Chinese-German Journal of Clinical Oncology

May 2011, Vol. 10, No. 5, P286–P289

DOI  10.1007/s10330-011-0728-4

The significance and features of apoptosis and proliferation of NK/T cell lymphoma* Dabin Wang1, Meng Ming1, Junhua Liu1, Jianhua Yi2, Dianding Zou3   Department of Pediatrics, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, China   Department of Pathology, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, China 3   Department of Pediatrics, Zhongnan Hospital, Wuhan University, Wuhan 430071, China 1 2

Received: 26 October 2010 / Revised: 21 December 2010 / Accepted: 25 February 2011 © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2011 Abstract  Objective: The aim was to study the features and clinical significance of cell apoptosis and proliferation of NK/T cell lymphoma. Methods: TdT-mediated dUTP nick end labeling and immunohistochemical Streptavidin-peroxidase method were used to study cell apoptosis and the expression of proliferation cell nuclear antigen in 25 NK/T cell lymphoma and 10 reactive lymphoid tissues. Results: Apoptotic index (AI) and proliferative index (PI) averaged (1.92% ± 0.86%) and (41.48% ± 5.10%) respectively in the 25 NK/T cell lymphomas and (6.70% ± 1.89%) and (20.10% ± 2.77%) in the 10 reactive lymphoid tissues. Compared with reactive lymphoid tissues, AI was significantly reduced in NK/T cell lymphoma (t = 10.80, P < 0.01) while PI significantly increased (t = 12.39, P < 0.01). In addition, in NK/T cell lymphoma, AI and PI were positively related (r = 0.69, P < 0.01). Conclusion: In NK/T cell lymphoma, cell apoptosis is reduced while cell proliferation increased. The imbalance between cell apoptosis and cell proliferation is closely related to the development and progression of NK/T cell lymphoma. Key words   NK/T cell lymphoma; apoptosis; proliferation cell nuclear antigen

NK/T cell lymphoma is a rare primary extranodal nonHodgkin’s lymphoma, a therioma which causes progressive destruction to nasal and facial midline. The incidence of NK/T cell lymphoma in China is the highest in the world, but its’ occurrence and development mechanism is not fully understood. Apoptosis is a programmed cell death (PCD) controlled by genes, the imbalance of apoptosis and proliferation are closely related to the occurrence and development of tumor. TdT-mediated dUTP nick end labeling (TUNEL) and immunohistochemical streptavidin-peroxidase method (SP) were used to study cell apoptosis and the expression of proliferation cell nuclear antigen (PCNA) in 25 NK/T cell lymphoma and 10 reactive lymphoid tissues, to explore the role of apoptosis and proliferation in NK/T cell lymphoma occurrence and development.

Correspondence to: Dabin Wang. Email:[email protected] * Supported by a grant from the Top Researches Foundation from Hubei Provincial Education Office (No. B200624014).

Materials and methods General information Samples were taken from the archiving wax block in the Department of Pathology, Yunyang Medical College Hospital, China, from January 1998 to June 2003, samples were all fixed with 10% formalin, paraffin-embedded, HE stained, undergone the analysis of histologically forms and CD45RO, CD3ε, CD56, CD20 immunohistochemistry, and then undergone TIA-1, Granzyme B immunohistochemical staining, EBER1/2 in situ hybridization, they were all clearly diagnosed according to the WHO new classification in 2000 [1]. Sixteen males and 9 females aged from 8 to 67 were included, no radiotherapy or chemotherapy had ever been done before the diagnosis. The most common site was nasal cavity, with 19 cases (76.00%), followed by the nasopharynx with 3 cases, 2 cases of palate, 1 of oropharynx. Another 10 cases of nasopharyngeal lymphoid tissue of reactive hyperplasia were taken as control. Experimental methods Immunohistochemical streptavidin - peroxidase method (SP) CD45RO, CD56, CD20, Granzyme B monoclonal antibody, CD3ε polyclonal antibody, mouse anti-human

