Andrew Schache

Evaluation of Human Papilloma Virus Diagnostic Testing in Oropharyngeal Squamous Cell Carcinoma: Sensitivity, Specificity, and Prognostic Discrimination

Purpose: Human papillomavirus-16 (HPV16) is the causative agent in a biologically distinct subset of oropharyngeal squamous cell carcinoma (OPSCC) with highly favorable prognosis. In clinical trials, HPV16 status is an essential inclusion or stratification parameter, highlighting the importance of accurate testing. Experimental Design: Fixed and fresh-frozen tissue from 108 OPSCC cases were subject to eight possible assay/assay combinations: p16 immunohistochemistry (p16 IHC); in situ hybridization for high-risk HPV (HR HPV ISH); quantitative PCR (qPCR) for both viral E6 RNA (RNA qPCR) and DNA (DNA qPCR); and combinations of the above. Results: HPV16-positive OPSCC presented in younger patients (mean 7.5 years younger, P = 0.003) who smoked less than HPV-negative patients (P = 0.007). The proportion of HPV16-positive cases increased from 15% to 57% (P = 0.001) between 1988 and 2009. A combination of p16 IHC/DNA qPCR showed acceptable sensitivity (97%) and specificity (94%) compared with the RNA qPCR “gold standard”, as well as being the best discriminator of favorable outcome (overall survival P = 0.002). p16 IHC/HR HPV ISH also had acceptable specificity (90%) but the substantial reduction in its sensitivity (88%) impacted upon its prognostic value (P = 0.02). p16 IHC, HR HPV ISH, or DNA qPCR was not sufficiently specific to recommend in clinical trials when used in isolation. Conclusions: Caution must be exercised in applying HPV16 diagnostic tests because of significant disparities in accuracy and prognostic value in previously published techniques. Clin Cancer Res; 17(19); 6262–71. ©2011 AACR.

Validation of a novel diagnostic standard in HPV-positive oropharyngeal squamous cell carcinoma

Background:Human papillomavirus (HPV) testing in oropharyngeal squamous cell carcinoma (OPSCC) is now advocated. Demonstration of transcriptionally active high-risk HPV (HR-HPV) in fresh tumour tissue is considered to be the analytical ‘gold standard’. Clinical testing has focused on formalin-fixed paraffin-embedded (FFPE) tissue at the expense of sensitivity and specificity. Recently, a novel RNA in situ hybridisation test (RNAscope) has been developed for the detection of HR-HPV in FFPE tissue; however, validation against the ‘gold standard’ has not been reported.Methods:A tissue microarray comprising FFPE cores from 79 OPSCC was tested using HR-HPV RNAscope. Analytical accuracy and prognostic capacity were established by comparison with the reference test; qRT–PCR for HR-HPV on matched fresh-frozen samples.Results:High-risk HPV RNAscope had a sensitivity and specificity of 97 and 93%, respectively, against the reference test. Kaplan–Meier estimates of disease-specific survival (DSS, P=0.001) and overall survival (OS, P<0.001) by RNAscope were similar to the reference test (DSS, P=0.003, OS, P<0.001) and at least, not inferior to p16 immunohistochemistry +/− HR-HPV DNA-based tests.Conclusion:HR-HPV RNAscope demonstrates excellent analytical and prognostic performance against the ‘gold standard’. These data suggest that the test could be developed to provide the ‘clinical standard’ for assigning a diagnosis of HPV-related OPSCC.

HPV Specific Testing: A Requirement for Oropharyngeal Squamous Cell Carcinoma Patients

Human papillomavirus (HPV) testing is now recommended as part of the work up for patients with oropharyngeal squamous cell carcinoma (OPSCC) and those patients with cervical lymph node metastasis of unknown origin. The laboratory testing strategy should accurately assess the presence or absence of oncogenic HPV infection in routinely collected tumour samples that are subject to standard fixation protocols, alcohol-fixed cytological preparations and formalin-fixed tissue samples. The HPV status should correlate with biologically relevant outcome measures such as overall, disease-specific and disease-free survival. Whilst increased expression of p16 by immunohistochemistry is considered to be a surrogate marker of oncogenic HPV infection and is a validated independent prognostic biomarker, only HPV specific tests provide definitive evidence of the aetiological agent. We provide an overview of HPV testing in OPSCC, justifying the use of HPV specific tests. We examine the analytical accuracy of HPV specific tests against the ‘reference’ test—high risk HPV mRNA in fresh tissue—and contrast this with the performance of p16 immunohistochemistry as a stand alone test. We highlight the added value of HPV specific tests in prognostication, clinical trial design, and population-based disease surveillance. We consider that HPV specific testing is the starting point for developing increasingly informative biomarker panels in the context of ‘stratified medicine’. We briefly frame test information in the context of disclosure of HPV status to patients. We conclude that only a testing strategy that includes HPV specific tests can deliver more effective care for patients with OPSCC. The international head and neck oncology community should work together to clearly define the minimum requirements for assigning a diagnosis of HPV-related OPSCC in order to ensure consistent reporting of this emerging and increasingly prevalent disease.

