Detection Of Gene Rearrangement In Bone Marrow Of Non-Hodgkin's Lymphoma Patients And Its Clinical Significance

Background and ObjectivesIn recent years, the incidence of Malignant lymphoma (ML) is increasing, and the incidence of non-Hodgkin's lymphoma (NHL) is about 7 times over that of Hodgkin's lymphoma (HD) in China. In clinic, the whole diagnosis of ML including subclassification, subtypes, clinical stages, with or without B symptom and prognostic factors, need to be worked out before treatment so that doctors can choose proper regimen and evaluate prognosis. However, there is some difficulty in over-all diagnosing. The resones are, on one hand, at present that diagnosis mainly depended on morphological and was assisted with immunohistochemical, which cannot still be maked sure whether they were lymphoma and their subtypes in some cases. On the other hand, Malignant lymphoma with bone marrow infiltration (BMI) is regarded as stage IV, but detecting rate of BMI is low and false negative rate is high by microscopic analysis of bone marrow smear. Generally, there is some difficulty with routine diagnostic methods in diagnosing, staging, evaluating prognosis and determining whether and how long the patients should continue to be treated. Therefore, it is important to set up a comprehensive assessment system of diagnosis and treatment for NHL.A great progress has been maked in management of ML with the utilization of new approach of treatment and antitumor drugs. However, a part of cases relapsed after treatment, and a few cases resisted to chemotherapy at the beginning of treatment, which is called relapsed/refractory lymphoma. The major cause of relapse is that there are some residual malignant cells after complete response in clinic. This minimal residual disease (MRD) cannot be found by morphological means. So a more sensitive method shoud be set up to detect MRD.With the development of molecular biology, the techniques for analysis of illness in gene level are gradually applicated in clinic, which provides an effective tool for the diagnosis of lymphomas. The antigen receptors genes (Ig and TCR) of lymphocytes become functional genes by undergoing rearrangements from germ-line cells to mature cells because of antigene stimulation. The rearrangement styles of antigen receptors genes of every B or T lymphocyte are completely different. During the differentiation and development of lymphocytes, if a lymphocyte in some stages transforms into malignant cell and then proliferates monoclonally malignant disease will form at last. Antigen receptors genes of normal lymphocytes of bone marrow in human being are amplified by PCR and smear phenomenon is observed after electrophoresis because of multiclonal proliferation and the different length of the gene band. However, the genes of NHL in BMI are amplified by PCR and a clear band is detected after electrophoresis because of monoclonal proliferation (except oligoclonal/subclonal rearrangement). One malignant cell can be identified from 106 normal cells by PCR. Therefore, detecting gene rearrangement by PCR can dig out infiltration of lymphoma cells in bone marrow, which can guide clinical treatment, estimate efficacy and evaluate prognosis.This thesis explores the effect of gene rearrangement on bone marrow of NHL by immunoglobulin heavy chain (IgH) and T cell receptor- Y (TCR- Y ), which can provide the clinical value in diagnosing, staging, guiding treatment, estimating the efficacy and evaluating prognosis of NHL. Materials and Methods1 Specimens1.1 The specimens were collected from bone marrow of 52 patients with non-Hodgkin lymphoma from 2003-6 to 2004-10. For 52 pations: 10-75 years old, 32 cases were male and 20 cases were female. According to Ann Arbor stage, 21 cases were stage I / II and 31 cases were stage III/IV. According to WHO classification of histopathology in 2001 year, 7 cases were low grade of differentiation and 45 cases were intermediate-and-high grade of differentiation. 11 specimens of bone marrow were collected after clinical complete response. These patients came from the oncology department of the First Affiliated Hospital of Zhengzhou University.1.2 Five specimens of bone marrow of not identified cases were collected from the oncology department of the First Affiliated Hospital of Zhengzhou University.1.3 The peripheral blood of twelve healthy donors was taken for control. 