Establishment Of An Animal Model For Thymic Lymphoma Induced By N-methyl-N-nitrosourea In Inbred Mice

【Objective】To establish an animal model for thymic lymphoma in inbred mice induced by intraperitoneal injection of DNA alkylating agent N-methyl-N-nitrosourea (MNU), which would mimic this kind of human malignant lymphoma. Histopathologic, immunohistochemical, ultrastructural and nested PCR studies were performed to detect the the origin of tumor cells and to identify the subtype classification of induced tumors.【Methods】1. In the first part of our study, 52 male and female mice of the C57BL/6 strain at 6-8 weeks of age were randomly assigned to exposure and control groups, 40 and 12 animals respectively. Briefly, the exposure group were injected by the intraperitoneal route with MNU solution shortly after dissolution at a dosage of 50 mg/kg body weight. The control group received the same dosage of sterile 0.9% NaCl solution. The experimental animals were boosted 8 weeks later. Following injection of MNU, the mice were examined frequently. Seriously ill animals were killed and with mice found dead were completely autopsied. All mice were sacrificed by cervical dislocation for autopsy before the 22th experimental week. Complete gross examination was performed for detection of tumor masses.2. In the second part, thymic lymphomas as well as involved organs were fixed and routinely processed for light and electron microscopy examination to analyze histopathologic and ultrastructure features of the tumors. Sections were submitted to immunohistochemical staining with CD3, CD20, TdT and PCNA to identify the origin and subtype classification of the neoplasias.3. In the third part, 8 samples of thymic lymphoma and 4 samples of thymus tissue were frozen immediately in liquid nitrogen after removed and stored at -70℃. Genomic DNA was isolated from the thymic tumors and thymus tissues. Analysis with nested polymerase chain reaction(PCR) for TCRβand TCRγclonal gene rearrangement was carried out to determine the lineage and clonality of the T-cell lymphoma cells. The PCR products of TCRγgene rearrangement were purified and directly sequenced.【Results】1. The exposure group presented a mean body weight significantly lower than the control at the end of the experiment. At the 22th week, the proportion of MNU-treated animals developing thymic lymphoma after injection of MNU was 67.5%(27?40). No significant sex difference in the incidence was observed. Extensive invasion into other organs was found in 77. 8% of affected animal.2. Histopathologic study revealed that 27 cases of thymus became totally replaced by sheets of cells of the lymphoid series. The thymic tumors were densely infiltrated with relatively uniform, mitotically active, medium-sized cells with round to ovoid and sometimes convoluted nuclei that were surrounded by small amounts of cytoplasm. The chromatin pattern was finely granular and prominent nucleoli were present in most of the cells which had a high mitotic rate. There may be a"starry sky"pattern in some cases. All the tumors coexpressed CD3 and TdT. On transmission electron microscopic study, the mean diameter of the malignant lymphocytes was 7.02±1.12μm. Their nuclearcytoplasmic ratio was high, and their plasma membranes were smooth. The nuclear profiles were usually regular, with varying percentage of convoluted nuclei. Most nuclei contained one or two, usually eccentrically located nucleoli. Few cell organoids were observed in cytoplasm. Howere, apoptosis was common. 3. 8 cases of thymic lymohomas were all detected TCRβand TCRγclonal gene rearrangement. The positive rate was 100%. In the 4 cases of control group, no positive detected. DNA sequence analysis confirmed that the PCR products generated were indeed TCRγrearrangements.【Conclusions】1. As was confirmed, thymic lymohoma in mice can be induced by twice intraperitoneal administration of MNU. The biological behavior of the MNU-induced tumors resembled those of human thymic lymphomas derived from thymic T-cells.2. All the cases classified by histopathologic, immunohistochemical and ultrastructural studies as precursor lymphoblastic lymphoma were unquestionably related to the thymus origin and T-cell lineage.3. Just as determined by TCRβand TCRγgene rearrangement analysis and DNA sequence analysis, the MNU-induced thymic lymphoma in mice was of a T-cell origin and of monoclonal origin.In conclusion, MNU-induced thymic lymphomas in mice show strong parallels, all in their biological behavior and morphology and immunophenotype and genetics, to human thymic lymphoma. It confirmed that our cases may represent the murine counterpart of human tumor entity.