Application Of Polymerase Chain Reaction For Detection Of IgH And TCR Gene Rearrangements In Non-Hodgkin Lymphoma

Pathological diagnosis of lymphoma has been one of the most difficult fields in clinical pathology. The histopathological character of lymphoma is the basis of pathological diagnosis, and the immunohistochemistrical (IHC) detection is helpful of diagnosis and classification. However, the misdiagnositic rate remains relatively high combined of histopathological and IHC assay. Recently diagnostic accuracy has been improved with the application of molecular methods to clinical pathology and it is one of ways for detection of immunoglobulin heavy chain (IgH) gene and T cell receptor (TCR) gene rearrangements in non-Hodgkin's lymphoma (NHL) by polymerase chain reaction (PCR) method. The consensus V and J primers used to detect IgH are always designed directed to the relatively conservative nucleotide sequences of the FR1, FR2, FR3, and J region. The primers with the highest detection rate and the most popular choice are directed against a region termed FR3 region of the various VH genes. FR3-directed primers FR3A detect approximately 60% of B cell malignancies. The combination of other FR primers will improve the detection rate of IgH gene clonal rearrangement. TCR γ gene is rearrangement at early stage of T lymphoid development in both TCRαβ and TCRγδ. lineage, and the complete genomic structure has beenknown for many years, so it is a preferential target for clonality analyses. It contains a limited number of Vy and Jy segments. The amplification of all major Vy-Jy combinations is possible with 4 Vy and 3 Jy primers. In T-cell non Hodgkin's lymphoma (T-NHL), the detection rate of TCR Y gene clonal rearrangement is about 60%.The utility of PCR technique in the assessment of clonality in lymphomas has been increasing. With the data accumulated, the reliability and feasible of the molecular technique exist a series of questions which bother clinical pathologist, such as false positive, false passitive, the diagnosis of case of dual rearrangements and oligoclonal rearrangement and the feasibility and the reliability of clonal detection in small specimen.it had been reported of dual rearrangements in lymphoma long before. It means both IgH and TCR gene clonal rearrangements simultaneously occur in a simple. Furthermore, it is named biclonal rearrangement which refers to 2 alleles of IgH simultaneously rearrange in B-NHL, so is T-NHL. The notion of dual reaarngements in our study is the former. More attention is recently paid to dual reaarngements. Both at home and abroad there are little papers about whether lymphomas of dual rearrangements by PCR method take place dual rearrangements truly and what pathogenesis is.Clonal detection in small specimen is one of difficulty in molecular diagnosis of lymphoma. Since the use of PCR IgH gene analysis is being increasingly advocated for analyzing clonality in small tissue biopsies(such as gastroscopy speciemen), in fine needle aspiration specimens, and even in small groups of cells mocrodissected from glass slides of cytological material, some PCR studies have reported relatively high rates of B cell monoclonality in apparently reactive tissues. The problems of the reason of the phenomenon and how to decrease the false positive remain to be elucidated.As a uaual subtype of NHL, extranodal marginal zone B-cell lymphoma of MALT type is a series of extranodal marginal zone lymphoma in mucosa and epithelium tissue, particularly common in gastric and intestines. With reactive follicles and no separate immune marker, it is different from other lymphoma and the clinical pathologist need to diagnosis in gastroscopy and intestroscopy speciemens. So pathologist commits himself to search for simple and convenient molecular marker. Moreover it might provide valuable data for the diagnosis of small specimen.PCR analysis of IgH and TCR Y gene rearrangement had been carried out in 82 cases of NHL to evaluate detection rate of clonality gene rearrangement in our lab. Cases of dual rearrangements of electrophoresis were