Studies On Detection Of Bone Marrow Involvement By Flowcytometry And Polymerase Chain Reaction And Their Clinical Significance

B cell lymphoma is one kind of non-Hodgkin lymphoma(NHL) and malignant neoplasm originated from B lymphocyte. Patients with B cell lymphoma commonly present with painless peripheral lymphadenopathy. Compared with Hodgkin lymphoma, they tend to invlove extranodal organ and tissue and also accompany with systemic symptoms, such as unexplained fever, night sweats and weight loss. Based on the World Health Orginazation classification of haematological and lymphoid tumors in 2000, B cell lymphoma has been classified into more than 10 subtypes. In different subtypes of B cell lymphoma, they have variable clinical behaviors and high incidence of bone marrow involvement in their advanced stages. According to Ann Arbor staging system, patients with bone marrow involvement (BMI) were considered D stage. In International Prognostic Index (IPI) system for predicting prognosis for aggressive lymphomas, the number of extranodal involvement sites is an important component and significantly impacts on survival. The diagnosis of BMI for patients with malignant lymphma remains centered on 3 primary aspects: (1) further recognition and diagnosis of disease; (2) providing information regarding disease stage; and (3) providing prognostic indications that predict the risk from disease.Bone marrow biopsy and bone marrow aspirate are effective methods which they are widely used for evaluating bone marrow involvememt in clinical work. Flow cytometry and molecular diagnostic technique provide new directions for diagnosis of bone marrow involvement. In western countires, these two techniques were intensively investigatedin diagnosis of BMI in B cell lymphoma. Our studies focus on the application of new methods for detection of BMI in Chinese B cell lymphoma. The current research is comprised of the following three parts.Part 1 Application of flowcytometry in the detection of bone marrow involvement in patients with B cell lymphomaObjective To explore feasibility and effectiveness in detection of bone marrow involvement of B-cell lymphoma by flow cytometry (FCM) and to evaluate the value of clinical pathology.Methods 79 bone marrow samples from B-cell lymphoma patients were detected by EPICS-XL(?) flow cytometry using CD45-SSC gating strategy combined with 3 different fluorescence labeled anti CD45, CD5, CD10,CD19, CD20, CD22, CD23, CD43, FMC-7,λ and κ monoclonal antibodies, compared with cytomorphology of bone marrow aspiration .Results (1)35 cases (44.3 % ) were demonstrated bone marrow involvement in 79 B-cell lymphoma patients, while only 16 cases (20.3%) were detected by analysis of cytomorphology of bone marrow. There is a significant difference between two methods (P<0.05) ; The agreement level was 75.9%.(2) Diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL) and small lymphocytic lymphoma (SLL) at the time of diagnosis have different incidence of bone marrow involvement by using FCM, showing 20.3%,43.8% and 100.0%in DLBCL, FL and SLL respectively. (3)For the patients without definitive pathological type, the immunophenotype detected by FCM can be helpful for diagnosis.(4)FCM had a good specificity and found no abnormal immunophenotype in non lymphatic leukemia.The sensitivity of this method was about 15%.(5)Rate of bone marrow involvement by FCM in patients with nodal and nodal plus extranodal involvement, aggressive histological type, advanced stage by Ann Arbor system and high-mediate risk group were significantly higher than corresponding groups (P<0.001) , the rate of patients with B symptoms and pretreated history had slight increase but no statistical implication was shown(P>0.05). There is no correlation between bone marrowinvolvement and gender,age,LDH status or spleen involvement (P>0.05) .