Christine Weyn

Evaluation of Combined General Primer-Mediated PCR Sequencing and Type-Specific PCR Strategies for Determination of Human Papillomavirus Genotypes in Cervical Cell Specimens

ABSTRACT A strategy combining human papillomavirus general primer (mainly the PGMY primers)-directed PCR sequencing and type-specific PCR is presented. DNA samples were first tested in general primer-mediated PCR. The amplified fragments of positive samples after ethidium bromide-stained DNA gel analysis were further sequenced, and corresponding DNA samples were further analyzed by PCR using type-specific primers for human papillomavirus (HPV) types 16, 18, 31, and 52. The comparison of the results of 157 samples analyzed by this strategy in parallel with the Hybrid Capture 2 tests and with the HPV INNO-LiPA (Innogenetics line probe assay) shows that this method is suitable for HPV detection and genotyping in cervical cell samples. Although the PCR sequencing method is as sensitive as the HPV INNO-LiPA for HPV detection, our method allows the identification of a broader range of HPV types. In contrast, the HPV INNO-LiPA was less time-consuming and better identified coinfections.

Effects of HPV-16 E5, E6 and E7 proteins on survival, adhesion, migration and invasion of trophoblastic cells

Among high-risk human papillomaviruses (HPV), HPV-16 infection is the most prevalent causative factor for cervical cancer. Beside other mucosal targets, HPV-16 was reported to infect the placenta and to replicate in trophoblastic cells. Since these cells share invasive properties of tumoral cells, they represent an ideal model to investigate several oncogenic processes. In the present work, we analyzed the impacts of HPV-16 E5, E6 and E7 oncoproteins on the trophoblastic model. Our results showed that E5 impaired the viability of trophoblastic and cervical cell lines but E6 and E7, favoring cell growth, neutralized the E5 cytotoxic effect. In addition, E5 decreased the adhesiveness of trophoblastic cells to the tissue culture plastic and to endometrial cells similarly as described previously for E6 and E7. E5 and E6 plus E7 increased also their migration and their invasive properties. Cells expressing HPV-16 early proteins under the control of the long control region endogenous promoter displayed growth advantage and were also more motile and invasive compared with control cells. Interestingly, the E-cadherin was downregulated in trophoblastic cells expressing E5, E6 and E7. Nuclear factor-kappaB and activator protein-1 activities were also enhanced. In conclusion, HPV-16 early proteins enhanced trophoblastic growth and intensify the malignant phenotype by impairing cell adhesion leading to increased cellular motile and invasive properties. HPV-16 E5 participated, with E6 and E7, in these changes by impairing E-cadherin expression, a hallmark of malignant progression.

Evidence of Human Papillomavirus in the Placenta

Human papillomavirus (HPV) is an epitheliotropic virus typically infecting keratinocytes but also possibly epithelial trophoblastic placental cells. In the present study, we set out to investigate whether HPV can be recovered from transabdominally obtained placental cells to avoid any confounding contamination by HPV-infected cervical cells. Thirty-five placental samples from women undergoing transabdominal chorionic villous sampling were analyzed, and we detected HPV-16 and HPV-62 in 2 placentas. This study suggests that HPV infection of the placenta can occur early in pregnancy. The overall clinical implication of these results remains to be elucidated.

Detection of ALK expression in non‐small‐cell lung cancer with ALK gene rearrangements – comparison of multiple immunohistochemical methods

Testing for ALK rearrangements in advanced, non‐squamous non‐small‐cell lung cancers that are wild‐type for activating EGFR mutation has become standard care. Fluorescence in‐situ hybridization is considered the gold standard for this evaluation. Pre‐screening with immunohistochemistry has been suggested, to reduce testing costs and to make testing more widely available. By analysing the sensitivity and specificity of different ALK immunohistochemical assays, we aimed to identify the most reliable assay to detect ALK rearrangement.

Detection of HPV and the role of p16INK4A overexpression as a surrogate marker for the presence of functional HPV oncoprotein E7 in colorectal cancer.

