC,BCL2,BCL6 and Ki-67 in SLBCL was observed under IHC microscope.EBV-encoded smalRNA(EBER)probe was used to detect Epstein-Barr virus(EBV).3.FISH detection of SLBCL molecular changes: MYC,BCL2 and BCL6 use the two-color separation probe of Locus Specific Identifier(LSI);PD-L1/L2 uses LSI two-color amplification probe.4.Sanger sequencing was used to detect mutations in the 5-10 region of TP53 exon.5.Seven cases of SLBCL were sequenced in the second generation,and compared with the common DLBCL gene changes in COSMIC database,SLBCL gene differences were found.6.The prognostic factors of patients with SLBCL were analyzed by one-way ANOVA;The clinicopathological differences between SLBCL and DLBCL(n=40 cases)were compared.Result1.All 17 SLBCL tumors occurred in the lymph nodes,including 11 in the neck,2 in the groin,2 in the supraclavicular,1 in the submandibular and 1 in the axilla.The tumor cells in all cases showed an intra-sinus growth pattern,and the distribution of tumor cells in the lymphatic sinus ranged from 30% to 100%.2.IHC showed that all cases were strongly positive for CD20 and negative for CD5 and CD15.All 17 cases were negative for EBV RNA.According to Hans classification,16cases(94.1%)showed non-GCB immunophenotype,and 1 case(5.9%)showed GCB immunophenotype.Ki-67 ranges from 70% to 90% with a median of 80%.CD30 expression was positive in 16 patients,ranging from 23 to 90%;the median was 68%.13/15 cases(86.7%)were positive for BCL2,13/14 cases(92.9%)were positive for MYC,and 12/14 cases(85.7%)were both positive for BCL2 and MYC.10(62.5%)samples were positive for P53,of which 4 patients had diffuse strong positive expression.11/14 cases(78.6%)were PD-L1 positive,of which 5 cases(45.5%)had PD-L1 TPS scores ranging from 1 to 49%,and 6 cases(54.5%)had PD-L1 TPS scores ranging ≥50%.3.12/16 cases(75%)had MYC copy number amplification.BCL2 abnormalities were found in 12/16 cases(75%),including signal dissociation in 1 case and copy number amplification in 11 cases.BCL6 copy number amplification was found in 12 of 16(75%)patients,of which 6 patients had both segregated signal and copy number amplification.Seven patients had PD-L1/L2 abnormalities,including 4 with amplification and 3 with polyploidy.In addition,10/15(66.7%)patients had concurrent MYC,BCL2,and/or BCL6 abnormalities,and 9/15(60%)patients had triple abnormalities in MYC,BCL2,and BCL6.4.Exons 5-10 of TP53 were successfully amplified by PCR in 13 cases.Among them,8cases of TP53 gene missense mutations were detected,(1 case carried 2 gene changes,2 missense mutations occurred in Loop-L2 region,2 missense mutations occurred in Loop-L32,and 2 missense mutations occurred in Loop-L32.LSH helix motif region).The expression of P53 was correlated with TP53 missense mutation(r=0.854,P=0.0002< 0.001).5.The next-generation sequencing of 7 cases of SLBCL showed that the mutation frequency of TP53,MYD88,KMT2 D,CREBBP and PIM1 increased compared with the common DLBCL gene changes in the COSMIC database.6.Univariate analysis showed that the OS of patients with IPI score greater than or equal to 4 points and MYC,BCL2 and/or BCL6 abnormalities at the same time was significantly lower than that of negative patients.7.Compared with ordinary DLBCL patients,SLBCL patients were more likely to have B symptoms,high tumor stage,high IPI score and elevated serum LDH level(P<0.05).SLBCL cases were mostly non-GCB immunophenotype and expressed CD30,MYC,BCL2 and PD-L1(P<0.05).SLBCL more frequently had TP53 mutations and were more likely to have MYC,BCL2 and/or BCL6 abnormalities(P<0.05).The OS rate of SLBCL patients was significantly lower than that of ordinary DLBCL patients(P<0.001).Conclusions1.SLBCL exhibits many fundamentally different genetic changes and biological characteristics from ordinary DLBCL.The majority of SLBCL showed non-GCB immunophenotype,high expression of CD30,PD-L1,MYC and BCL2,high frequency of TP53 mutation,more prone to MYC,BCL2 and/or BCL6 abnormalities,PD-L1/L2 amplification and/or or increased frequency of polyploidy.2.Second-generation sequencing showed higher mutation frequencies of TP53,MYD88,KMT2 D,CREBBP and PIM1 in SLBCL.3.Most SLBCL patients have an aggressive course and poor prognosis. |
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