Deregulation of the RB pathway is shared by most human malignancies. Components upstream of the retinoblastoma tumour suppressor (pRB), namely the INK4 family of cyclin-dependent kinase (CDK) inhibitors, the D-type cyclins, their partner kinases CDK4/CDK6, and pRB as their critical substrate, are differentially targeted in diverse types of cancer. An 'unorthodox' spectrum of defects within this cascade occurs in testicular germ cell tumours (TGCTs), including silencing of pRB transcription, overexpression of cyclin D2, and loss of p18INK4c. To improve understanding of the role of this pathway in spermatogenesis, and its subversion in TGCTs, we examined immunohistochemical expression patterns of CDK4, p16INK4a, p15INK4b, and pRB, and established an in situ assay for cyclin D-mediated phosphorylation of serine795, a phosphorylation event critical for neutralization of pRB's growth-restraining ability. pRB was expressed throughout adult spermatogenesis and was detectable in teratomas, but was absent or grossly reduced in carcinoma in situ (CIS) and most seminomas and embryonal carcinomas. Unexpectedly, we also found that pRB was absent from fetal human gonocytes, the candidate target cell for all types of TGCTs. Thus, rather than a tumorigenesis-promoting loss of pRB, the lack of pRB in TGCTs likely reflects its developmental control. Widespread expression of p15INK4b, found in normal testes, was preserved in TGCTs. In contrast, p16INK4a was lost or reduced in large subsets of TGCTs. CDK4 was expressed in normal spermatogonia, CIS, and invasive TGCTs, as was serine795-phosphorylated pRB. Our data on expression of pRB support the plausible origin of TGCTs from fetal gonocytes, and the serine795 phosphorylation demonstrates that the cyclin D-dependent kinases are active, and neutralize pRB in spermatogonia and in those TGCTs that express pRB. We hope that this study will inspire further immunohistochemical applications of phosphospecific antibodies in pathology, and examination of the RB pathway defects in relation to curability of TGCTs. Copyright 2003 John Wiley & Sons, Ltd.

Department of Cell Cycle and Cancer Institute of Cancer Biology Danish Cancer Society Copenhagen Denmark