Detection And Significance Of IgH/CCND1 In Mantle Cell Lymphoma

BackgroundLymphomas,especially non-Hodgkin's lymphomas(NHL) are kinds of neoplasm with complicate morphology and variable categories,and different kings of lymphomas show enormous variation in clinical behavior and response to therapy,so the pathologic diagnosis of lymphoma is one of the difficulties and significances in clinical pathology,and also the focus of investigation that lots of people have been to dedicate themselves to it.Nowadays,with the gradually increase of understanding, many kinds of types lymphomas have been distinguished,while the mantle cell lymphoma(MCL) has been remained the dead zone of people's recognition.MCL is a rare,specific subtype of lymphoma and accounts for about 3%to 10% of NHL,which is most commonly observed in the male population ranging from 50 to 70 years of age.Patients are characterized by disseminated disease when they went to see the doctor(about 80%patients were at clinicalⅢ/Ⅳstages),usually involving bone marrow and peripheral blood.Though its morphology is very similar to other small B-cell lymphomas(such as small lymphocytic lymphoma[SLL],follicular lymphoma[FL],and marginal zone B-cell lymphoma[MZL]),it is more aggressive than other small B-cell lymphomas,with the worst prognosis of all low-grade NHL, and was divided to be the aggressive subtype in the new WHO classification.So the definite diagnosis and distinction from other small B-cell lymphomas is very important,which has significance in patient's prognosis and therapy.The most direct and fundamental pathology diagnosis is base on the morphology, while the difficulties that resulted from inconect treatment of samples and atypical histological feature are very common,so often need to draw support of other techniques.At present,the detection of immunological markers is very important in lymphoma diagnosis,which does some favor to distinguish benign tissues from malignant lymphomas;moreover,specific markers can be used to make detail classification.CyclinD1 over-expression is the predominant diagnosis and discrimination marker.However,the prophase treatments and fixation types of samples may result in depression and inactivation of antigen presentation,the detection rate of CyclinD1 is low.Over the past years,molecular biology techniques developed fast,especially the gene and chromosome abnormalities detection techniques,which have extensive application in the lymphoma diagnosis.Immunoglobulin heavy chain(IgH) gene rearrangement is the prominent character of lymphocytes,benign tissues show polyclonal rearrangement and malignant lymphomas show monoclonal or oligoclonal rearrangement,and special kinds of lymphomas have distinct rearrangement. Detection of IgH gene rearrangement can make help to distinguish benign tissues from malignant lymphomas,and may give some clue to the origin of different types of lymphoma.The chromosome t(11;14)(q13;q32) translocation is the most specific cytogenetic alteration of MCL,which results in cyclinD1 over-expression,and almost all the patients have this abnormality.According to the WHO classification,the best diagnosis of malignant lymphoma should be based on cytogenetic or molecular data, detection of t(11;14)(q13;q32) translocation at genetic level is more sensitive and specific for the diagnosis of MCL.In this study,we had developed our investigation on MCL and other small B-cell lymphomas(including SLL,FL,MZL,MALT) by morphologic,immunologic,IgH gene rearrangement and t(11;14)(q13;q32) translocation analysis,expecting to find a sensitive and specific method for the diagnosis of MCL,and to provide some guidance to the prognosis assessment and clinical therapy. Methods1.Samples collection,morphology and phenotypic features analysis412 cases of NHL with definite diagnosis from Nan fang hospital between 2002 and 2006 were organized and reclassified.Of which 57 cases with MCL and other small B-cell lymphomas with similar MCL morphology were picked out,and 61 cases with small B-cell lymphomas were collected from Guangdong province Lymphoma Study Group simultaneously,so the totally 118 cases(39 with MCL,19 with SLL,24 with FL,11 with MZL,25 with MALT) were reviewed on the basis of morphologic features and phenotypic features.2.Detection of IgH gene rearrangement73 cases with affluent tissues(31 with MCL,13 with SLL,9 with FL,2 with MZL,8 with MALT,10 with chronic tonsillitis) were picked out for IgH gene rearrangement analysis by