testified whether dual rearrangements actually would appear by DNA sequencing. We performed FR3A PCR analysis on serially diluted DNA samples from 5 B-NHLs, 6 reactive lymph nodes, 6 peripheral blood specimens and sought to determine likely mechanism of false positive. We investigated 14 chronic gastritis and 10 gastric MALT-MZL specimens and tried to find out whether IgH PCR study is helpful for the differential diagnosis of MALT-MZL and benign case. The last part was a dissented case which was performed with polymerase chain reaction for detection of IgH and TCR gene rearrangements.Materials and Methods1. Materials(1) NHL specimens: 82 cases of NHL were obtained from our affiliated hospitals and other local hospitals from 1997 to 2005. 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 divided into T- and B- NHL on the basis of their immunophenotypes and morphologies according to the WHO classification. Of 82 cases of NHL there were 56 B-NHLs and 26 T-NHLs.(2) Control specimens: 6 cases of reactive lymph nodes, 5 peripheral blood and 14 chronic gastritis specimens with follicle hyperplasia were obtained from our affiliated hospitals. Of 14 chronic gastritis specimens, there were 4 gastroscopy cases, the other were non tumor of gastric lymphoma's verge Methods(1) DNAs from paraffin embedded tissues were prepared by phenol-chloroform extraction method. DNA was extracted from peripheral blood specimen by high salt method.(2) Our study analyzed the rearrangement pattern of IgH and TCR gene with primers of FR3A, FR2A and TCR Y in a group of 82 NHLs. PCR products were evaluated using firstly 2% agarose gel and then 8% denaturing polyacrylamide gel for electrophoresis. DNA sequencing was performed on PCR products of dual rearrangement of electrophoresis.(3) FR3A PCR analysis was performed on serially diluted DNA samples from 5 B-NHLs, 6 reactive lymph nodes, 6 peripheral blood specimens. The PCR products were applied onto firstly 2% agarose gel then 8% denaturing polyacrylamide gel for electrophoresis.(4) The rearrangement patterns of IgH gene in a group of 14 chronic gastritis specimens were performed. Electrophoresis was the same as the above.Results1. The detection rate of IgH gene clonality was 64.3%( 36/56) by primer FR3A in 56B-NHL;using the combination of primer FR3A and FR2A, the detection rate of IgH clonality increased to 78.9%(44/56).2. The detection rate of TCR Y clonality in 26 cases of T-NHL was 46.2%( 12/26).3. The combination of the two IgH gene primers with the multiplex TCR y gene PCRallowed detection of dual rearrangements in 6.0%( 5/82) of NHL, and verified bysequencing. The cases of dual rearrangement were B cell immunophenotype by IHC. Of 5 cases, there were 2 DLBCLs, 2 MALT-MZLs and 1 FL.4. All 5 B-NHLs yielded single discrete band, which were maintained in all dilutions.By contrast, target DNA of 6 reactive lymph nodes and 6 blood specimens showed a polyclonal pattern at starting template concentration and at dilutions ranging from 1:2 to 1:3;however, serial dilution showed strong pseudomonoclonal bands at dilutions of 1:10 and above.5. The detection rate of IgH gene clonality was 60% by primer FR3A in 10 MALT-MZLs;with the combination of primer FR3A and FR2A, the detection rate of IgH clonality increased to 70%. Chronic gastritis specimens did not show monoclonal bands.Conclusions1. The detection rate of IgH gene clonality is 64.3 % by primer FR3A in B-NHL;with the combination of primer FR3A and FR2A, the detection rate of IgH clonality increases to 78.9%.2. The detection rate of TCRy clonality in T-NHL is 46.2 %.3. The combination of the two IgH gene primers with the multiplex TCR Y gene PCR allowed detection of dual rearrangements in 6.0% of NHL. Pathological types are DLBCL, MALT-MZL and FL.4. IgH gene and TCR gene rearrangements must be detected simultaneously in a suspect sample in order to true and integrity result.5. Serial dilution of DNA is one of ways to discriminate the false positive of IgH clonal detection.6. Clonal detection is a useful assay for the differential diagnosis of gastric MALT-MZL and chronic gastritis.