BackgroundBased on the well-recognized etiological role of human papillomavirus (HPV) in cervical, anogenital and oropharyngeal carcinogenesis, a potential role of HPV in colorectal carcinogenesis has been suggested. For that reason, the aim of the present study was to investigate the presence of HPV DNA in colorectal carcinomas (CRC) and to study overexpression of p16INK4A as a marker for the presence of an active HPV oncoprotein E7. These findings were correlated with clinical and pathological prognostic factors of CRC.MethodsThe presence of HPV was assessed using a multiplex PCR system of 10 non-biotinylated primers. The amplified fragments of HPV positive samples were further analyzed by a highly sensitive, broad spectrum SPF10 PCR and subsequently genotyped using reverse hybridization in a line probe assay.P16INK4A protein expression was investigated in a subset of 90 (30 HPV positive and 60 HPV negative) CRC samples by immunohistochemistry.ResultsHPV DNA was found in 14.2% of the CRC samples with HPV16 as the most prevalent type. No significant differences in clinical and pathological variables were found between HPV positive and negative CRCs, except for age. HPV positive patients were significantly younger (p = 0.05). There was no significant correlation between the presence of HPV and overexpression of p16INK4A (p = 0.325).ConclusionsIn conclusion, the presence of oncogenic HPV DNA in a small cohort of CRC samples may suggest that HPV may be involved in the carcinogenesis of some CRC. However, contrary to what has been observed in head and neck squamous cell cancer and cancer of the uterine cervix, p16INK4A does not seem to be a surrogate marker for an active HPV infection in CRC. Therefore, further functional analyses are necessary to elucidate the role of HPV in CRC.

Regulation of human papillomavirus type 16 early gene expression in trophoblastic and cervical cells

We compared the outcome of different cellular and viral factors on the regulation of the HPV-16 early viral gene expression in trophoblastic and cervical cancer cells. A high variability of the long control (LCR) activity was observed, prompting us to evaluate the role of secreted factors in the control of the early gene expression in trophoblastic cell lines. Endogenous progesterone and exogenous dexamethasone were found to activate LCR driven transcriptional activity. Since host cells express HPV early proteins to regulate LCR activity, we investigated the effect of the combined HPV-16 early proteins on the LCR driven transcription and the possible involvement of E2. A physiological level of HPV-16 early proteins expression strongly induced the LCR driven reporter activity. According to mutational analysis, E1 and E2 proteins, indispensable for viral replication, were not involved in LCR extrachromosomal transcriptional regulation. This suggests that E5 and/or E6 and/or E7, consequently, activated viral transcription.

Clinical performance evaluation of a sensitive, rapid low-throughput test for KRAS mutation analysis using formalin-fixed, paraffin-embedded tissue samples

BackgroundTesting for KRAS mutations in metastatic colorectal cancer (mCRC) on formalin-fixed, paraffin embedded (FFPE) tumor tissue has become standard of care. Different molecular methods exist to determine hotspot KRAS mutations in exon 2, 3 and 4, but testing is often limited by the sensitivity and the speed of analysis. The aim of this retrospective study was to establish the clinical performance of the Idylla™ KRAS Mutation Test on FFPE tumor samples of patients with mCRC.MethodsKRAS mutation analysis was performed using the therascreen KRAS on the RotorGene Q platform (CE-IVD; Qiagen) and results were subsequently compared to the Idylla™ KRAS Mutation Test. Discordant result testing was performed with massive parallel sequencing or alternative routine approaches.ResultsData from 182 samples were used to show that the overall agreement between the two methods for mutation characterization was 96.7% [95%CI: 93.0%-98.5%]. Six out of 182 samples (3.3%) showed true discordant results.ConclusionThe Idylla™ KRAS Mutation Test allows for a fast and reliable analysis of FFPE samples with a turnaround-time of two hours without the need of molecular infrastructure or expertise in order to guide the personalized treatment of colorectal cancer patients.

