Bringing India to LSE: India Week 2013

As India Week 2013 draws to a close, Arisa Manawapat describes how Spice brought India to LSE

TMEM45A, SERPINB5 and p16INK4A transcript levels are predictive for development of high-grade cervical lesions

Women persistently infected with human papillomavirus (HPV) type 16 are at high risk for development of cervical intraepithelial neoplasia grade 3 or cervical cancer (CIN3+). We aimed to identify biomarkers for progression to CIN3+ in women with persistent HPV16 infection. In this prospective study, 11,088 women aged 20-29 years were enrolled during 1991-1993, and re-invited for a second visit two years later. Cervical cytology samples obtained at both visits were tested for HPV DNA by Hybrid Capture 2 (HC2), and HC2-positive samples were genotyped by INNO-LiPA. The cohort was followed for up to 19 years via a national pathology register. To identify markers for progression to CIN3+, we performed microarray analysis on RNA extracted from cervical swabs of 30 women with persistent HPV16-infection and 11 HPV-negative women. Six genes were selected and validated by quantitative PCR. Three genes were subsequently validated within a different and large group of women from the same cohort. Secondly, Kaplan-Meier and Cox-regression analyses were used to investigate whether expression levels of those three genes predict progression to CIN3+. We found that high transcript levels of TMEM45A, SERPINB5 and p16INK4a at baseline were associated with increased risk of CIN3+ during follow-up. The hazard ratios of CIN3+ per 10-fold increase in baseline expression level were 1.6 (95% CI: 1.1-2.3) for TMEM45A, 1.6 (95% CI: 1.1-2.5) for p16INK4a, and 1.8 (95% CI: 1.2-2.7) for SERPINB5. In conclusion, high mRNA expression levels of TMEM45A, SERPINB5 and p16INK4a were associated with increased risk of CIN3+ in persistently HPV16-infected women

Identification and characterisation of potential biomarkers for persistence and progression of a HPV16-infection