Chinese-German J Clin Oncol, May 2011, Vol. 10, No. 5

PCNA monoclonal antibody (ready to use) and SP immunohistochemistry kit was purchased from Fuzhou Maixin Biotechnology Development Company (China); TIA-1 monoclonal antibody were products of DAKO, purchased from Gene Company Limited (Denmark). Methods: 4 μm sections of paraffin were routinely dewaxed to water; 3% H2O2 blocking endogenous peroxidase for 10 min; microwave boiled to repair antigen; 10% normal goat serum sealed for 10 min; adding the primary antibody, kept overnight at 4 ℃; then added the secondary antibody of Biotechnologylabeled incubated at room temperature for 30 min; plus enzyme labeled streptavidin horseradish avidin, incubated at room temperature for 10 min; DAB coloration, hematoxylin, routine dehydration, transparent, sealed. Positive film provided by the kit company was taken as the positive control, instead of primary antibody, PBS was taken as negative control. TUNEL apoptosis detection TUNEL kit was purchased from Beijing Zhongshan Biotechnology Co., Ltd (China). The 4 μm sections of paraffin were routinely dewaxed to water; 3% H2O2 sealed for 30 min; proteinase K (20 μg/mL dissolved in Tris/HCL with pH7.4) incubated for 20 min at 37 ℃; incubated in TUNEL reaction mixture at 37 ℃ for 60 min; peroxidaseanti-fluorescein antibody incubated at 37 ℃ for 30 min; DAB coloration, hematoxylin, routine dehydration, transparent, sealed. Nucleotide mixture contained reaction solution, instead of TUNEL reaction mixture was taken as a negative control. EBER1/2 in situ hybridization Fluorescein isothiocyanate (FITC) labeled EBV peptide nucleic acid (PNA) probe (EBER-PNA, DAKO Y5200) and in situ hybridization detection kit (DAKO K5201) were purchased from Gene Co. Limited (Denmark). 4 μm paraffin routinely dewaxed to water; 3% H2O2 processed for 10 min at room temperature; protease digestion for 20 min; add drops of FITC labeled EBER-PNA probe, covered with silica coverslip (Sigma, USA); oven incubated for 2 h at 55 ℃; adding rabbit Anti-FITC/alkaline phosphatase (AP) labeled antibody, kept for 30 min at room temperature; penta-tetrachlorodibenzo-Three indole phosphate/nitrogen blue tetrazolium (BCIP/NBT) coloration for 1 hour, stained again with methyl green; routine dehydration, transparent, plugging and sealing. Positive and negative control probes were used in the known EBV-positive T-cell lymphoma. Result judgments CD45RO, CD56, CD20 membrane protein positive tumor cells showed brown staining; CD3, TIA-1, Granzyme B protein positive tumor cells showed brown cytoplasmic fine granular staining; EBER1/2 positive signals were in the nucleus, showed a purplish blue color, no stained cy-

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toplasm and cell membrane. Apoptosis and proliferation were positive when brown or brown granules appeared in the nucleus. A high magnification view of 5–10 was selected fort each case, randomly count the number of PCNA and apoptosis positive cells in 1000 tumor cells, with its percentage as a cell proliferation index (PI) and apoptosis index (AI), the data was expressed with χ ± s. Statistical analysis SPSS 10.0 was employed for the t-test, and Fisher exact test.

