HPV and potential prognostic markers in primary vaginal carcinoma

Abstract

The overall aim of this PhD thesis is to evaluate HPV status and immunohistochemical expression of different biomarkers, including tumor suppressor p16, proliferation marker Ki67, molecular markers in the LRIG family, WRAP53ß, and dyskerin as well as immune markers CD4+ and CD8+ TILs, and their correlation to clinical manifestations and survival as part of a search for potential prognostic factors in women diagnosed with primary vaginal cancer.

Paper I evaluates the presence of HPV in vaginal cancer tumor samples, as well as immunohistochemical expression of p16 and Ki-67. This study includes 68 short-term and long-term survivors diagnosed with vaginal cancer. The results, which have been correlated with both clinical parameters and survival, show presence of HPV in 43% of patients, with HPV16 found in 63% of the HPV-positive cases. HPV-positivity did not correlate with improved survival but did correlate with low histopathological grade. High expression of p16 was found in 54% of cases and correlated with low histopathological grade (p=0.004), HPV-positivity (p=0.032) and long-term survival (p=0.047). High expression of Ki-67, found in only 34% of patients, correlated negatively with histopathological grade (p<0.001) and tumor size (p=0.047). The results suggest that evaluation of p16 and Ki67 may be of value in tumor grading, while expression of p16 may also serve as a possible marker for HPV-positivity. In this study, high p16 expression, in contrast with positive HPV status and presence of Ki-67, was associated with improved survival in the univariate analysis, whereas multivariate analysis indicated that only histopathological grade and tumor size remain as independent prognostic factors. Paper II focuses on the LRIG (leucine-rich repeats and immunoglobulin-like domains) proteins – LRIG1, LRIG2 and LRIG3. Expression of these three proteins is often altered in cancer and has significance for cancer progression. The LRIG1 protein acts as a tumor suppressor, while the function of the remaining two is still unclear. We evaluated immunohistochemical expression of LRIG1, LRIG2, and LRIG3 in tumor samples from a cohort of 70 patients, diagnosed with vaginal cancer between 1975 and 2002, in order to find out whether such expression relates to clinical manifestations and survival. Our results show high (>50% of the cells) expression of LRIG1 and LRIG2 in 72% of tumors, but conversely, little or no expression of LRIG3. The latter two markers did not correlate with any clinical manifestations or survival, while high expression of LRIG1 correlated with HPV positivity and with improved cancer-specific survival (HR 0.35: 95% CI 0.68- 0.73) in vaginal cancer patients. Paper III addresses the molecular factors dyskerin and WRAP53ß in vaginal cancer. These two proteins play a role in the telomerase holoenzyme complex and are upregulated in different cancers. Expression of dyskerin and WRAP53ß was assessed by immunohistochemistry in 68 tumor samples drawn from the same study population as in study II. Most of the tumor samples demonstrated low to moderate expression of dyskerin. This protein is associated with shorter survival time and worse cancer-specific survival (HR 3.701: CI 95% (1.094-12.517). WRAP53ß was also expressed in most cells from the tumor samples, albeit without any association to clinical manifestations or prognosis. Paper IV is concerned with immune response as it relates to presence of CD4+ (Tumor Infiltrating Lymphocyte) TILs and CD8+ TILs in vaginal cancer tumor samples and also addresses the potential association between TILs, p16 expression and HPV status with clinical manifestations and survival. Once again, immunohistochemistry staining was used to evaluate CD4+ and CD8+ TIL infiltration along with p16expression in 69 tumor samples from the same study cohort used for the two previous studies. The results howed higher density CD4+ and CD8+ TIL infiltration in both HPV-positive and p16-positive tumors. High infiltration of CD4+ and CD8+ TILs in tumor samples implies better prognosis. Tumors demonstrating p16 overexpression in addition to high CD8+ TIL infiltration were associated with statistically significant (p=0.033) improvement in prognosis. In contrast, absence of p16 in HPV-negative tumors correlates with a substantially worse prognosis (p= 0.010).

In summary, the studies in this thesis, which is concerned with exploring potential prognostic markers in vaginal cancer, identify p16 as a prognostic marker of interest, especially when considered in light of HPV status. Moreover, LRIG1 and dyskerin may be novel prognostic markers of potential interest, while LRIG2, LRIG3, and WRAP53ß appear to fall short in this regard. Furthermore, CD8+ TILs may also be of interest as a prognostic factor, especially when considered together with HPV status and p16 expression. Although this thesis implicates p16 expression together with HPV status as clinically relevant prognostic factors in vaginal cancer, future studies, using larger study cohorts, will be needed to validate these results for improved diagnostics and treatment strategies for women diagnosed with vaginal cancer.