BMC Cancer 2008, 8:379 http://www.biomedcentral.com/1471-2407/8/379 the three transcription factors (E2A, EBF, and PAX5) to be expressed, and E2A together with EBF regulates the expres- sion of PAX5.[3,4] The inhibitor of DNA binding ID2 (inhibitor of DNA binding 2 or inhibitor of differentia- tion, ID2), may bind and negatively regulate E2A and PAX5[5,6] by direct interaction. ID2 expression in devel- oping hematopoietic cells seems to repress B-cell develop- ment and B-cell-specific gene expression and to favor development of other lineages, [7-11] whereas in mature B cells, ID2 is up-regulated during plasma cell differentia- tion with concomitant loss of expression of several B-cell genes.[12] Furthermore, the balance between ID2 and E2A, and between ID2 and PAX5 seem to be important for B-cell differentiation.[13,14] ID2 has been found strongly and uniformly expressed in the HRS cells of classic Hodg- kin Lymphoma and probably represses B-cell-specific gene expression by inactivation of E2A (and perhaps also PAX5). [14] Expression of two other members of the ID (inhibitor of differentiation) family of proteins, ID1 and ID3, was found to be induced in the presence of latent membrane protein 1 (LMP1), the Epstein-Barr virus onco- protein, by the activation of NF-kappaB, phosphatidyli- nositol 3-kinase, mitogen-activated protein kinase, and c- Jun N-terminal kinase signaling[15,16]. However, there is to date no study on the relationship between ID2 and EBV-LMP1 in Hodgkin lymphoma. Overexpression of P16(INK4A)[17] has been used as a diagnostic adjunct in premalignant and malignant humanpapilloma virus (HPV) lesions in gynecologic pathology[17] in recent years. Previous research [18] also suggested that EBV- LMP1 and P16(INK4A) had a role in the carcinogenesis of classical Hodgkin lymphoma, but there was no corre- sponding investigation of associations between ID2, EBV- LMP1 and p16(INK4A), which might provide further understanding of the mechanism. Therefore, additional studies are needed to identify whether ID2, EBV-LMP1 and P16(INK4A) have similar diagnostic value and to explore the possible relationship between them in classi- cal Hodgkin lymphoma. We used the ID2, EBV-LMP1 and P16(INK4A) antibodies to investigate the possible role of the expression of the three proteins in 60 cases of Chinese classical Hodgkin lymphoma. It is hoped that the study will give information on the pathogenesis of this disease. Methods Patients Sixty patients (36 men, 24 women) with histologically confirmed classical Hodgkin lymphoma were recruited into the study. Ethical approval for this study was not required by our institute as the experiments carried out did not relate to patient's privacy, impairment or treat- ment. The Hodgkin lymphomas were classified as nodular sclerosis (n = 29), mixed cellularity (n = 11), lymphocyte- rich (n = 16) and lymphocyte-depleted (n = 4) variants according to the World Health Organization recommen- dations [1]. Immunohistochemistry A formalin-fixed, paraffin-embedded block was selected from each patient with Hodgkin lymphoma for immuno- histochemical study. Paraffin sections, 4 μm in thickness, were cut, dewaxed in xylene and rehydrated in a graded ethanol series. Then sections were immersed in 3% hydro- gen peroxide in methanol for 10 minutes to block endog- enous peroxidase activity and rinsed in running water. Subsequently they were immersed in boiling 0.01 M cit- rate buffer (pH 6.0) in a pressure cooker, which was then sealed and brought to full pressure for 2 minutes. It was then de-pressured and cooled under running water. The lid was then removed, and the hot buffer was flushed out with cold water from a running tap. The cooled sections were washed twice in phosphate buffered saline (PBS) before immunocytochemical staining. The sections were then covered the primary antibodies. These polyclonal rabbit antibody against human ID2 pro- tein (Santa Cruz, CA, USA), mouse monoclonal antibody to Latent membrane protein 1 of the Epstein-Barr virus (EBV-LMP1) (CS.1–4, DAKO) and mouse monoclonal antibody against human P16(INK4A) protein (Zymed, South San Franciso, CA, USA), all diluted 1 in 100. After exposure to primary antibody for 1 hour, the sections were allowed to react with poly peroxidase-anti-mouse/ rabbit IgG for 20 minutes by the standard PV-6000 Poly- mer Detection System (Zymed, South San Franciso, CA, USA). The system is a non-biotin detection system, thus, avoiding non-specific staining due to endogenous biotin. The sections were then washed in water, counter-stained with Mayer's haematoxylin for one minute at room tem- perature, dehydrated, cleared and mounted. Human cervical squamous cell carcinoma tissues were used as a positive control for P16(INK4A), smooth muscle cells of vessels were used as an internal positive control for ID2 and human nasopharyngeal carcinoma was used as a positive control for EBV-LMP1. Negative controls were sections treated as above but with the primary antibody replaced by 0.01 M PBS. Expression of P16(INK4A), ID2 and EBV-LMP1 was considered positive when at least 10% of HRS cells were stained. In addition, 10 B-cell lympho- mas, 8 T-cell lymphomas and 10 reactive lymph nodes (4 lymph nodes sampled during resection of carcinomas and 6 lymphadenitis) were used for comparison. The B-cell lymphomas comprised 5 diffuse large B-cell lymphomas, 4 extranodal marginal zone B-cell lymphomas (MALT lymphomas) and 1 Burkitt lymphoma. The T-cell lym- phomas were 2 anaplastic large cell lymphomas, 2 extran- odal NK/T-cell lymphomas of nasal type, 2 Page 2 of 5 (page number not for citation purposes)