Results NK/T cell lymphoma histological and immunological phenotypes All 25 cases of lesions had different degrees of coagulation necrosis, inflammatory cell infiltration, tumor cell was medium or large sized and of pleomorphic nature, nuclear shape was distorted. Immune markers: CD45RO, CD3ε and TIA-1 positive in 25 cases (100%), granzyme B-positive in 22 cases (88%), CD20 negative in all (100%). Among 25 cases of lesions, 21 were EBER1/2 positive (84%). Lymphoma cells apoptosis of NK/T cell In NK/T cell lymphoma, TUNEL positive cells were individually scattered, often accompanied by some morphological changes typical of apoptosis such as cell shrinkage, cytoplasmic condensation, nuclear margination of chromatin condensation and apoptotic body formation (Fig. 1). In reactive proliferation of lymphoid tissues, the TUNEL positive cells mainly scattered in lymphoid follicle germinal center, and was rarely found in the mantle zone and interfollicular areas (Fig. 2). AI averaged (1.92 ± 0.86)% in 25 cases of NK/T cell lymphoma tissues, and in 10 cases of reactive hyperplasia of lymphoid tissue, the AI averaged (6.70 ± 1.89)%, so they were of statistically significant difference (t = 10.80, P < 0.01). Lymphoma cell proliferation of NK/T cell In NK/T cell lymphoma tissue, PCNA-positive cells were of diffused or scattered distribution (Fig. 3). In reactive lymphoid hyperplasia, a high positive rate of PCNA was found in germinal centers of lymphoid follicles, and negative in mantle zone cells, PCNA-positive cells scattered in interfollicular areas (Fig. 4). PI averaged (41.48 ± 5.10)% in 25 cases of NK/T cell lymphoma tissues, in the reactive lymphoid hyperplasia PI averaged (20.10 ± 2.77)%, the difference was of statistical significance (t = 12.39, P < 0.01).

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Fig.  1 Nasal NK/T cell lymphoma, TUNEL marked positive nucleuses were in brown color and individually scattered, often accompanied by some morphological changes typical of apoptosis such as cell shrinkage, cytoplasmic condensation, nuclear margination of chromatin condensation (TUNEL × 400) Fig.  2 Chronic amygdalitis, TUNEL marking the positive cells mainly scattered in lymphoid follicle germinal center Fig.  3 Nasal NK/T cell lymphoma, PCNA immune detection, nucleuses in proliferation were positive, and the positive cells were of diffused distribution (SP × 400) Fig.  4 Chronic amygdalitis, PCNA immune detection, a high positive rate of PCNA was found in germinal centers of lymphoid follicles, and negative in mantle zone cells, PCNA-positive cells scattered in interfollicular areas (SP × 400)

The correlation of lymphoma cell apoptosis and cell proliferation of NK/T cell The linear correlation analysis indicated that the AI and PI of NK/T cell lymphoma in 25 cases were significantly positively correlated (r = 0.69, P < 0.01).

Discussion NK/T cell lymphoma refers to the peripheral T and NK cell lymphoma established in the new WHO classification of lymphoma in 1997 [1, 2], it is a highly invasive therioma which associates with the EB virus (Epstein-Barr virus, EBV). Its tumor cells could express a number of cytotoxic factors (TIA-1, Granzyme B, perforin), it usually expresses both the differentiation antigen of T-cell (UCHL1, CD3, CD8, etc.) and the NK cell associated antigens (CD16, CD11b, CD56, CD57, etc.), but its exact properties of the cells are still controversial. In the patient group above, there were 25 cases of CD45RO, CD3ε and TIA-1 positive (100%), 22 cases of Granzyme B positive (88%), 25 cases of CD20 negative (100%), suggesting that tumor cells derived from the cytotoxic T lymphocytes or NK cells. In 25 cases of lesions, there were 21 cases of EBER1/2 positive (84%), confirmed that NK/T cell lymphoma was a therioma highly associated with EB virus. In the organic body, the dynamic balance of apoptosis and proliferation of cells is an important means to maintain the steady state of the body, the imbalance between the two will lead to the accumulation of genetic variation, extension of cell growth cycle, eventually leads to cancer. Not only the indefinite proliferation but also the apoptosis of cells exists in the tumor, abnormal regulation, less active proliferation and weakened apoptosis of the two are important reasons for the occurrence and development of tumor [3]. Apoptosis, as an important form of programmed cell death, its form and characteristics of biochemical change are represented by nuclear chromatin condensation, disappearance of cell morphology, and the formation