Eine persistente Infektion mit einem Hochrisiko (HR)-Typ humaner Papillomviren (HPV) ist ein notwendiger Risikofaktor für die Entstehung von zervikalen Krebsvorstufen und eines Zervixkarzinoms. Infektionen mit humanen Papillomviren sind häufig, jedoch verlaufen die meisten HPV-Infektionen transient und heilen spontan aus. In weniger als 30% der Fälle gelingt es dem Virus, sich langfristig zu etablieren und die Infektion bleibt bestehen. Eine Progression dieser persistenten Infektionen kann in ca. 50% dieser Fälle beobachtet werden. In prä-invasiven Läsionen und Zervixkarzinomen wird HPV16 am häufigsten gefunden, jedoch erkranken nicht alle Frauen mit einer HPV16-Infektion. Dies lässt darauf schließen, dass weitere Faktoren einen Einfluss auf den Ausgang einer Infektion mit einem HR-HPV Typ haben. Ziel dieser Arbeit ist die Identifikation solcher potentiellen Biomarker, welche nach einer einmaligen Bestimmung eine Aussage über das Progressionsrisiko einer Frau mit einer persistenten HPV-Infektion treffen können. Mit Hilfe solcher Marker kann die Lebensqualität infizierter Frauen gesteigert, und die wirtschaftliche Belastung reduziert werden. Mit Hilfe einer ca. 11 000 Frauen umfassenden dänischen Kohorte („Junge Dänische Kohorte“) konnten zwei neue prädiktive Biomarker für die Entwicklung einer schweren Dysplasie oder eines Karzinoms in persistent HPV16-infizierten Frauen identifiziert werden: TMEM45a und p16/CDKN2A zeigen eine signifikant höhere Expression in Frauen, welche innerhalb einer medianen Nachbeobachtungszeit von 12,9 Jahren eine hochgradige zervikale Läsion oder schwerer (CIN3+) entwickelten, jedoch zum Zeitpunkt der Probenentnahme eine normale Zytologie aufwiesen. Zuvor publizierte Ergebnisse, dass es sich bei TMEM45A um ein durch Hypoxie reguliertes Gen handelt, konnten bestätigt werden. Eine Induktion der TMEM45A mRNA unter hypoxischen Bedingungen konnte jedoch ausschließlich in HPV-haltigen Zellen beobachtet werden, in HPV-negativen Zelllinien blieb die Menge konstant oder wurde sogar reduziert. Ebenso konnten morphologische Zellveränderungen, Ablösen von Zellen und Zellsterben nach einem „Knock-down“ von TMEM45A durch siRNA nur in HPV-haltigen Zellen beobachtet werden. Eine anti-apoptotische Funktion wurde bereits beschrieben, jedoch konnte der Mechanismus, über den die anti-apoptotische Funktion vermittelt wird, nicht geklärt werden. Mittels RT-PCR wurde eine neue Transkriptvariante von TMEM45A identifiziert und es konnte gezeigt werden, dass sich das Protein in zytoplasmatischen Vesikeln befindet. Der Proteinnachweis von TMEM45A mittels Immunoblot war durch das Auftreten mehrerer unterschiedlich großer Banden erschwert. Nach der Analyse mehrerer kommerziell erhältlicher Antikörper, sowie der Herstellung eines weiteren anti-TMEM45A-Antikörpers konnte eine Bande identifiziert werden, deren Intensität sich durch Überexpression und siRNA „Knock-down“ spezifisch veränderte. Als mögliche Marker für die Persistenz und Progression einer HPV16-Infektion werden weiterhin sowohl die Viruslast als auch der Integrationsstatus des viralen Genoms diskutiert. Beide Marker wurden in Proben der bereits erwähnten Dänischen Kohorte, sowie einer zweiten, ca. 42 000 Frauen umfassenden Kohorte („LBC-Kohorte“), mit Hilfe der quantitativen Real Time PCR (qRT PCR) bestimmt. Es konnten in der Jungen Dänischen Kohorte signifikante Unterschiede der Viruslast und des Integrationsstatus zwischen transienten und persistenten Infektionen, jedoch nicht zwischen nicht-progredierenden und progredierenden HPV16-Infektionen gefunden werden. Für die Validierung dieser Resultate wurde eine Multiplex-PCR zur simultanen Bestimmung der beiden viralen Proteine E2 und E6, sowie eines Referenzgens zur Zellzahlbestimmung, etabliert. Die Ergebnisse dieser Messungen in der LBC-Kohorte zeigten jedoch überraschenderweise keine signifikanten Unterschiede der potentiellen Marker zwischen transienten und persistenten Infektionen. Aufgrund der Inkonsistenz der Ergebnisse der Viruslast und des Integrationsstatus kann deshalb keine eindeutige Aussage über deren Rolle als Marker für die Persistenz einer HPV16-Infektion getroffen werden.A persistent infection with a high risk (HR)- human papillomavirus (HPV) is a necessary risk factor for the development of cervical precancer and cancer. Most infections with human papillomaviruses can be cleared by the immune system. However, in less than 30% of the cases the virus establishes a persistent infection. HPV16 is found in most of all pre-invasive lesions and cervical cancers, however, progression of persistent infections can be observed in approximately 50% of all HPV16-infections. This suggests that more modifiers exist that influence the outcome of an infection with a HR-HPV type. The aim of this work is the identification of potential biomarkers with the ability to predict the risk of progression of persistently HPV16-infected women. Quality of life of infected women may increase and economical strain may be reduced by identifying such markers. By means of a large population-based cohort study („Young Danish Cohort“), comprising approx. 11 000 women, two predictive biomarkers for the development of high grade cervical lesions or cervical cancer were identified: The expression of TMEM45a and p16/CDKN2A was significantly higher in women who developed a high-grade cervical lesion or worse within a median follow-up time of 12.9 years, while having a normal cytology at baseline. In addition, previously published results about TMEM45A as a novel hypoxia regulated gene, were confirmed. However, induction of TMEM45A mRNA under hypoxic conditions could only be observed in HPV-containing cells; the amount of TMEM45A mRNA showed no increase but rather decrease in HPV-negative cell-lines. HPV-positive cells showed morphological changes including detachment and cell death after knock-down of TMEM45A by specific siRNAs. An anti-apoptotic function of TMEM45a was previously described, but the mechanism remains unclear. By means of RT-PCR a new transcript variant of TMEM45a was identified and we demonstrated that the protein is located in cytoplasmic vesicles. Detection of TMEM45a protein by western blot was aggravated due to the appearance of several bands at different sizes. After analysis of various commercially available antibodies, as well as the production of an additional anti-TMEM45a-antibody, it was possible to identify a band that specifically changed after TMEM45A overexpression and siRNA knock-down. Furthermore, viral load and physical state of the viral genome are discussed to be potential markers for persistence and progression of a HPV16-infection. Both potential markers were determined in samples of the Young Danish Cohort and in the LBC-Cohort, a second cohort comprising approx. 42 000 women, using quantitative real-time PCR. In the Young Danish Cohort, viral load and physical state showed significant differences between women with transient and persistent HPV16-infections but not between non-progressors and progressors. To validate these results, a multiplex-PCR for simultaneous detection of the viral proteins E2 and E6, as well as a reference gene for normalisation, was established. Surprisingly, no significant differences in viral load and physical state between transient and persistent HPV16-infections were observed in the LBC-cohort. Due to these inconsistencies concerning viral load and physical state, it is not possible to make a clear statement about their role as markers for persistence of a HPV16-infection

Physical state and viral load as predictive biomarkers for persistence and progression of HPV16-positive cervical lesions: results from a population based long-term prospective cohort study

Persistent infection with a high risk (hr) human papillomavirus (HPV) has been established as the main cause of cervical cancer and high-grade cervical intraepithelial neoplasia (CIN3). Because most infections are transient, testing for hrHPV lacks specificity and has a low positive predictive value. It has been suggested that additional parameters like viral load and physical status of the viral genome could improve the effectiveness of HPV-based screening. We investigated the association between HPV16 viral load and physical state with viral persistence or risk of incident CIN3 or worse in a population-based prospective cohort study comprising 8656 women (20-29 years). All participants had two gynecological examinations two years apart and were followed through the nationwide Danish Pathology Data Bank (median follow-up: 12.9 yrs). Seventynine cervical swabs from women with a persistent HPV16 infection were available for analysis. For comparison we selected a random age-matched sample of transiently HPV16 infected women (N=91). Persistently infected women with incident CIN3 or cancer (CIN3+; N=31) were compared to women with normal cytology during follow up (non-progressors; N=39). Quantitative real-time PCR for HPV16E6, E2 and IFNb1 was done to determine the HPV16 viral load and the E2/E6 ratio was used as a surrogate marker for integration. Women with normal cytology who became persistently HPV16 infected had a significantly lower HPV16 load at baseline than women who cleared the infection (median 4.72 copies/cell versus median 20.0 copies/cell, respectively; p=0.0003). There was no difference in viral load at enrollment between women who progressed to CIN3+ and women who stayed cytologically normal (p=0.85). At the second examination viral load tended to be higher in women who progressed, but the difference was not statistically significant (p=0.39). The E2/E6 ratio was shown to be lower in the persistently infected group (p<0.0001) already at the first examination, but no difference between non-progressors and CIN3+ cases was observed at any of the two examinations (p=0.61 and 0.86). Lower viral load and integration of the viral genome are predictive for the persistence of HPV16 DNA, but not for the progression of a persistent HPV16 infection to CIN3+ in women with normal cytology