HPV strain prevalence and HPV-related biomarker expression in vulvar carcinoma at Tygerberg academic hospital.

Petersen, Nadine Samantha (2019-12)

Thesis (MMed)--Stellenbosch University, 2019.

Thesis

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.

AFRIKAANSE OPSOMMING: Die toename in die voorkoms van karsinoon van die vulva in jonger vroue kan moontlik aan 'n verhoogde infeksie met menslike papillomavirus (MPV) toegeskyf word. ‘MPV subtipes wat met vulvêre karsinoom geassosieer word, verskil in verskeie liggings. In Suid-Afrika word die histologiese subtipes van die karsinoom nie altyd gespesifiseer nie en die MPV-voorkoms in indringende vulvar plaveiselkarsinoom (iVPK) is grootliks onbekend. Die hoë voorkoms van MIV kan ook problematies wees, aangesien ‘n verswakte immuuunsisteem een van die risikofaktore vir die ontwikkeling van MPV-afhanklike vulvêre kanker is. Die doel van hierdie studie is dus om, met behulp van kwantitatiewe “real-time” polimerase kettingreaksie (kPKR), die MPV genotipes in formalin gefikseerde, paraffienwas-ingebede vulvêre intraepiteel neoplastiese (VIN) en iVPK weefsel te identifiseer. Die voorkoms van MPV subtipes word met MIV-status, sowel as kliniese en patologiese kenmerke asook die uitdrukking van biologiese merkers gekorreleer. ‘n Totaal van 72 VIN en 68 iVPK gevalle wat tussen Januarie 2003 en Junie 2018 by Tygerberg Akademiese Hospitaal gediagnoseer was, was in die studie ingesluit. DNS en RNS was geekstraeër en die kwaliteit en kwantiteit daarvan was met behulp van fluorometrie en spektrofotometrie bepaal. Die integriteit van die DNS was deur die kPKR amplifisering van 'n 205 bp fragment van die menslike ß-globien-geen, terwyl die van RNS met behulp van die RIN-nommer en die DV200-telling van die Agilent 2100 Bioanalyzer geanaliseer was. Die teenwoordigheid van MPV DNS was deur middel van kPKR en die gebruik van “primers” wat die E6 / E7-gene van MPV 16, 18, 11 en 35 teiken, ondersoek. Die uitdrukking van die MPV 16 en 18 E7 mRNA-transkripsies is met spesifieke “primers en probes” deur middel van kPKR ondersoek. Die resultate van MPV tipering is met histologiese diagnose en subgroepe sowel as MIV-status vergelyk. Verder is weefsel “microarrays” (WMA) ook saamgestel uit monsters met p16INK4a blok positiewe immunohistochemie en die kleurpatroon was tussen die WMAs en die volledige weefsel skyfies vergelyk. Resultate toon dat die grootste deel van ons studiepopulasie uit pasiënte tussen die ouderdomme van 36 tot 59 jaar wat gediagnoseer was met VIN en iVPK in die Tygerberg Akademiese Hospitaal tussen Januarie 2003 en Junie 2018, bestaan het. Boonop is daar gevind dat 'n beduidende deel van die pasiënte jonger as 59 jaar oud en MIV-positief was, ongeag van die diagnose, met 'n groot gedeelte selfs tussen 18 en 35 jaar oud. Oor die algemeen was DNS van voldoende gehalte en integriteit verkry en die menslike ß-globiengeenfragment is in alle vulvêre monsters geamplifiseer, wat daarop dui dat die DNS geskik was vir kPKR-ontleding. Net so was RNS uit alle geskikte monsters geekstraeër en dit was van voldoende gehalte vir die gebruik in kPKR, soos aangedui deur die DV200-tellings, RIN-getalle en die uitdrukking van GAPDH in monsters wat omgeskakel was na komplimentêre DNS. MPV-DNS is in 77,8 % van die VIN en 66,2 % van die iVPK-monsters opgespoor, met MPV 16 as die mees algemene MPV tipe wat by albei diagnose gevind was. Die meerderheid van die pasiënte waarvoor MPV-DNA gevind was, was ook met MIV geïnfekteer. Toe histopatologiese subtipes met die MPV DNA bevindings vergelyk was, was daar getoon dat 82 % van die VIN II / III (HSIL) en 37,5 % van die VIN I (LSIL) monsters positief vir MPV getoets het. 'n Verhoogde voorkoms van MPV was ook in die keratiniserende variante van die iVPK monsters gevind. Wat die analise van mRNA-transkripsie betref, was die proporsie van monsters waarin MPV 16 en MPV 18 mRNA uitgedruk was, effens minder as vir die waar MPV-DNA gevind was. Daar was egter 'n hoë ooreenkoms tussen die twee MPV-ontledingsmetodes gevind en 'n hoë sensitiwiteit en spesifisiteit was ook getoon. Twee WMA’s, een met MIV-positief en die ander met MIV-negatiewe monsters, was saamgestel uit monsters wat blok-positief vir p16INK4a IHC-kleuring was. Die spesifisiteit en sensitiwiteit van die p16INK4a IHC-kleuring op volledige skyfies en die TMA-skyfies was vergelyk en ‘n goeie ooreenkoms tussen die uitkoms van IHC kleuringspatroon was getoon. Ten slotte, hierdie bevindings het getoon dat VIN en iVPK by baie jonger vroue in ons studiepopulasie gediagnoseer was, in vergelyking met meer ontwikkelde lande. 'n Hoë voorkoms van MIV was opvallend in MPV-positiewe monsters, met MPV 16 die algemeenste subtipe wat gevind was. Resultate het ook aangedui dat die keratiniserende iVPK variante grootliks met MPV in ons studiepopulasie geassosieer was, wat ook teenstrydig is met wat gerapporteer word. Die bevindings van hierdie studie bied 'n mate van insig in die voorkoms van vulvêre karsinoom en hul voorloper letsels , wat die sterk assosiaise tussen MPV en die ontwikkeling van die kanker in ons omgeweing uitbeeld. Laastens is die gevolgtrekking gemaak dat WMA's gebruik kan word vir aanvullende toetsing van vulvêre monsters, met die doel om die tegnologie se talle voordele te benut, veral in die konteks van pre-infiltrerende MPV-geassosieerde navorsing. Die identifisering van nuwe biomerkes, wat die vordering vanaf voorloper letsels na infiltrerende siekte is steeds deurlopend en onbeslis, daarom word verdere navorsing egter benodig om die etiologie en patofisiologie van vulvêre kanker in Suid-Afrika beter te verstaan.

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