Extrachromosomal HPV-16 LCR transcriptional activation by HDACi opposed by cellular differentiation and DNA integration

Histone deacetylase inhibitors (HDACi) have been shown to render HPV-carrying cells susceptible to intrinsic and extrinsic apoptotic signals. As such, these epigenetic drugs have entered clinical trials in the effort to treat cervical cancer. Here, we studied the effect of common HDACi, with an emphasis on Trichostatin A (TSA), on the transcriptional activity of the HPV-16 Long Control Region (LCR) in order to better understand the impact of these agents in the context of the HPV life cycle and infection. HDACi strongly induced transcription of the firefly luciferase reporter gene under the control of the HPV-16 LCR in a variety of cell lines. In the HaCaT keratinocyte cell line undergoing differentiation induced by TSA, we observed a reduction in LCR-controlled transcription. Three major AP-1 binding sites in the HPV-16 LCR are involved in the regulation by TSA. However, whatever the status of differentiation of the HaCaT cells, TSA induced integration of extra-chromosomal transfected DNA into the cellular genome. Although these data suggest caution using HDACi in the treatment of HR HPV infection, further in vivo studies are necessary to better assess the risk.

Multidisciplinary molecular tumour board: a tool to improve clinical practice and selection accrual for clinical trials in patients with cancer

Background The complexity of delivering precision medicine to oncology patients has led to the creation of molecular tumourboards (MTBs) for patient selection and assessment of treatment options. New technologies like the liquid biopsy are augmenting available therapeutic opportunities. This report aims to analyse the experience of our MTB in the implementation of personalised medicine in a cancer network. Materials and methods Patients diagnosed with solid tumours progressing to standard treatments were referred to our Phase I unit. They underwent comprehensive next generation sequencing (NGS) of either tumour tissue or cell-free circulating tumour DNA (ctDNA) or both. The MTB expressed either a positive or negative opinion for the treatment of the patients with discovered druggable alterations inside a clinical trial, in an expanded access programme, with a compassionate use. Afterwards, discovered alterations were matched with OncoKB levels of evidence for the choice of alteration-specific treatments in order to compare MTB outcomes with a standardised set of recommendations. Results NGS was performed either on ctDNA or tumour tissue or in both of them in 204 patients. The MTB evaluated 173 of these cases. Overall, the MTB proposed alteration-specific targeted therapy to 72 patients (41.6%). 49 patients (28.3% of the total evaluated) were indicated to enter a clinical trial. In 29 patients with matched liquid biopsy NGS (lbNGS), tumour tissue NGS (ttNGS) and MTB evaluation, the MTB changed the treatment strategy coming from standardised recommendations based on lbNGS and ttNGS alone in 10 patients (34.5%), thanks to the evaluation of other clinical parameters. In our cohort, lbNGS was more likely, compared with ttNGS, to detect point mutations (OR 11, 95% CI 2.9 to 24.1, p<0.001) and all-type alterations (OR 13.6, 95% CI 5.5 to 43.2, p<0.001) from the same genes of matched patients. Conclusions Our MTB allows patients with refractory cancer to be included in clinical trials and improves the precision of clinical decisions compared with a standardised set of mutation-driven recommendations.

Inter-laboratory variability in the presence of human papillomavirus in normal and abnormal cervical cytology samples.

INTRODUCTION Treatment of cervical carcinoma is affected by the stage of the disease, being most likely curable with early detection. High-risk human papillomavirus (HPV) infection and persistency are necessary to the development of precancerous and cancerous lesions leaving the possibility to detect in time cells progressing in at risk behaviour. METHODS This study documents the proportion of HPV DNA positivity in 906 samples with cytological result negative for intraepithelial lesion and malignancy (NILM), 220 atypical squamous cells of undetermined significance (ASC-US) samples and 211 low grade intraepithelial lesions (LSIL) samples collected from various pathological laboratories in Brussels, Belgium. RESULTS The proportion of samples harbouring one or more HPV types was 10.8% (95% confidence interval, 95% CI: 8.8-12.8) in NILM, 34.5% (95% CI: 27.6-40.3) in ASC-US, 54.3% (95% CI: 47.5-61.1) in LSIL, with significant variability of HPV proportion in ASC-US and LSIL between laboratories. CONCLUSION This study provides an on-site picture, confirming an added value of HPV DNA detection.