Identi®cation of novel molecular markers which correlate with HPV-induced tumor progression Matthias Nees 1,3 , Erwin van Wijngaarden 1,4 , Evi Bakos 1 , Achim Schneider 2 and Matthias DuÈrst 1,5 1 Deutsches Krebsforschungszentrum, ATV 0615, Im Neuenheimer Feld 242, D-69120 Heidelberg; 2 Abteilung Frauenheilkunde, Friedrich-Schiller-Universita Èt, Bachstr. 18, D-07740 Jena, Germany High risk HPVs (human papillomaviruses) are causally involved in the development of cervical cancer. However, other factors, such as somatic genetic alterations also play a decisive role in malignant conversion of cervical keratinocytes. Mutations and chromosomal aberrations are known to in¯uence the pattern of gene expression. Therefore we used the recently developed RT ± PCR based method of dierential display of mRNAs to detect dierences in gene expression which correlate with tumorigenicity. Non-tumorigenic HPV16-immortalized human foreskin keratinocytes (HPK IA) were compared with their tumorigenic counterparts and 49 dierent genes were identi®ed which were either up- or down- regulated. The identi®ed genes encode proteins of the cytoskeleton and the extracellular matrix, the nuclear splicing apparatus, transcription regulators and mem- brane-associated proteins. The expression pattern of all genes was also examined by RNA ± RNA in situ hybridization in biopsies of normal cervical epithelium, precancerous lesions and cancers. Two genes, C4.8 and C21.7, are of particular interest because their expression is upregulated in a subset of high grade precancerous lesions and in over 58% of cancers. These two genes may therefore be considered as putative progression markers. C4.8 is a new member of the transmembrane 4 (TM-4) protein family which includes tumor-associated antigens such as CD63 and TAPA-1, whereas C21.7 shows no signi®cant homologies to any known genes or proteins. Keywords: HPV (human papillomavirus); cervical cancer; prognostic markers; dierential display; tumor progression Introduction The world wide incidence of cervical cancer is in the range of 5 ± 48 per 100 000 women. The lowest and highest rate is observed in Spain and Columbia respectively (Muir et al., 1987; Ponten et al., 1995). It is generally accepted that cervical cancer arises from histopathologically de®ned precursor lesions referred to as high grade intraepithelial lesions (HSIL). These precursors lesions may either arise from human papillomavirus-(HPV) induced low grade lesions (LSIL) or directly from the HPV-infected epithelium (Kiviat et al., 1992; Koutsky et al., 1992; Schiman, 1992). Clearly, infection with high-risk HPV types is a sine qua non component for the development of cervical cancer but the carcinogenic process itself is of multistep nature. This is not only evident by the long delay between HPV infection and malignant conversion of the target cell but also by the accumulation of cytogenetic abnormalities such as chromosomal imbal- ances, allel loss and structural aberrations which are characteristically observed in cervical cancer. These genetic alterations have also been observed in HSIL, albeit to a much lesser extend (Heselmeyer et al., 1996). The clinical value of HPV detection for screening for SIL has been shown in several studies (Reid et al., 1991; Cuzick et al., 1995; Schneider et al., 1996). However, the majority of SIL identi®ed in screening programs regress spontaneously and identi®cation of progression markers is mandatory in order to avoid overtreatment. Viral markers such as presence of high risk HPVs (Herrington et al., 1996); virus load (Cuzick et al., 1996; Nindl et al., 1996), pertinent HPV detectability (Hildesheim et al., 1994; Londesborough et al., 1996), or the presence of speci®c HPV genotype variants (Xi et al., 1995; Londesborough et al., 1996) are clinically not useful for predicting the progressive potential of SIL due to low positive predictive value. Genetic hallmarks prognostic for the development of cervical cancer may be more valuable in this context. Mutations and chromosomal aberrations may in¯uence the expression of as yet unknown cervical cancer- related genes. To take advantage of this possibility, we decided to use the recently developed RT ± PCR based method for dierential display of mRNAs to detect stable altered expression patterns which correlate with dierent stages of HPV-induced disease. By the use of several combinations of short arbitrary primers for reverse transcription and PCR, theoretically all mRNA species of a particular tissue or a given cell can be ampli®ed (Liang and Pardee, 1992; Liang et al., 1992, 1993). The procedure is semi-quantitative and permits the detection of dierentially expressed genes simply by comparing the PCR products of two RNA extracts. In recent years several cell culture systems have been introduced to study the mechanism of the multistage carcinogenic progression of cervical cells. Oncogenic genital HPV types are capable of immortalizing human primary epithelial cells in culture (DuÈrst et al., 1987; Pirisi et al., 1987; Tsutsumi et al., 1992; Woodworth et al., 1988). This property is not shared with HPV6 or HPV11 which generally induce benign lesions only (Schlegel et al., 1988; Woodworth et al., 1989). HPV- immortalized keratinocytes can acquire a malignant phenotype after transfection with an activated ras- Correspondence: M DuÈrst Current addresses: 3 NCI, Laboratory of Biology, Bethesda, MD 20892, USA; 4 Netherlands Cancer Institute, Division of Molecular Carcinogenesis, Plesmanlaan 121, NL-1066 CX Amsterdam, Netherlands; 5 Abteilung Frauenheilkunde, Friedrich-Schiller- UniversitaÈt, Bachstr. 18, D-07740 Jena, Germany Received 19 September 1997; revised 9 December 1997; accepted 11 December 1997 Oncogene (1998) 16, 2447 ± 2458  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc