Diagnostic Cytology
Fig. 7.49 Human papillomavirus (HPV)
of the cervix. These smears have been
stained with rabbit anti-HPV antibodies using
immunoenzymatic reaction (immunoenzymatic
stain with chromogen DAB x HP).
investigated. Other oncogenic HPV types (31, 33, 35, 39, 45, 51,
52, 56, 58, 59, and 68), in conjunction with HPV types 16 and
18, are responsible for more than 95% of cervical squamous
carcinoma and its precursors and in excess of 90-95% cervical
adenocarcinoma and its precursor adenocarcinoma in situ. HPV
types 16 and 18 have also been found in several human cervical
cancer cell lines, including the famous HeLa cell. Success has
been achieved in transformation experiments conducted using
HPV subgenomic particles and human epidermoid cell lines.
Additional molecular evidence linking HPV to cervical cancer
has been demonstrated by experimental data showing that the
viral genes E6 and E7 of high-risk HPVs can extend the life span
of genital epithelial cells. E7 can disrupt the cell cycle by bind-
ing to pRB with up-regulation of cyclin E and p16INK4. E6 can
interrupt cell death by binding to p53 and preventing replicative
senescence by up-regulation of telomerase. Both genes have
been shown to induce centrosome duplication and genomic
instability.73,79-83 More recently, the strong association of HPV
with cervical cancer has resulted in the development of vaccines
directed against the most common oncogenic HPV types 16 and
18.108 These vaccines have been shown to prevent infection and
the development of precancerous lesions due to these subtypes,
and one has been FDA approved for use in girls aged 9-26.
Historical Perspective
The typical cytomorphologic changes
now associated with HPV infection were first documented by
Ayre in 1949.85 Papanicolaou, in his
Atlas of Exfoliative Cytology
published in 1954, presented magnificent illustrations depict-
ing cellular changes currently recognized as HPV-associated.86
Koss and Durfee in 1956 used the term koilocytic atypia to
describe the surface epithelial changes of the cervix and its rela-
tionship with cancer.87 Naib and Masukawa in 1961 published a
paper entitled "Identification of Condyloma Acuminata Cells in
Routine Vaginal Smears."88 Additional papers on the same topic
were published by Ayre,89 Sagiroglu,90 and De Girolami.91 Ayre
for the first time suggested a possible viral etiology of cervical
dysplastic lesions.89 In the mid-1970s, Meisels and Fortin, and
Purola and Savia independently defined a set of cellular changes
that are associated with cervical condylomatous lesions and
HPV infection.72,92 Also, flat condyloma, indistinguishable from
cervical dysplasia, was recognized as a separate viral-related
entity. Laverty and co-workers93 demonstrated the presence of
virus particles within the koilocytes in cervical tissues ultrastruc-
turally. In 1980, Woodruff and colleagues demonstrated the
presence of HPV capsid antigen in genital condylomas using
polyclonal rabbit antihuman wart virus antibodies.94 Gupta
and associates employed Papanicolaou and immunoenzymatic
stained cells in cervical smears and demonstrated viral particles
in cervical dysplastic cells identified by the viral antigen detec-
tion system.95, 96
Immunoenzymatic studies may reveal the presence of HPV
antigens in 0.5-5% of the cells in cervical smears. Abundance of
HPV infection, accompanying inflammatory reaction (G.
and the degree of dysplastic changes determine the propor-
tion of the antigen-positive cells. In up to 80% of these cases,
corresponding cervical biopsy tissue may reveal identical results.
Antigenically stained cells may include parabasal, intermedi-
ate, and metaplastic types (Fig. 7.49). Antigen positivity has no
relationship to age, but is instead dependent on the degree and
depth of condylomatous change in the squamous epithelium.
Nearly 90% of the cases with two-thirds thickness involvement
may reveal the HPV antigen. Condylomatous and low-grade
lesions (CIN grade I) reveal HPV antigens more commonly.
Nucleic acid hybridization was described in 1975 by South-
ern.97 It has been used extensively for study of HPV DNA in
vaginal secretions and infected cells, both in the smears and in
tissues. Brahic and Haase developed in situ hybridization, which
permits localization of labeled DNA or RNA probes within the
cellular preparations and tissue sections.98 In 1987, Gupta and
co-workers used radiolabeled HPV DNA probes and established
the presence of viral genomes in infected dysplastic cells in
cervical smears96 (Fig. 7.50). RNA probes have been developed
and used by Stoler and Broker among others.99 Radiolabeled
probes have more recently been replaced by immunoenzymatic
techniques. Filter dot and PCR techniques have been used to
investigate the presence of extremely small quantities of the viral
genome in infected specimens and to identify latent infection.
This has also allowed investigation of the relationship of specific
viral types to human disease. More recently, a signal amplifi-
cation hybrid capture technique was developed by Lorincz to
detect the presence of HPV in liquid cytology samples.100,101
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