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CHARACTERIZING HIGH-RISK HPV E7 ONCOPROTEIN AMONG PATIENTS WITH OROPHARYNGEAL CANCER (ID 1213)
Abstract
Introduction
High risk type human Papillomavirus (hrHPV), particularly HPV16 accounts for approximately half of all oropharyngeal carcinomas (OPC). Due to the high prevalence and the transient nature of HPV infections, the detection of hrHPV genotypes in samples obtained from OPC tissues does not necessarily speak of an HPV-driven tumor. Detection of high level of hrHPV oncoproteins may be helpful in optimally distinguishing HPV-driven oropharyngeal cancers from HPV-positive but HPV-independent OPCs. With this study, we aim to characterize an ELISA-based detection of hrHPV E7oncoprotein among patients with histologically confirmed OPC.
Methods
Tumor surface brush samples (digene® HC2 DNA Collection Device, Qiagen, Hilden, Germany) were immersed in a 20ml vial containing the fixative medium PreservCyt (Hologic, Bedford, MA). For the detection of HR-HPV E7-oncoprotein a sandwich ELISA tests system (recomWell HPV 16/18/45, Mikrogen, Neurid, Germany) was used. Results are presented as optical density (OD) with a limit of detection set at 0.5pg of protein per well. P16 immunohistochemistry was also conducted.
Results
A total 46 patients – median age 64,9 years, 87% males – with histology confirmed OPC were included in the study. Twenty-one patients (45,7%) were positive for E7 oncoprotein. Twenty (57.1%) out of 35 individuals with valid P16 immunohistochemistry results showed p16 overexpression. E7 oncoprotein positivity was significantly associated with p16 positivity: OR (95% CI) = 5.12 (1.18-22.2), P= 0.03.
Conclusions
A sensitive detection hrHPV oncoprotein speaks specifically of HPV-associated pathology as compared to p16 detection which is rather a sign of cell cycle deregulation.The significant association between high level of hrHPV E7 oncoprotein positivity and p16 overexpression indicates that ELISA-based high-risk HPV oncoprotein detection may be an easy and cost-effective option in precisely identifying HPV-driven OPCs. Since HPV-driven and HPV-independent OPCs are two different entities with different therapeutic and prognostic consequences, this preliminary finding seems to be of non-negligible clinical value.
EVALUATION OF AUTOMATED CLINICAL AND DIAGNOSTIC HUMAN PAPILLOMAVIRUS (HPV) TESTING BY RNA ISH AGAINST COMBINED P16 IHC AND DNA ISH IN OROPHARYNGEAL SQUAMOUS CELL CARCINOMA (ID 888)
Abstract
Introduction
Current diagnostic methods for detecting high-risk HPV in oropharyngeal squamous cell carcinoma (OpSCC) is via a two-stage algorithm, namely p16 immunohistochemisty (IHC) followed by HPV DNA in-situ hybridisation (ISH) if the former is positive. Within this context, there have been recent calls for a single-step HPV laboratory diagnostic test with a view to greater clinical effectiveness. This study evaluated the clinical utility of automated RNAISH as a single-step alternative to the two-stage algorithm within a routine diagnostic histopathology setting.
Methods
38 p16+/DNAISH+, 42 p16- and 20 p16+/DNAISH- were randomly selected from the diagnostic archive of the Head & Neck Pathology Department, Guy’s Hospital. High-risk HPV RNAISH was undertaken on all cases on an automated clinical platform. Manufacturer recommended and on-slide p16/HPV positive and negative controls were used (Figure 1). Test quality assurance and diagnostic RNAISH were independently assessed by two observers. Initial discordant cases were re-evaluated. A consensus diagnosis was reached in the presence of a third observer on remaining discordant cases. All RNAISH results were then correlated against p16 +/- DNAISH status.
Results
12 cases required re-testing and initial evaluation revealed 16 discordant cases. The 16 disagreements along with 12 randomly selected additional cases were reassessed leaving 5 discordant cases (Table 1, kappa=0.90, p<0.001). There was full concordance between RNAISH and p16 +/- DNAISH status (Figure 2a-c). Of the 20 p16+/DNAISH- tumours, RNAISH was positive in 18 and negative in 2 cases (Figure 2d-f).
| Observer1 positive | Observer1 negative | |
|---|---|---|
| Observer2 positive | 57 | 0 |
| Observer2 negative | 5 | 38 |
Conclusions
Automated RNAISH is a viable alternative to current two-stage HPV testing for OpSCC in routine diagnostic histopathology and is likely to increase clinical effectiveness. Our study also indicates diagnostic RNAISH interpretation requires a degree of training and experience. Furthermore, on-slide HPV analyte controls are important for interpretation of RNAISH staining.
ASSESSMENT OF ISOTHERMAL AMPLIFICATION AMPFIRE ASSAY FOR DETECTION AND GENOTYPING OF HPV IN FORMALIN-FIXED PARAFFIN-EMBEDDED HEAD AND NECK CANCER SAMPLES (ID 165)
Abstract
Introduction
Infection with high risk HPV is etiologically linked to a group of oropharyngeal squamous cell carcinomas (OPSCCs) that show better clinical outcome and response to treatment. The combination of p16INK4A overexpression and HPV-DNA testing increases diagnostic accuracy and have a better prognostic value. The aim of this study was to assess the AmpFire HPV Multiplex and Genotyping Tests, for formalin-fixed paraffin-embedded (FFPE) samples.
Methods
Extracted DNA of the entire collection of OPSCC FFPE specimens collected between 2014-2019 at Hospital de Bellvitge summing a total of 160, plus 23 samples of other head and neck (HN) localizations were routinely tested using the Linear Array HPV Genotyping Test (LA). Additionally, viral DNA was detected and 15 HR- HPV types genotyped using the AmpFire HPV Multiplex and Genotyping Tests (Atila Biosystems). The DNA was amplified and real-time fluorescent detected using a 60ºC isothermal reaction for 74 min.
Results
LA and AmpFire HPV Multiplex tests showed, for total samples and OPSCC, a positive agreement of 98.89% and a 99.36% and a kappa index of 0.972 and 0.984, respectively. An overall concordance of 100% for the presence of HPV16 and 18 was observed. Amongst the samples where the test detected “Other HR-HPV genotypes”, the AmpFire Genotyping test was performed. The main disagreement was found for genotypes HPV45 and HPV52. It is worth mentioning that LA is unable to identify HPV52 alone in samples containing HPV33, HPV35, and/or HPV58 as a cross-reactive probe for all these genotypes is used. Moreover, 145 OPSCC samples had the p16INK4A IHC test done. The agreement observed between positives and negatives for HPV-DNA and p16 INK4A was 93.8% and a ki of 0.848.
Conclusions
The AmpFire HPV Tests are simple sample-to-answer and low cost assays for detection and genotyping of HPV in HN FFPE samples.
ETIOLOGICAL FACTORS IN OROPHARYNX AND ORAL CAVITY SQUAMOUS CELL CARCINOMA DIAGNOSED AT YOUNG AGE: A SPANISH COHORT AND AN USA CASE-CONTROL STUDY. (ID 1271)
- Miren Taberna (Spain)
- Jitesh Shewale (United States of America)
- Sara Tous (Spain)
- Maria Nieva (Spain)
- Carles Fabregat (Spain)
- Marisa Mena (Spain)
- Xavier Leon (Spain)
- Marta Guix (Spain)
- Teresa Bonfill (Spain)
- Alicia Lozano (Spain)
- Alex Teulé (Spain)
- Ricard Mesía (Spain)
- Maura L. Gillison (United States of America)
- Laia Alemany Vilches (Spain)
Abstract
Introduction
There is a gap in knowledge in the etiological factors in head and neck cancers diagnosed at young age. The aim of this study is to compare the demographic, toxic habits and HPV status between a Spanish retrospective cohort of oropharyngeal cancer (OPC) patients (≤45 years old vs >45 years). Furthermore, we have analyzed the toxic habits status between young cases and controls from an USA case-control study (including OPC and oral cavity cancers-OC).
Methods
We have reanalyzed data from a retrospective cohort of patients diagnosed with OPC in four Catalonian hospitals from 1990 to 2017 and a case-control study in USA from 2011 to 2014 at The Ohio State University Medical Center. In the OPC cohort unconditional logistic regression was used to compare age groups. Differences in characteristics between cases and controls were compared using the Pearson’s Chi2 test.
Results
49 young and 816 old OPC patients were included from the Spanish cohort and 23 young cases and 46 controls from the USA study. Spanish cohort characteristics comparing young and old OPC patients are described in Table 1. No significant differences were reported in gender, toxic habits or HPV status (positivity: 12.2% vs 7.5% in young and old OPC patients, respectively). There were more non-smokers HPV-related OPC patients than HPV-negative ones (p-value<0.001) in both age groups. A descriptive of the toxic habits of the patients with OC stratified by case or control in the USA study is describe in table 2. There were no statistically significant differences between cases and controls for all toxic habits evaluated; 57.8% of cases (24) were HPV-positive.
Conclusions
No significant differences were reported in gender, toxic habits or HPV status between young and old OPC patients; neither between young cases and controls regarding tobacco, alcohol or marijuana use.
DETECTION OF HPV 16/18 E6 ONCOPROTEINS IN CARCINOMA OF THE OROPHARYNX AND IN NECK CARCINOMA OF UNKNOWN PRIMARY (ID 1223)
- Anna Menegaldo (Italy)
- Lea Schroeder (Germany)
- Dana Holzinger (Germany)
- Giancarlo Tirelli (Italy)
- Elisa Dal Cin (Italy)
- Margherita Tofanelli (Italy)
- Monica Mantovani (Italy)
- Marco Stellin (Italy)
- Annarosa Del Mistro (Italy)
- Stefania Rigo (Italy)
- Angelo Paolo Dei Tos (Italy)
- Angela Guerriero (Italy)
- Monia Niero (Italy)
- Jerry Polesel (Italy)
- Maria Cristina Da Mosto (Italy)
- Michael Pawlita (Germany)
- Tim Waterboer (Germany)
- Paolo Boscolo-Rizzo (Italy)
Abstract
Introduction
The human papillomavirus (HPV) oncogenic role in patients with oropharyngeal squamous cell carcinoma (OPSCC) and neck lymph node metastasis of squamous cell carcinoma from unknown primary tumor (NSCCUP) is increasingly acknowledged and rapidly rising in Western Countries. The identification of clinically relevant transforming HPV infections is crucial for staging, prognostication and enrollment in clinical trials. The aim of this study was to determine the feasibility and accuracy of the detection of HPV 16/18 E6 oncoprotein in cytological specimens from primary carcinomas and neck metastases.
Methods
In addition to tissue biopsies for histological diagnosis, cytological specimens from primary tumors and neck metastases were collected by fine needle aspiration from 34 patients with OPSCC with lymph node involvement or NSCCUP and tested with a commercial lateral flow test (OncoE6, Arbovita) detecting HPV16 and 18 E6 oncoproteins. Sera were also collected at diagnosis. Results were compared to presence of HPV-DNA together with p16INK4a overexpression or HPV DNA together with HPV E6 seropositivity as reference method.
Results
Eighteen of 29 OPSCC (62%) and 3 of 5 NSCCUP (60%) were HPV-driven according to our reference method. The HPV 16/18 E6 oncoprotein test had a sensitivity of 94% (95% CI: 70%-100%) and a specificity of 100% (95% CI: 66%-100%) on primary tumor, and a sensitivity of 88% (95% CI: 64%-99%) and a specificity of 100% (95% CI: 74%-100%) on neck metastases. Test agreement between the E6 oncoprotein lateral flow test and the clinical reference method was excellent both for primary lesion and neck metastases (Cohen’s kappa value 0.92 and 0.88 respectively).
Conclusions
In this study, we found the detection of HPV 16/18 E6 oncoproteins to be a feasible, highly reliable and low-invasive method to assess HPV status in OPSCC and NSCCUP.
A SIMPLE, RAPID, MULTIPLEX, AMPFIRE ISOTHERMAL AMPLIFICATION ASSAY FOR DETECTION AND GENOTYPING OF HUMAN PAPILLOMAVIRUSES IN FORMALIN-FIXED PARAFFIN-EMBEDDED OROPHARYNGEAL CANCER TISSUES (ID 1034)
Abstract
Introduction
Rapid and accurate detection and identification of human papillomavirus (HPV) is important for both clinical management and population screening. Detection of HPV DNA from formalin-fixed paraffin-embedded (FFPE) specimens has been a challenge as it usually requires lengthy and inefficient sample process. This presentation describes the simplest and fastest HPV detection methods from FFPE oropharyngeal cancer tissue samples
Methods
The AmpFire HPV assay (Atila Biosystems Inc, Mountain View, CA, USA) incorporates a multiplex isothermal amplification to detect and genotype 15 high-risk (HR) HPV genotypes directly from raw FFPE samples without needing to extract or purify the DNA. The whole detection process requires couple pipetting steps and can be completed within 2.5 hours. We performed analytic validation of Atila AmpFire Multiplex HPV assays on FFPE cervix/vulva and oropharynx diagnostic tissue samples.
Results
Limits of detection determined by plasmids cloned with HPV genotype-specific sequences were 2 copies/reaction for HPV16, HPV18, and some HR HPV genotypes, and 20 copies/reaction for the remaining HR HPV genotypes. The performance of the AmpFire assays in clinical samples was evaluated using 214 FFPE specimens. The AmpFire assay failed in one clinical specimen for an invalid rate of 0.5%. The AmpFire assay detected HPV in clinical samples with positive percent agreements of 100.0% for HPV16, 100.0% for HPV18, and 94.7% for non-16/18 HR-HPV, and 100% negative percent agreements for HPV16, HPV18 and non-16/18 HR-HPV. Qualitative detection agreement was obtained in the reproducibility study.
Conclusions
In summary, the Atila AmpFire HPV assay demonstrated excellent analytic sensitivity and specificity for detection and genotyping of 15 HR HPV genotypes. Assay parameters of simple specimen processing, small sample size requirement, rapid turnaround time and being near instrument-free render it well suited for HPV detection and genotyping in FFPE specimens.