Detection Of The Cloning Gene Rearrangement And T-cell-associated Chemokine Receptor Expression Patterns In T-cell Non-Hodgkin's Lymphoma

Pathological diagnosis of lymphoma has been acknowledged as the most difficult fields in clinical pathology.Diagnosis of lymphomas is only based on histomorphology and immunohistochemical techniques,which results in misdiagnosis and missed diagnosis on occasion.With the development of molecular biology,clonality analysis of lymphoid cell populations plays an increasing role in the primary diagnosis as well as follow up of lymphomas and gradually matures.Overseas,clonality analysis especially in immunoglobulin gene cloning,has been used as routine pathological diagnosis in B-cell non-Hodgkin's lymphoma(B-NHL).T-cell non-Hodgkin lymphoma(T-NHL)is an uncommon lymphoma in human,which demonstrates high heterogeneity.Currently,the pathological diagnosis of T-NHL is not very optimistic in the domestic and foreign studies.There are two problems need to be solved in diagnosis of T-NHL lymphoma:first, yes or no? Second,which type? That is the about diagnosis and classification issues of T-NHL.To the first question,because of appearance of T-NHL is changeable and without cloning markers on immunology,it is advocated that all suspectable T-cell proliferations (especial T-cell-rich B-NHL)need diagnosis of molecular cloning gene rearrangement. The diagnosis of lymphoid malignancies can be supported by clonality assessment based on the fact that,in principle,all cells of a malignancy have a common clonal origin.Now,in clinical practice of molecular diagnosis in lymphoma,PCR-based clonality analyses methods practically has replaced Southern blotting,becauces besides high speed,simple,cost effectiveness,cheap and high sensitivity,the great advantage of the PCR techniques is that they do not require intact DNA.The highest sensitivity of PCR detection is generally expected on DNA isolated from fresh or frozen samples,while using templates from FFPE(formalin fixed and paraffin embedded,FFPE)tissue samples may impair performance.Unfortunately,in the majority of the diagnostic cases and in retrospective studies,only FFPE tissue samples are available for molecular genetic studies.DNA recovered from FFPE tissues generally consists of fragmented small target sequences with chemical alterations,it is disadvantageous for molecular studies,so,how to elevate the detect sensitivity of highly degraded samples is a problem researcheres confront with.On the other hand,the patterns applied in defferent literatures,such as primers,PCR running parameter and the way of PCR products analysis are not all the same,resulting the defferent toutcomes.So,how to introduce a suitable and standardized process into the routine clinical pathological diagniosis is another problem the researcheres confront with.In fact,clonality analysis is not easy on FFPE samples,it requires even more experience than that of performed on fresh samples or is more complicated than most genomic PCR amplifications for somatic genes.Although PCR-based clonality analyses are not novel methods,there is still paucity of published studies that analyze the necessary changes in the PCR parameters while amplifying DNA templates isolated from FFPE tissue samples.To the second question,differentiation marker expression studies have shown that the majority of T-cell lymphoma developed from the malignant transformation of the T cells after thymus.So,these malignant tumor is known as peripheral T-cell lymphoma (periferal T cell lymphomas,PTCLs).PTCLs are a kind high heterogeneity of malignant lymphoma.Theirs diagnosis,classification and studies of the molecular pathogenesis of tumor pathology have been one of the most difficult and most controversial areas. According to 2001 WHO classification,T-NHL has divided a number of independent existence of the tumor entities,such as enteropathy type T cell lymphoma(ETTCL), anaplastic large cell of the T-cell lymphoma(ALCL),angioimmunoblastic T-cell lymphoma(AITL),nose-T / NK cell lymphoma,as well as liver and spleen yδT-cell lymphoma,etc.In addition,a new separation of PTCLs was that periferal T cell lymphomas,unspecified(PTCL-U),but the term only used to describe a class of the heterogeneity of lymphoma,which have different clinical lymphoma features,histological features,genetic change,and treatment response and the corresponding prognosis.So the detailed classification of PTCLs,especially PTCL-U,remain to be clarified.To date, the classification of T-cell lymphoma has been largely based on morphologic or clinical criteria.Recent studies show that the expression pattern of chemokine receptor on normal T-cell subsets is related to the cytokines from the cell subsets,CCR3 selective expression in Th2 cells of the human or mice,CXCR3 selective expression in Th1 cells.For the above reasons,we select TCRγgene rearrangement in FFPE samples as the research object and choose the TCRγmulti-primers in our study.Through optimizing the extraction of DNA and the multi-parameter of PCR,we analysis TCRγgene rearrangement on FFPE samples in T-NHL,in order to establish the significance of different variables on performance of PCR amplification.Another pair of consensus primers of TCRβgene was applied as the complement and control of TCRγgene primers.Meanwhile,higher rate of detection and the most popular choice FR3A and FR2A consensus primers detect the lgH gene rearrangement in 53 cases of T-NHL to evaluate detection rate of clonality gene rearrangement in our lab.We studied the expressions of chemokine receptors,Th1-associated CXCR3 and Th2-associated marker CCR3 in 45 cases of T-NHL to evaluated the relationship with clinical Pathology.Materials and Methods1 Materials(1)T-NHL specimens:53 cases of NHL were obtained from our affiliated hospitals and other local hospitals from 1997 to 2008.All specimens were fixed in formalin and then embedded in paraffin.Sections were stained by hemotoxylin and eosin and by IHC.All cases were divided into different subtypes on the basis of their immunophenotypes [LCA(2B11,DAKO),CD45RO(UCHL1,DAKO),CD20(L26,DAKO)CD30(Ber-H2)]; morphologies and clinical information according to the 2001 WHO on the lymphatic and hematopoietic tumor classification standards;meantime all cases were categorized into two different histological types according to the Keil criteria of 1992.