(6) BMI determined by FCM has a strong correlation with complete response(CR) after therapy, the possibility of CR in negative patient by FCM is higher than positive individual and the difference has statistical significance (P＜0.05 ) .No impact of BMI by FCM on short-term survival of BMI was observed .Conclusions Flow cytometry is an effective method for detection of bone marrow involvement in B-cell lymphoma and is superior to cytomorphology; Bone marrow involvement detected by FCM can be useful for helping diagnosis. The sensitivity of FCM is about 15%.Positive rate in patients with advanced stage by Ann Arbor system and indolent lymphoma were higher than the corresponding groups. The chance of achieved CR after therapy in FCM positive group is lower than FCM negative one.Further observation after longer follow-up period will be needed to determine the effect on survival.Part 2 Study of polymerase chain reaction (PCR) in the detectionof bone marrow immunoglobulin heavy chain gene rearrangement inpatients with B cell lymphomaObjective To explore feasibility and effectiveness in detection of bone marrow involvement of B-cell lymphoma by PCR and to evaluate the value of clinical pathology.Methods Clonal gene rearrangement of immunoglobulin heavy chain was detected by seminested PCR in 105 bone marrow samples from B-cell lymphoma patients, compared with cytomorphology of bone marrow aspiration .Results (1) 48 cases (45.7% ) were demonstrated bone marrow involvement in 105 B-cell lymphoma patients, while only 22 cases (21.0%) were detected by analysis of cytomorphology of bone marrow. There is a significant difference between two methods (P<0.05) ; The agreement level was 71.4%.(2) Diffuse large Bcell lymphoma (DLBCL), follicular lymphoma (FL) and small lymphocytic lymphoma (SLL) at the time of diagnosis have different incidence of bone marrow involvement by using FCM, showing 30.8%,25.0% and 100.0%in DLBCL, FL and SLL respectively.(4)PCR had a high sensitivity which was about 2%-4%. (5)Rate of bone marrow involvement by PCR in patients with advanced stage by Ann Arbor system were significantly higher than corresponding groups (P=0.02) .Positive rate of PCR in patients with more than 60 years,male,pretreated history, normal LDH level and indolent lymphoma were higher than corresponding groups, but no statistical significance was shown(P>0.05). There is no correlation between bone marrow involvement with involvement sites ,B symptoms or spleen involvement (P>0.05) .(6) The result determined by PCR has a strong correlation with complete response(CR) after therapy, the possibility of CR in negative patient by PCR is higher than positive individual and the difference has statistical significance (P<0.05) .No impact of BMI by PCR on short-term survival was observed. Conclusions PCR is an effective method for detection of bone marrow involvementin B-cell lymphoma and is superior to cytomorphology. The sensitivity of PCR is about 2-4%.Positive rates in patients with advanced stage by Ann Arbor system were higher than the corresponding groups. The chance of achieved CR after therapy in PCR positive patient is lower than PCR negative one.Further observation after longer follow-up period will be needed to determine the effect on survival.Part 3 A comparison of bone marrow cytomorphology,immunophenotypic analysis by FCM, gene rearrangementdetected by PCR in the detection of bone marrow involvement in Bcell lymphomaObjective To compare diagnostic value of cytomorphology of bone marrow aspirates, immunophenotype detected by flowcytometry and immunoglobulin heavy chain gene rearrangement detected by polymerase chain reaction in bone marrowinvolvement in B cell lymphoma.Methods Bone marrow cytomorphology, FCM and PCR were simultaneously carried out in 75 bone marrow of B cell lymphoma and compared with each other.Results (1)16 were demonstrated by cytomorphology, 36 by FCM, 33 by PCR. The positive rate were 21.3%, 48% and 44% respectively. The differences among these methods have statistical significance (P<0.05) .(2) The concordance rate of these methods was 64% (48/75). There had 6 cases with FCM positive, cytomorphology and PCR negative; 2 cases with FCM and cytomorphology positive,but PCR negative; 14 cases with FCM and PCR positive,cytomorphology negative;5 cases with PCR positive,FCM and cytomorphology negative.(3) The concordance rate between FCM and PCR was 82.7%(62/75).Conclusions Both FCM and PCR are better than cytomorphology observation of bone marrow aspiration for the detection of bone marrow involvement and helpful complementary methods for BMI detection. The result of FCM and PCR are helpful for predicting treatment response. To validate the effect on outcome of disease by the different results which detected by these 3 methods, longer follow-up period for prognsis will be needed.