Expression of Cell Cycle–Related Gene Products in Langerhans Cell Histiocytosis Bart Schouten, M.D., R. Maarten Egeler, M.D., Ph.D., Pieter J.M. Leenen, Ph.D., Antonie H.M. Taminiau, M.D., Ph.D., Lambert J.C.M. van den Broek, and Pancras C.W. Hogendoorn, M.D., Ph.D. Background: The pathogenesis of Langerhans cell histiocytosis (LCH), a disease characterized by an abnormal accumulation of the dendritic Langerhans cells, is still unknown. Based on the monoclonality of the CD1a+ cell and reports of familial clustering, it is hypothesized that a genetic alteration at a cellular level may be causative. This genetic change may have an effect on the cellular mechanisms controlling proliferation and apoptosis. Materials and Methods: LCH-lesions were studied for the ex- pression of Ki-67, present in the nucleus of proliferating cells. Furthermore, the expression of cell cycle-related gene products TGF- receptor I and II, MDM2, p53, p21, p16, Rb, and Bcl2 were studied. The TGF-R genes play a role in tumor suppression, whereas Bcl2 inhibits apoptosis. The remaining genes are part of either the p53-p21 and/or p16-Rb pathways, which induce cell cycle arrest or apoptosis in response to DNA damage. Results: In 30 biopsies the diagnosis of LCH could be confirmed on the basis of CD1a positivity (27 bone and 3 skin). All cases showed scattered nuclear-positive staining for the proliferation marker Ki-67. In more than 90% (n 27) of these cases, expres- sion of TGF receptor I and II, MDM2, p53, p21, p16, Rb, and Bcl2 was detected in lesional LCH cells. The overexpression was in general heterogeneous, ranging from limited focal staining of scattered cells within the lesion to strong diffuse staining. Conclusions: These findings suggest that the cellular mechanisms that sense and respond to DNA-damage, namely the p53-p21 path- way and the p16-Rb pathway, are activated. The expression of Ki-67 indicates that the cells in LCH are proliferating. The ob- served overexpression of Bcl2 may play a role in the activation of p53 and p16 and/or the arrest of apoptosis. Key Words: Langerhans cell histiocytosis—Cell cycle gene prod- ucts (Ki-67, TGF  R, p53, MDM2, p16, Rb, p21, Bcl2)— Apoptosis—Proliferation. L angerhans cell histiocytosis (LCH) is a poorly under- stood and occasionally aggressive disorder that features lesional cells akin to Langerhans cells. Although these LCH cells seem to be key players, T-cells, macrophages, and eosinophils are also usually seen within the LCH-lesions (1). The LCH cells, in all clinicopathologic forms except the smoking-related adult pulmonary LCH, have been proven to be clonal (2–5). This has been demonstrated in multiple patients, as well as in biopsies of multiple sites. The clonal- ity of the LCH cells indicates that a genetic basis for LCH is likely. Furthermore, familial cases of LCH have been documented (6). Although such familial LCH cases are rare, these cases add to the concept of an underlying genetic defect. Genetic alterations at the cellular level might cause disruptions controlling proliferation and apoptosis (7). Until now, few studies of the various factors involved in cell cycle by LCH cells have been reported. Only p53, MDM2, and Bcl2 expression have been investigated in small numbers of LCH cases (8,9). Both studies showed upregulation of p53 and Bcl2 expression, but no mutations or genomic rear- rangements could be detected using the single strand con- formational polymorphism (SSCP) technique (8,9). Expres- sion of MDM2, a regulator of p53 activity, could not be detected (8). Disturbances of cell proliferation and apoptotic pathways may be fundamental in the development of LCH. Thus, we investigated the expression of a number of key factors in the complex system that controls proliferation and apoptosis, namely TGF receptor I and II, p21, p16, Rb. Together with MDM2, p53, and Bcl2, we aimed to assess various factors involved in regulating LCH cell multiplica- tion and survival. MATERIALS AND METHODS Patients Between February 1986 and December 2000, 80 patients were diagnosed with LCH at the Leiden University Medical Center (LUMC), which acts as a reference center for bone tumors. Of 33 patients, sufficient representative formalin- fixed, paraffin-embedded material was available for the present study. In 30 patients the disease was located in the bone; three patients had a lesion of the skin. In all cases the diagnosis was confirmed by immunohistochemistry for S100 and CD1a, together with its characteristic light micro- scopic characteristics as specified by the Writing Group of the Histiocyte Society (10). Submitted for publication November 12, 2001; accepted February 15, 2002. From the Departments of Pathology (B.S., L.V.D.B., P.C.W.H.), Pe- diatrics (B.S., R.M.E.), and Orthopedics (A.H.M.T.), Leiden University Medical Center, Leiden, The Netherlands; and Department of Immunol- ogy, Erasmus University and Academic Hospital Rotterdam, Rotterdam, the Netherlands (P.J.M.L.). Address correspondence and reprint requests to R. Maarten Egeler, M.D., Ph.D., Immunology, Hematology, Oncology, Bone Marrow Trans- plantation and Autoimmune Diseases, Department of Pediatrics, Rm J6- 222, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. E-mail: R.M.Egeler@lumc.nl. Journal of Pediatric Hematology/Oncology, Vol. 24, No. 9, December 2002 © 2002 Lippincott Williams & Wilkins, Inc. 727