(2)Control specimens:4 cases of reactive proliferation of lymphoid tissue,(1 case of lymph node,3 cases of hyperplastic tossils)(3)Jurkat cell:select Human T-cell leukemia cell lines as positive control,,the culture medium RPMI 1640(including 10%super fetal calf serum),cultured in water Jacketed CO₂ Incubater which have 5%CO₂,temperature is 37℃.After cell counted,to adopt a centain number cells for the next experiment.2 Methods(1)SP immumohistochemistry method was applied to detect markers of lymphocyte immunophenotypes:[LCA(2B11,DAKO),CD45RO(UCHL1,DAKO),CD20(L26, DAKO),CD30(Ber-H2)].(2)Selected 6 cases of T-NHL and 4 cases of the reactive proliferation of lymphoid tissue,according to different dewaxing time and dewaxing temperature divided into two groups.Thern,DNA extraction from tissue was prepared by phenol-chloroform extraction method.We selected TCRy gene rearrangement in FFPE samples and choosed the TCRγmulti-primers as the research object,and optimized the cycle parameters of PCR.(3)Using PCR method,our study analyzed the rearrangement patterns of TCR and IgH gene with primers of TCRγ,TCRβ,FR3A and FR2A in 53 cases of T-NHLs.PCR products were evaluated using firstly 2%agarose gel and then 8%non-denaturing polyacrylamide gel for electrophoresis.(4)Select cytokines receptors Th1-associted CXCR3 and Th2-associted CCR3 detected the expression levels in 45 T-NHL by immunohistochemistry technique to assess its relationship with clinical pathology.3 Results(1)According to immunophenotypes and clinical information,in the 53 cases of T-NHL, 27 cases were periferal T cell lymphomas,unspecified(PTCL-U),12 cases were enterophathy-type T-cell lymphomas(ETTCL),8 cases were anaplastic large-cell lymphomas(ALCL),6 cases were subcutaneous panniculitis-like T-cell lymphomas lymphomas(SCPTCL).(2)DNA quality and higheryield were better in the group of a longer time and a higher temperature of dewaxing process.(3)For TCRγmultiple primers amplification in FFPE samples:the best amplification effect of TCRγA,B tubes was at the 40 cycles;the annealing temperature at 50.9℃-65.5℃for TCRγA tube can be amplified to achieve satisfactory results,and the TCRγB tube required at 50.9℃-56.7℃;TCRγA,B tubes of the necessary primers of were above 50 pmol,the final concentration of Mg²⁺were 1.25-1.75mM,the final concentration of dNTPs were 100 200uM.(4)Using our improved protocol of the TCRγmulti-primers,the detection rate of the cloning TCRγgene rearrangement in 53 cases of T-NHL was 62.3%(33/53);the detection rate of TCRβD2-J2 consensus primers was 41.9%(23/53);the combination TCRγof the multi-primers and TCRβconsensus primers,the total detection rate was 66.0%(35/53).(5)The combination the TCRγand the FR3A,FR2A primers,7.6%(4 / 53)T-NHL detected double rearrangement,the results of immumohistochemistry suggested that this group of four cases were T-cell phenotype.Pathological types were 3 cases periferal T cell lymphomas,non-specific(PTCL-U)and 1 case anaplastic large cell of the T-cell lymphoma(ALCL).(6)Immunohistochemical detection the CCR3 and CXCR3 expression in 45 cases of T-NHL,found that the overall expression rate of the two cytokines receptors is 24.4%. There was no significant difference in the expression of CCR3 and CXCR3 in different pathological types(P＞0.05).4 Conclusion(1)Extending the time and raising the temperature of dewaxing can contribute to elevate the quality and yield of DNA extraction from FFPE samples.(2)For TCRγmultiple primers,the increasing in the number of PCR cycle will help improve PCR amplification on poor DNA;a light decrease in the concentration of primers in combination with a slight increase in MgCl₂ as well as a standard amount of dNTPs can be the modifications of choice while adjusting TCRγclonality tests on poor quality DNA from FFPE sample.(3)Our experience may be of help during the optimization process of TCRγmultiple primers in technically difficult cases as well as to determine which parameters and how should be changed to minimize false-negative and false-positive results.(4)Using the optimal TCRγA,B tubes of multiple primers,the detection rate of cloning of TCRγgene rearrangements in T-NHL is higher than that of TCRβconsensus primers D2-J2,but the TCRβconsensus primers D2-J2,can increase the detection rate.(5)Gene rearrangement and the immune phenotype of lymphoma cells are not consistent.Through gene rearrangements can not determine T,B cell lines sources, unless the lg and TCR gene rearrangements must be detected simultaneously and rule out one of its cell lines to determine the source.(6)According to whether or not expression of CXCR3 or CCR3 to further classification T-cell lymphoma,needs to further experimental verification.