of DNA fragments, the presence of apoptotic cells could be observed in tissue sections with the technique of DNA fragments in situ end labeling (TUNEL). In the process of apoptosis, genomic DNA will produce double-stranded break, low molecular weight DNA fragments and high molecular weight single-stranded DNA ends (gap), these gaps of DNA chain can be identified by the approach of enzyme labeling nucleotides 3’ ends. In the kits employed in this study, the terminal deoxynucleotidyl transferase (TdT) catalyzed the free 3’ terminal nucleotide polymerization in non-catalytic template-dependent manner, eventually labeled the gap of DNA chain ends. PCNA (proliferating cell nuclear antigen, PCNA), also known as cyclin (cyclin), is a 36 kD nuclear antigen, it is a subsidiary protein of DNA polymerase δ, and is necessary for cell proliferation and DNA synthesis. The emergence of PCNA signifies that the cells are in active proliferative state, it is an objective indicator to reflect cell proliferation status. The emergence of PCNA is significantly related with cell proliferation, in quiescent cells, there’s few of it, in the later period of G1, it starts to increase, then reaches the peak at S phase, and significantly decreased at G2 and M phase. PCNA is a good marker of proliferated cell nuclear, also a good secondary indicator of cell proliferation activity, it is helpful for the identification of a variety of benign and malignant tumors, the classification and grading of histological type and patient prognosis. Leoncini et al studies [4] indicated that the expression level of PCNA is related to the degree of malignancy of non-Hodgkin’s lymphoma (non-Hodgkin’s lymphomas, NHL), the higher the degree of malignancy, the higher the cell proliferation activity was. In China, He et al studies [5, 6] have also confirmed that the expression level of PCNA can reflect the malignant degree and prognosis of NHL in a fairly objective manner. In this experiment, the application of TUNEL assay of apoptosis and immunohistochemical assay of PCNA expression could reflect the level of tumor cell apoptosis

Chinese-German J Clin Oncol, May 2011, Vol. 10, No. 5

and proliferation in a relatively objective and sensitive manner. The results indicate that in NK/T cell lymphoma, apoptosis index (AI) averages (1.92 ± 0.86)%, cell proliferation index (PI) averages (41.48 ± 5.10)%; and compared with reactive lymphoid tissue hyperplasia, NK/T cell lymphoma is significantly lower at AI, but significantly higher at PI, and the differences are significant (t = 10.80, t = 12.39, P < 0.01), suggesting a decrease of cell apoptosis in NK/T cell lymphoma, and an active cell proliferation, inhibition of tumor cell apoptosis is accompanied by excessive cell proliferation, the imbalance of apoptosis and cell proliferation might play an important role in the occurrence and development of NK/T cell lymphoma. The imbalance between cell apoptosis and proliferation is closely related with the occurrence and development of various tumors, in the study a correlation analysis is conducted between the apoptosis index (AI) and cell proliferation index (PI) of 25 cases of NK/T cell lymphoma. The results indicate a positive correlation between the AI and PI of NK/T cell lymphoma (r = 0.69, P < 0.01), the higher rate of apoptosis rate, the higher rate of proliferation rate accordingly. This may be related with the choice mechanisms occurred when the different cell clone subsets in the same tumor competing with each other, some tumor cells such as clone subsets of low-proliferative potential or cell subsets at the end of proliferation may die when they are induced by clone subsets of high proliferative potential, so the latter can get better conditions for the rapid growth of the tumor. Leoncini et al [4, 7, 8] studies have indicated that, there’s a non-linear correlation between NHL cell proliferation and apoptosis, that is to say, the high portion of cell proliferation is often accompanied by a high portion of cell apoptosis but has nothing to do with histological type. The results of this research are consistent with those reported above, suggesting that cell proliferation and apoptosis, the two pathological processes which have opposite effects on tumor, are not independent but influencing each other; they jointly influenced the occurrence and development of nasal NK/T cell lymphoma through a particular mechanism. The limitations of material, necrosis, inflammatory

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background, small amount and no obvious atypia of tumor cells, as well as the lack of colonel immune phenotype make the diagnosis of NK/T cell lymphoma a very difficult job, adding up to the condition and economic constraints, some detection such as EB virus in situ hybridization, TCR gene rearrangement analysis are hard to be implemented in routine work. The immunohistochemistry detection of PCNA expression and TUNEL detection of apoptosis is a simple and economic method with a high specificity and sensitivity. Combined detection of tumor cells in AI and PI to reflect the growth kinetics and biological behavior of tumor characteristics can be important indicators of a NK/T cell lymphoma clinical diagnosis and prognosis.

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