2 Methods2.1 Every specimen was studied by microscopic analysis of bone marrow smear before chemotherapy. After DNA was extracted from 2ml bone marrow that was taken from every case the gene rearrangement was detected. Then the differences of above-mentioned two methods were investigated. The differences of the gene rearrangement positive rate of NHL between early stage(stage I / II) and advanced stage(stageIII/IV) and between low grade of differentiation and intermediate-and-high grade of differentiation were observed. Positive control (human B-cell lymphoma cell line Raji or human T-cell lymphoma cell line Jurkat; or P -globin) and negative control (DNA of the peripheral blood of normal human being or non-DNA) were set up.2.2 The gene rearrangement was detected by polymerase chain reaction including semi-nest PCR and multi-primers PCR, and then the products of PCR were electrophoretic through 8% polyacrylamide and then the gels were stained with sliver.2.3 The assessing standard for PCR results2.3.1 The wide of bands was not more than lmm, and the edges were tidy.2.3.2 The band was in ranges of expecting bp.2.3.3 PCR was failure but not false negative result if there was neither smear phenomenon nor primer dimer.2.3.4 If there was a distinct main band from others in ranges of expecting bp and the location of the band was correct, this band was also regarded as positive band. 2.4 The Technological RouteBone marrow → Extracting DNA → PCR amplification Analysis←Sliver staining ←8%polyacrylamide gel electrophoresis3 StatisticalThe SPSS statistical package program 10.0 was used for analyses. The experimental data was analyzed with chi-square test. Statistical significant level was considered as "a equals 0.05". Results1 When the specimens of NHL were correctly diagnosed, the positive rate of gene rearrangement of bone marrow detected by PCR could reach 84.62%(44/52). The BMI positive rate by microscopic analysis of bone marrow smear was 30.77%(16/52). There was significant difference between above-mentioned two methods (P<0.001). Detected by PCR, the positive rates of BMI for patients in early stage(stage I / II) and in advanced stage(stageIII/IV) were 71.43%(15/21) and 93.55%(29/31), respectively. There was no significant difference between different stages (F>0.05). Detected by PCR, the positive rates of BMI for patients in low grade of differentiation and in intermediate-and-high grade of differentiation were 85.71 %(6/7) and 84.44%(38/45), respectively. There was no significant difference between different grades of differentiation (F>0.05).2 Five specimens of bone marrow of not identified cases were detected by PCR and four of them have smear phenomena. Two cases ware diagnosed as lymphadenitis through consultation of several pathological departments in Henan province. After flow cytometorical analysis two cases are acute monocyte leukemia. TCR- Y rearrangement was found in one case who was diagnosed as peripheral T cell lymphoma(NHL) by pathologists of our hospital. During chemotherapy, he was consulted by Xiehe Hospital in Beijing, and diagnosed as Hodgkin's lymphoma. For making sure the diagnosis, he wasconsulted again by a hospital in America. As a result, he was also diagnosed as NHL and TCR- Y rearrangement was found from wax-embedded tissue, just similar as what we found from bone marrow.3 The gene rearrangement of the bone marrow of 11 patients with lymphoma before and after chemotherapy was surveyed. Before chemotherapy, the rearrangements were found in all patients. The patients had complete response in clinic after chemotherapy, the rearrangements were found in five cases. One patient then was continuously treated with high-dose chemotherapy. After that, the gene rearrangement was turned negative by next checking up. The patient has been alive for 1.5 years without relapse. Disease of two patients rapid relapsed after therapy, one patient was died during the followed second-line scheme therapy, and the other patient who was treated with the second-line scheme and intrathecal injection is alive up to now. Continuously treated with single-drug, two patients of low-grade of differentiation NHL with positive band are alive up to now. The patients with negative rearrangements after chemotherapy are alive without relapse. Conclusions1 The positive rate of detected gene rearrangement by PCR is significant higher than that of cheked by microscopic analysis of bone marrow smear in bone marrow of NHL. There was no significant difference between different clinical stages and between different grades of differentiation.2 Gene rearrangements analyses in bone marrow of NHL could contribute to staging exactly and assisting in diagnosing.3 To monitor MRD by detecting gene rearrangement could guide clinical treatment and estimate efficacy.