Anatomic Pathology / SURVIVIN EXPRESSION IN CERVICAL NEOPLASIA

Survivin is an inhibitor of apoptosis protein (IAP) that is expressed in fetal development and in cancer. Survivin expression in premalignant lesions remains undefined. We obtained 73 samples of cervical squamous tissue, including 31 normal, 17 lowand 15 high-grade squamous intraepithelial lesions (LSILs, HSILs), and 10 squamous cell carcinomas (SCCs) from cone biopsy and hysterectomy specimens, and stained for survivin using an immunoperoxidase method. Nuclear staining was detected in normal mucosa, LSILs, and HSILs; staining intensity was greatest in cases with morphologic evidence of human papillomavirus (HPV) infection. In situ hybridization of serial sections demonstrated colocalization of HPV DNA and survivin. Cytoplasmic staining was observed in immature squamous metaplasia and in SCCs. Survivin expression in immature metaplastic squamous mucosa may reflect a role for survivin in normal squamous differentiation. However, the histologic correlation between nuclear staining and HPV infection suggests involvement of survivin in HPV-mediated disruption of normal cellular maturation. The most common fatal malignancy of women in many developing countries is cervical cancer, while in the United States, there has been a tremendous reduction in cervical cancer mortality owing to the success of diagnostic cytopathology.1-3 The majority of cervical cancers are squamous cell carcinomas (SCCs), and most cases are preceded by intraepithelial precursor lesions that can be divided into low-grade squamous intraepithelial lesions (LSILs), including condyloma/mild dysplasia, and high-grade squamous intraepithelial lesions (HSILs), comprising moderate dysplasia, severe dysplasia, and carcinoma in situ.4,5 While high-grade lesions seem to progress to invasive cancer at a higher rate than low-grade lesions, it is not possible to determine the risk of progression in individual SILs. Molecular markers of malignant potential may one day have an important role in the detection of lesions that have the greatest potential for progression to cancer and may also have a role in increasing the sensitivity of current diagnostic techniques. The 2 major classes of apoptosis inhibitors are the Bcl-2 family and the inhibitor of apoptosis protein family (IAP). The first IAP was identified in a baculovirus,6 and in recent years, many others have been identified in various mammalian species, including humans.7 Survivin seems to exert its primary antiapoptotic effect by binding to, and inhibiting, the proapoptotic group of proteases known as caspases (cysteine-containing aspartate-specific proteases), in particular, caspases 3 and 7.8 It also has been shown that survivin associates with microtubules of the mitotic spindle and that disruption of this interaction leads to a loss of survivin’s antiapoptotic function and increased caspase-3 activity.9 Survivin is unique among the human IAPs because it is expressed in most cancers but has been described only rarely in corresponding normal adult somatic tissues.9,10 Anatomic Pathology / ORIGINAL ARTICLE Am J Clin Pathol 2002;117:738-744 739 © American Society for Clinical Pathology However, the role of survivin expression in malignant transformation has not yet been evaluated. Furthermore, the expression of survivin protein in cervical cancer has not been reported. The purpose of the present study was to determine whether survivin is expressed in benign, potentially premalignant, or malignant lesions of the cervical mucosa. For this purpose, the immunohistochemical localization of survivin protein was evaluated in normal cervical squamous mucosa, LSIL, HSIL, and invasive SCC. Materials and Methods Production and Characterization of the Antisurvivin Antibody Antiserum to survivin was produced by immunization of New Zealand white rabbits with an amino-terminal survivin peptide sequence (PTLPPAWQPFLKDHRI) linked to keyhole limpet hemocyanin, as detailed by Ambrosini et al.11 Western blot analysis of the antiserum against 20 μg of total protein HeLa cell extract, using an immunoperoxidase method with enhanced chemiluminescence (Amersham Pharmacia, Buckinghamshire, England), showed reactivity of the antiserum with a single protein of approximately 16.5 kd, consistent with the published molecular weight of survivin. By contrast, reaction of the Western blot with preimmune serum showed no immunoreactivity. Immunoglobulins from the immunized rabbit and the preimmune serum were purified by affinity chromatography on a peptide-sepharose matrix, coupled with Protein A (Pierce, Rockford, IL). The specificity of the immunoglobulins for survivin was characterized by immunocytochemical evaluation of survivin expression in COS cells. A human complementary DNA (cDNA) corresponding to the full length of survivin was subcloned into the expression vector pSG5 (Stratagene, La Jolla, CA), to produce a survivin fusion protein bearing a Cterminal vesicular stomatitis virus glycoprotein epitope tag. The resultant plasmid vector was then expressed transiently into COS cells. Immunocytochemical analysis of the antisurvivin immunoglobulins against COS cells transfected with survivin cDNA using an immunoperoxidase detection method showed positive staining. By contrast, no staining was seen when COS cells transfected with expression vector lacking survivin cDNA were substituted for COS cells expressing survivin.