Masters Degrees (Anatomical Pathology)

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Browsing Masters Degrees (Anatomical Pathology) by Subject "Biomarkers"

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    HPV strain prevalence and HPV-related biomarker expression in vulvar carcinoma at Tygerberg academic hospital.
    (Stellenbosch : Stellenbosch University, 2019-12) Petersen, Nadine Samantha; Sanderson, Micheline; Razack, Rubina; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Anatomical Pathology.
    ENGLISH ABSTRACT: The incidence of vulvar carcinoma in younger women has increased, possibly due to augmented human papillomavirus (HPV) infection. Variable geographic prevalence of HPV that are associated with vulvar carcinoma exists. In South Africa, histological subtyping is not always specified and the HPV prevalence in invasive vulvar squamous cell carcinoma (iVSCC) is largely unknown. High HIV prevalence could also be problematic as one of the risk factors for HPV-dependent vulvar cancer is impaired immunological status (Saidu 2016). The objectives of this study are to identify HPV subtypes in vulvar intraepithelial neoplasia (VIN) and iVSCC FFPE tissue using quantitative real-time polymerase chain reaction (qPCR) and to correlate HPV strain prevalence results with HIV status, clinicopathological features and biomarker expression. The study samples comprised of FFPE vulvar tissue from patients diagnosed with VIN and iVSCC at Tygerberg Academic Hospital between January 2003 and June 2018. Altogether, 72 VIN and 68 iVSCC FFPE tissue samples were included. DNA and RNA were isolated from FFPE vulvar tissues and the quantity and purity were assessed using fluorometry and spectrophotometry, respectively. The integrity of the derived DNA was determined through amplification of a 205 bp fragment of the human β-globin gene, while that of RNA was analysed with the RIN number and DV200 score of the Agilent 2100 Bioanalyzer. The presence of HPV DNA was investigated using qPCR employing primers targeting the E6/E7 gene of HPV 16, 18, 11 and 35. The expression of the HPV 16 and 18 E7 mRNA transcripts was examined with specific primers and probes using qPCR. HPV DNA detection results were correlated with histological diagnosis and subgroup as well as HIV status. Furthermore, tissue microarrays were also constructed of p16INK4a block positive immunohistochemistry samples and the staining pattern between the TMA and full sections were compared. Results show that patients between the ages of 36 to 59 years diagnosed with VIN and iVSCC at Tygerberg Academic Hospital between January 2003 and June 2018 were the largest proportion of our study population. Additionally, a significant proportion of the patients who were younger than 59 years, irrespective of diagnosis, were found to be HIV positive, with a large portion even being between 18 and 35 years old. Total DNA and RNA were extracted from the included FFPE vulvar tissue blocks. Overall, DNA of sufficient quality and integrity was obtained as a 205 bp human β -globin gene fragment was amplified in all FFPE vulvar samples, suggesting that the DNA was fit for qPCR analysis. Similarly, RNA was extracted from all applicable samples and was of adequate quality for use in qPCR as indicated by the DV200 scores, RIN numbers and the expression of GAPDH in samples converted to cDNA. HPV DNA was detected in 77.8 % of VIN and 66.2 % of iVSCC samples with HPV 16 being the most prevalent strain detected in both diagnoses. The majority of patients for which HPV DNA were detected were found to be co-infected with HIV. When histopathological subtypes were compared with HPV DNA detection, results showed that 82 % of VIN II/III (HSIL) and 37.5 % of VIN I (LSIL) samples tested positive for HPV. Notably, a high HPV prevalence was also found in the keratinizing variants of the samples representative of iVSCC. Regarding mRNA transcript analysis, overall, the proportion of samples in which HPV 16 and HPV 18 mRNA was expressed were slightly less than for HPV DNA detected. However, a high concordance was found between the two HPV detection methods, with high sensitivity and specificity obtained. Two tissue microarrays, one with HIV positive and the other with HIV negative samples, were constructed using samples that were block positive for p16INK4a IHC staining. When comparing the specificity and sensitivity of the p16INK4a IHC staining on full sections slides to that of TMA slides, results showed good agreement between scoring. In conclusion, these findings showed that VIN and iVSCC were diagnosed in women of much younger age in our study population compared to more developed countries. A high HIV prevalence was noticeable in HPV positive samples, for which HPV 16 was the most common subtype detected. Results also indicated that iVSCC keratinizing variants were largely associated with HPV in our study population which were also in contrast to what is reported. The findings of this study provide some insight into the prevalence of vulvar carcinoma and its precursor lesions, emphasizing the strong association of HPV in disease development in our setting. Lastly, it was also concluded that TMA’s may be utilized for ancillary testing of vulvar specimens, with a view of exploiting this technology for its multitude of advantages, especially in the context of pre-invasive HPV associated research. The search for new biomarkers, to predict progression from precursor lesions to invasive disease is still ongoing and inconclusive, therefore, further studies are needed to aid the better understanding of the aetiology and pathophysiology of vulvar cancer in South Africa.