Application Of BIOMED-2 Protocols To Analyze Clonal Rearrangement In B-NHL

Backgrounds and AimsNon-Hodgkin lymphomas(NHLs)represent 50%of all hematological malignancies;they constitute a heterogeneous group of lymphoproliferative malignancies.Their diagnosis and classification are often complex.NHLs arise from the unusual proliferation of B or T lymphocytes at various differentiation stages.About 90-95%of the lymphoid malignancies have a B-cell origin.B-cell development mostly occurs in the secondary follicles of lymphoid organs.Lymphoproliferations are generally diagnosed via histomorphology,cytomorphology,immunohistochemistry and cytometric immonophenotyping.Although mostly conclusive,occasionally the differential diagnosis between reactive lesions and malignant lymphomas is difficult.In such cases molecular clonality studies of immunoglobulin(Ig)/T-cell receptor(TCR) rearrangements can be useful.For a long time,Southern blot analysis has been the gold standard technique for molecular clonality studies.Despite the high reliability of Southern blot analysis,it is increasingly replaced by PCR techniques,because of several inherent disadvantages: Southern blot analysis is time-consuming,technically demanding,requires 10-20 ug of high-quality DNA,and has a limited sensitivity of 5-10%.The main advantages of PCR techniques are their speed,the low amounts of DNA required,the possibility to use DNA of lower quality,and the relatively good sensitivity of 1-5%,for some types of rearrangements even＜1%.Consequently,PCR techniques allow the use of small biopsies(eg fine-needle aspiration biopsies),or the use of formaldehyde-fixed paraffinembedded samples,which generally results in DNA of lower quality,that is, partly degraded into smaller fragments.Therefore archival material may also be used,if needed.Polymerase chain reaction(PCR)assessment of clonal immunoglobulin(Ig)and T-cell receptor(TCR)gene rearrangements is an important diagnostic tool in mature B-cell neoplasms.However,lack of standardized PCR protocols resulting in a high level of false negative and false positive results have hampered comparability of data in previous clonality studies.In order to address these problems and introduce reliable and easy PCR technology for routine clonality diagnostics in suspect lymphoproliferations, A total of 47 institutes from seven European countries participated in the BIOMED-2 Concerted Action.Multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin(Ig)and Tcell receptor(TCR)genes and the chromosome aberrations t(11;14)and t(14;18).This has resulted in 107 different primers in only 18 multiplex PCR tubes:three VH-JH,two DH-JH,two Ig kappa(Igκ),one Ig lambda(Igλ),three TCR beta(TCRβ),two TCR gamma(TCRγ),one TCR delta(TCRδ),three BCLI-Ig heavy chain(IGH),and one BCL2-IGH.Fresh/frozen tissue is considered to be the ideal sample type for the extraction of DNA for use in PCR-based clonality analysis.However,fresh/frozen material is not always available to diagnostic laboratories and in many laboratories,paraffin-embedded tissue samples constitute the majority of diagnostic biopsies submitted for analysis. DNA extracted from paraffin-embedded material is often of poor quality and so PCR protocols need to be evaluated for use with these sample types before they can be widely used in diagnostic laboratories.We can observe the cases' Ig VH gene mutation through the TA clone and sequencing.The detection of Ig VH gene mutation is a useful approach to track the stage of lymphomas development.Aims:Application of BIOMED-2 PCR assay detects of B-cell clonality in paraffin-embedded tissue;and to evaluate the usefulness of gene rearrangements analysis by BIOMED-2 PCR assays on DNA from paraffin-embedded tissues for the diagnosis of B-cell lymphomas.To detect the Ig VH gene mutation by T-A clone and Sequencing,During the peroid of follow-up,to detect mininal residual disease and the developing of disease through the length and the sequence of the PCR products.Materials and MethodsMaterials(1)NHL specimens:75 cases of B-NHL were obtained from our affiliated hospitals and other local hospitals from 1997 to 2008.All specimens were fixed in 10% formalin and then embedded in paraffin.Sections were stained by hemotoxylin and eosin and by IHC.All cases were confirmed as B- NHL on the basis of their immunophenotypes and morphologies according to the WHO classification.IHC contain LCA,CD3,CD20,kappa light chain和lambda light chain。(2)Control specimens:Negative control:1 cases of reactive lymph nodes from our affiliated hospitals.Positive control:DNA extracted from Raji cell lines and Jurkat cell lines.Methods(1)DNAs from paraffin embedded tissues were prepared by phenol-chloroform extraction method.The qulity of DNA is evaluated through control PCR(β-2 microglobulin).(2)Our study analyzed the rearrangement pattern of complete IgH,Igκand TCR gene with BIOMED-2 PCR protocols in a group of 75 B-NHLs.PCR products were evaluated using firstly 2%agarose gel and then 8%nondenaturing polyacrylamide gel for electrophoresis. (3)TA clone and sequencing were performed.Results1.The detection rate of gene clonality was 85.3%(64/75)by BIOMED-2 primer. The detection rate of IgH gene clonality is 78.7%(55/75),using the combination of Igκprimer,the detection rate of clonality increased to 85.3%(64/75).2.The results of three cloning positive cases:one of the two DLBCL cases is VH4 gene,the other is VH3.The two cases are of somatic hypermutation.,but don't have intraclonal hypermutation.The intractable case uses VH3 gene and one clone's sequences of the two incidences are the same;the second incidence has another clone. So the second incidence is the recurrence of the disease,and the disease of the second incidence is developing.Conclusions1.IgH gene and TCR gene rearrangements must be detected simultaneously in a suspect sample in order to fidelity and integrity result.2.The BIOMED-2 protocols work well with DNA extracted from paraffin-embedded material.3.Because most of amplified products of DNA extracted from paraffin-embedded samples are less than 300bp,we should select complete IGH-FR2(tube B),complete IGH-FR2(tube C),IGK(tube A),IGK(tube B),TCRG(tube A)and TCRG(tube B).4.Further modifications of the original BIOMED-2 protocal can increase in the clonality detection rate.5.The mutation of IgVH gene can be analyzed by sequencing,which can also analyze the molecular characteristics of B-NHL.