semi-nested PCR.We used commercial Kit,simple digestion boiling and nucleus boiling for DNA extraction,and adopted agar gel electrophoresis(AGE) and Capillary electrophoresis(CE) to detect the products.3.Detection of t(11;14)(q13;q32) translocationWe made some refining to improve the nucleus extraction and nuclei micro-array making.In this study,water bath heating for dew-axing and pointing nuclei on the slides directly were used to substitute the prior methods,in which dew-axing was performed at common temperature and a paraffin mold was used to make nuclei micro-array.Common PCR,semi-nested PCR and FISH were applied to the detection of t(11;14)(q13;q32) translocation,and then the results from different methods were compared.4.Statistical AnalysisThe x~2 test was used to examine relationships between variables,one-way ANOVA was used to compare means,and multiple comparisons method was used to examine relationships between different variables.Analyses were carried out by using SPSS13.0 software. Results1.Samples collection,morphology and phenotypic features analysis results(1)Of the 412 cases with NHL,62 cases were small B-cell lymphomas, accounting for 15.05%;While there were only 4 cases with MCL,accounting fbr 0.97%.(2)Our results showed that the detection rate of CyclinD1 could be improved by performing antigen retrieval with boiling in EDTA and by predigesting in 0.4% pepsin before antigen retrieval.The positive rate of CyclinD1 was 76.47%in MCL, which was significantly higher than in other small B-cell lymphomas.2.Detection of IgH gene rearrangement results(1)The concentration of DNA extracted by nucleus boiling was the highest of all; Though the purity of DNA extracted by nucleus boiling was lower than that of commercial Kit,it was higher than that of simple digestion boiling.AGE analysis showed that the integrity of DNA extracted by commercial Kit was the best(longer than 5Kb),and the length of DNA extracted by nucleus boiling was longer than 2Kb with clearly single strap,while the length of DNA extracted by simple digestion boiling was small with smear strap.PCR forβ-globin gene amplification showed that the positive rate of DNA extracted by nucleus boiling was 87.50%,though it was lower than that of commercial Kit,it was higher than that of simple digestion boiling.(2)The positive rate of IgH gene rearrangement detected by CE was 90.32%, there were 1 case with B-SLL,3 cases with FL and 3 cases with chronic tonsillitis were positive for IgH gene rearrangement when using AGE,while they were all polyclonal rearrangement when detected by CE.(3)The positive rate of IgH gene rearrangement was higher in MCL and B-SLL, which were 90.32%and 76.92%respectively;While it was lower in FL,MALT and MZL,which were 44.44%,42.86%and 50.00%respectively.3.Detection of t(11:14)(q13;q32) translocation results(1)The positive rate of t(11;14)(q13;q32) translocation was 25.81%by common PCR,while it was 35.48%by semi-nested PCR,which was higher than common PCR. (2)The nucleus extracted by water bath heating for dew-axing were good with little foreign materials,and the nuclei micro-array made by pointing nuclei on the slides directly was fine with clear background and steady nucleus.(3)The positive rate of t(11;14)(q13;q32) translocation was 93.10%by FISH with low background and strong signals pattern.Conclusions1.CyclinD1 is a predominant diagnosis and discrimination marker for MCL and the detection rate of it can be improved by performing antigen retrieval with boiling in EDTA and by predigesting in 0.4%pepsin before antigen retrieval.2.Nucleus boiling for DNA extraction is a convenient,effective,low cost and high yield method.3.Detection of PCR products by CE is more sensitive and specific than by AGE.4.The modified method for Nucleus extraction and nuclei micro-array making that we established in this study is better than the prior method.5.t(11;14)(q13;q32) translocation is a special diagnosis and discrimination marker for MCL,and detection of it by FISH especially by nuclei micro-array FISH,having convenient,effective and economic features,is more sensitive and specific than by PCR.Innovations of this research1.We established Nucleus boiling method for paraffin embedded tissues DNA extraction,and got satisfied results when adopted it in this study.2.Detection of PCR products by CE,improving the sensitivity and specificity.3.Modified the Nucleus extraction and nuclei micro-array making method,improving the quality and simplifying the process.