The need for an immunohistochemical marker of HPV-related precancer that is likely to progress is indisputable. The majority of the biomarkers used in pathologic diagnosis of squamous intraepithelial lesions (SILs), including p16, PCNA, MCM, ProEx C, reflect the presence of a high-risk HPV-related lesion, but are not specific for identification of progressive disease with potential to develop into an invasive carcinoma. It has been suggested that patterns of viral protein expression may be used to differentiate between self-limited productive viral infection and a true precancer. (6
Immunohistochemical detection of viral protein expression has been described in prior studies in cervical specimens. (14
) Kurman et al. reported immunohistochemical expression of PV (papillomavirus) common (structural) proteins in 43% of mild dysplasia, 15% of moderate dysplasia, and only in a rare case of severe dysplasia. (16
) In one study, immunohistochemical expression of L1 and L2 in two cases of condyloma and a CIN 2 lesion was identified in nuclei restricted to the upper epithelial layers. (14
A number of more recent studies have evaluated the expression of major capsid protein L1 in cytologic specimens. It has been shown that 30% to 75% of LSILs and 33% to 40% of HSILs express L1 in cytology specimens. (8
) Some of these studies evaluated the prognostic utility of L1 immunocytochemistry on a Pap sample in predicting lesion behavior. Most of the studies that relied on “cytologic regression” with variable length of follow-up (up to 2 years) associated the presence of L1expression with clinical “regression”. However, lesion regression is difficult to reliably establish in the clinical setting because histologic evaluation of the entire cervix is not possible in most cases and all other measures of outcome (Pap cytology or colposcopy with cervical biopsies) have imperfect sensitivity for detecting significant lesions. In addition, the diagnostic biopsy itself in some cases may be curative and result in removal of the entire lesion.
Our data are consistent with previously published studies using histologic specimens that demonstrated L1 expression in 30–65% of LSIL/CIN1. (22
) A lower proportion of the positive cases in histology compared to cytology specimens is likely related to the fact that in the majority of lesions the expression of capsid proteins is restricted to the most superficial layers of the epithelium. Thus, the keratinocytes in the superficial layers are most readily detached and harvested during sampling for a Pap test, but might also be inadvertently removed from the epithelial surface during colposcopy (mucous clearing etc.) and tissue handling during pathologic processing. A larger number of positive cases in cytology specimens compared to matched histologic preparations was previously described.(8
) Some studies evaluating the correlation of L1 and p16 expression in LSIL/CIN 1 have observed that lesions lacking L1 and demonstrating diffuse p16 expression are more likely to persist and progress.(25
) Diffuse p16 expression in these cases is most likely related to the presence of high-risk HPV within these lesions, most commonly HPV 16 and 18; these types are associated with lower regression rates compared to LSIL/CIN 1 harboring other HPV types,(28
) thus, the prognostic value of p16 expression is likely not independent of the HPV type implicated in the lesion development.
In our study, a significant fraction of LSIL/CIN 1 cases were L1 negative, but the value of L1 expression alone as a predictor of behavior in tissue specimens is unclear. The inability to detect L1 in these LSIL/CIN 1 cases may reflect inadequate sampling, given the extremely focal staining in some cases noted in this and other studies.(22
) Using cytology Pap samples likely provide a more representative sample of superficial lesional cells for analysis of L1 expression.
We have previously reported expression of L2 in 89% of LSIL/CIN 1 lesions and none of HSIL/CIN 2 and HSIL/CIN 3 in a study with a smaller number of cases.(10
) This data and our current data are different from what has been previously reported in another prior study that observed L2 expression in a significant proportion of moderate and severe dysplasias;(29
) this could be due to differences in histologic grading.
The discordant expression of L1 and L2 in the same lesion that was observed in 17 cases (43% of positive cases) in our study is surprising. This may be related to infection with multiple HPV types, with discordant expression related to antibody specificity in the setting of an HPV type other than 16 or 18. Thus, detection of both L1 and L2 proteins might not have added value if a more broadly HPV type-specific antibody for either L1 or L2 had been used.
All cases of HSIL/CIN3, either alone or with adjacent LSIL/CIN 1 or HSIL/CIN2, lacked= expression of capsid proteins. Statistically, expression of capsid proteins was negatively associated with the grade of the lesion (ptrend < 0.0001).
The absence of capsid protein expression observed in this study differs from data reported by Galgano and colleagues that recorded L1 expression in 16.5% of HSIL/CIN 3. (30
) These discordant findings could be related to fact that the current study was restricted to HPV16 and HPV18-related lesions, while the reported expression of L1 in HSIL/CIN 3 in the Galgano study was based on SILs irrespective of HPV type. Reported L1 staining in HSIL/CIN 3 in some other studies may also be related to the known world wide difference in histologic grading of SILs.
Recent studies evaluating methylation of HPV genes, specifically L1, found high levels of methylation of this gene associated with high-grade cytology. (31
) Consistent intense methylation of L1 gene was shown in HPV 18-related cervical carcinomas.(33
) These findings may provide further insight into the mechanisms of loss of capsid protein expression in higher grade SILs.
In our study, 62% of all LSIL/CIN1 lacked expression of both L1 and L2. The significance of this finding is not clear. Lack of detected expression in some of the negative cases could be a reflection of focality of staining and sampling. In other cases, it could represent a false negative result related to the presence of HPV types other than those that are covered by the antibodies used. Nonetheless, further investigation of the viral capsid protein expression as a marker of progression/persistence in LSILs in correlation with methylation analysis of the viral genome is warranted.
Eleven specimens contained discrete foci of both LSIL/CIN 1 and HSIL/CIN 3 (with or without HSIL/CIN 2). No expression of either L1 or L2 was observed in HSIL/CIN 3 foci. Interestingly, in these cases with multiple lesions/grades, the adjacent lower-grade components (LSIL/CIN 1 and HSIL/CIN 2) of these cases also lacked expression. The absence of capsid proteins expression in LSIL associated HSIL/CIN 3 may be related to molecular alterations that are associated with lesion progression but occur before the morphologic changes take place (e.g. integration, gene methylation, etc.). However, the exact mechanism responsible for the loss of L1 and L2 expression in these lesions is not known. It is also possible that these different grades of SIL represent independent lesions due to multiple HPV types that are not reactive with the antibodies used in this study. However, the number of specimens containing adjacent lesions of different grades is too small for drawing meaningful conclusions. Nonetheless, the hypothesis suggested by this observation, namely, that the presence of L1 and L2 expression might be used as a marker of absence of adjacent HSIL/CIN 3 in the same specimen, would be extremely useful clinically and warrants further examination.