The Expression Of ZAP-70 And CD38 In The B Cell Chronic Lymphocytic Leukemia And Its Clinical Significance

chronic lymphocytic leukemia(CLL)is one of the clonal lymphoproliferative tumors,more than 95%of that is B-cell CLL,less than 5%of that is T-cell CLL.The incidence ratio of CLL is 25%-30%of all leukemias in western countries,and about 10-30 times than which in India, China and Japan.The incidence of CLL is dormant,with the median age being 65～70 years,and the male/female ratio nearly being 2:1.The prognosis is overall good with the median survival around 10 years.Due to distinct clinical heterogeneity,the patients of CLL can be classified into two groups according to their good or bad prognosis.There have been intense rasearches for the best prognostic markers recently.ZAP-70(zata chain associated protein-70)is one of protein tyrosine kinases expressed in nomal T and NK-lymphocytes with 70KDa molecular quality,but absent in nomal B-lymphocytes.In B-CLL cell ZAP-70 can construct BCR signal transduction access replacing Syk,then stimulate cell into growth,proliferation and cancerization.CD38,one ofâ…¡type transmembrane glycoproteins,being a prognostic marker of CLL in early research,was disputed recently in its relationship with the disease course and the prognosis of CLL.In 2004 England CLL clinical guide and 2006 Amercian CLL clinical guide,gender,clinical staging,lymphocyte morphology,bone marrow infiltration,lymphocyte doubling time,serum markers,CD38,ZAP-70, cytogenetics,IgVH mutational status were all the prognostic factors.The IgVH mutation status was the most valuable prognostic factor,while unable to be performed for its difficulty and expensive.Recently the expression of ZAP-70 and CD38 of B-CLL have been researched more frequently in the foreign countries while lessly in our country for the low incidence.The two markers in B-CLL and their relationship with clinical stage,lymphocyte morphology,serum lactate dehydrogenase(LDH),clinical treatment and curative effect were assessed in this article.Materials and MethodsThe expression of ZAP-70 and CD38 were measured in 20 cases of B-CLL between November 2006 and August 2007 in our hospital.8 patientss were female,12 patients were male,whose ages ranged 47 to 76 years old.11 patients hadn't been treated before,9 patients had ever been treated with chemotherapy.The diagnosis of B-CLL was made according to《Diagnosis And Effect Criteria Of Blood Diease》,excluded other chronic B-cell lymphomas and leukemias.5 patients were in Binet A stage,8 cases were in Binet B stage,and 7 cases were in Binet C stage.ALL the patients were observed their clinical staging,lymphocyte morphology,serum LDH and the expression of ZAP-70 and CD38.The heparinised peripheral blood or bone marrow of 20 B-CLL patients were adjusted to 1×10⁶/ml cells,then among that 100ul was taken and added with 20ul CD3-APC,20ul CD19-FITC,20ul CD38-TC,then stained for 15 minutes at house temperature,,then added with Fix & Perm A 100ul,stained for 10 minutes at house temperature.The cells were washed with 2ml PBS,centrifugaled 1200 rpm for 5 minutes,then thrown away the upper liquids.The lower cells were added with 100ul Fix & Perm B and 5ul ZAP-70-PE,stained for 20 minutes at house temperature,then washed with 2ml PBS and centrifugaled 1200 rpm for 5 minutes,then thrown away the upper liquids.The lower cells were added with 1mL PBS,then performed using FACSCalibur^TM(BD Bioscience)flow cytometer.The dates were analyzed with Cell Quest software using K-S statistical test, comparing the D-value between B and T lymphocytes.The D-value＞0.84 was defined as ZAP-70 negative,and that of≤0.84 was positive.The positivity cut-off point for CD38 was≥20%,＜20%was CD38 negative.The serum LDH was analyzed with Full Automatic Biochemical Analysor(Beckman Co.)on peripheral blood,ALL the patients were divided into two groups according their LDH:≥225IU/L和＜225IU.A morphological evaluation of peripheral blood cells of all patients was performed on Wrigh's Staining films.In the presence of＞10%prolymphocytes or＞15%atypical cells (lymphoplasmocytoid cells and cleaved lymphocytes)CLL was defined as morphologically atypical.All the dates were analyzed by SPSS 10.0 software bag.Results1,The expression level of ZAP-70 in 20 cases of B-CLL.All the ZAP-70 D-value of the 20 cases were 0.72±0.16.There were the positive expression of ZAP-70 in 14 of 20 cases(70.0%).Among the positive patients 2 cases(10%)were strong positive whose D-value were≤0.50.There were the negative expression of ZAP-70 in 6 of 20 cases(30.0 %).2,The expression level of CD38 in 20 cases of B-CLL.All the CD38 expression percentage of 20 cases were 26.09±37.79%. There were the positive expression of CD38 in 9 of 20 cases(45.0%). There were the negative expression of CD38 in 11 of 20 cases(55.0%).3,The expression correlation between ZAP-70 and CD38 in 20 cases of B-CLL.There were no correlation between ZAP-70 and CD38 expression in 20 cases B-CLL(r=-0.340,P=0.142).The patients of ZAP-70 positive and CD38 negative were 8 cases(40%),that of ZAP-70 negative and CD38 negative were 5 cases(25%),that of ZAP-70 positive and CD38 negative were 6 cases (30%)in which D-values were 0.51～0.83 without strong positive.There was only 1 case with ZAP-70 negative and CD38 positive.4,The relationship of ZAP-70,CD38 and other clinical factors in 20 cases of B-CLLThe positive expression of ZAP-70 was no correlation with age and gender,but correlated with the clinical staging,the lymphocyte morphology and the serum LDH;That of CD38 was no correlation with age, gender,the clinical stage,the lymphocyte morphology and the serum LDH.5,The clinical treatment and actuality in 20 cases of B-CLLThe positive expression of ZAP-70 and CD38 were more high in Binet B+C patients in B-CLL,the chemotherapy mainly composed with fludarabine was effective in short-term,without long-term observation.The both positive expression of ZAP-70 and CD38 patients had worse prognosis.Summary1,The expression of ZAP-70 and CD38 can be performed using flow cytometer expediently,rapidly and cheaply. 2,A few B-CLL cases had positive expression of ZAP-70.The ZAP-70 positive was no correlation with age and gender.The positive cases were generally with high risk factors,such as in Binet B or C stage,with atypical lymphocyte morphology and higher serum LDH,all of that suggested bad prognosis.ZAP-70 may be used to judge the prognosis of B-CLL.3,A few B-CLL cases had positive expression of CD38.The CD38 positive was no correlation with age,gender,clinical stage,lymphocyte morphology and serum LDH.Farther researches between CD38 and the prognosis of B-CLL will be performed.4,There were no correlation between the expression of ZAP-70 and CD38 in B-CLL patientsThrough the mensuration of ZAP-70 and CD38 in B-CLL associated with other clinical indexes and literatures,We concluded that ZAP-70 may be a right prognositic marker of B-CLL,while CD38 may be disputed and require more researches with more clinical caese,more time,and right positive cut-off point.5,The positive expression of ZAP-70 and CD38 were more high in Binet B+C patients in B-CLL,the chemotherapy mainly composed with fludarabine was effective in short-term,without long-term observation.The both positive expression of ZAP-70 and CD38 patients had worse prognosis.