The epigenetic landscape of oral squamous cell carcinoma

Background:There is relatively little methylation array data available specifically for oral squamous cell carcinoma (OSCC). This study aims to compare the DNA methylome across a large cohort of tumour/normal pairs.Methods:DNA was extracted from 44 OSCCs and paired normal mucosa. DNA methylation analysis employed the Illumina GoldenGate high-throughput array comprising 1505 CpG loci selected from 807 epigenetically regulated genes. This data was correlated with extracapsular spread (ECS), human papilloma virus (HPV) status, recurrence and 5-year survival.Results:Differential methylation levels of a number of genes distinguished the tumour tissue sample from the matched normal. Putative methylation signatures for ECS and recurrence were identified. The concept of concordant methylation or CpG island methylator phenotype (CIMP) in OSCC is supported by our data, with an association between ‘CIMP-high’ and worse prognosis. Epigenetic deregulation of NOTCH4 signalling in OSCC was also observed, as part of a possible methylation signature for recurrence, with parallels to recently discovered NOTCH mutations in HNSCC. Differences in methylation in HPV-driven cases were seen, but are less significant than that has been recently proposed in other series.Conclusion:Although OSCC seems as much an ‘epigenetic’ as a genetic disease, the translational potential of cancer epigenetics has yet to be fully exploited. This data points to the application of epigenetic biomarkers and targets available to further the development of therapy in OSCC.

Squamous cell carcinoma of the head and neck outside the oropharynx is rarely human papillomavirus related

The incidence of human papillomavirus (HPV)‐driven disease beyond the oropharynx varies greatly in the reported literature.

HPV-Related Oropharynx Cancer in the United Kingdom: An Evolution in the Understanding of Disease Etiology.

A rising incidence of oropharyngeal squamous cell carcinoma (OPSCC) incidence has occurred throughout the developed world, where it has been attributed to an increasing impact of human papillomavirus (HPV) on disease etiology. This report presents the findings of a multicenter cross-sectional retrospective study aimed at determining the proportion of HPV-positive and HPV-negative OPSCC within the United Kingdom. Archival tumor tissue blocks from 1,602 patients previously diagnosed with OPSCC (2002-2011) were collated from 11 centers. HPV status was determined with three validated commercial tests to provide valid data for 1,474 cases in total. Corresponding national incidence data from the same decade were obtained from UK Cancer registries. The overall proportion of HPV+ OPSCC between 2002 and 2011 was 51.8% [95% confidence interval (CI), 49.3-54.4], and this remained unchanged throughout the decade [unadjusted RR = 1.00 (95% CI, 0.99-1.02)]. However, over the same period, the incidence of OPSCC in the broader UK population underwent a 2-fold increase [age-standardized rate 2002: 2.1 (95% CI, 1.9-2.2); 2011: 4.1 (95% CI, 4.0-4.3)]. Although the number of OPSCCs diagnosed within the United Kingdom from 2002 to 2011 nearly doubled, the proportion of HPV+ cases remained static at approximately 50%. Our results argue that the rapidly increasing incidence of OPSCC in the United Kingdom cannot be solely attributable to the influence of HPV. The parallel increase in HPV+ and HPV- cases we documented warrants further investigation, so that appropriate future prevention strategies for both types of disease can be implemented. Cancer Res; 76(22); 6598-606. ©2016 AACR.

Quantitative promoter methylation differentiates carcinoma ex pleomorphic adenoma from pleomorphic salivary adenoma

Background:Potential epigenetic biomarkers for malignant transformation to carcinoma ex pleomorphic adenoma (Ca ex PSA) have been sought previously with and without specific comparison with the benign variant, pleomorphic salivary adenoma (PSA). Previous analysis has been limited by a non-quantitative approach. We sought to demonstrate quantitative promoter methylation across a panel of tumour suppressor genes (TSGs) in both Ca ex PSA and PSA.Methods:Quantitative methylation-specific real-time polymerase chain reaction (qMSP) analysis of p16INK4A, CYGB, RASSF1, RARβ, human telomerase reverse transcriptase (hTERT), Wilms’ tumour 1 (WT1) and TMEFF2 gene promoters was undertaken on bisulphite-converted DNA, previously extracted from archival fixed tissue specimens of 31 Ca ex PSA and an unrelated cohort of 28 PSA. All target regions examined had formerly been shown to be hypermethylated in salivary and/or mucosal head and neck malignancies.Results:The qMSP demonstrated abnormal methylation of at least one target in 20 out of 31 (64.5%) Ca ex PSA and 2 out of 28 (7.1%) PSA samples (P<0.001). RASSF1 was the single gene promoter for which methylation is shown to be a statistically significant predictor of malignant disease (P<0.001) with a sensitivity of 51.6% and a specificity of 92.9%. RARβ, TMEFF2 and CYGB displayed no apparent methylation, while a combinatory epigenotype based on p16, hTERT, RASSF1 and WT1 was associated with a significantly higher chance of detecting malignancy in any positive sample (odds ratio: 24, 95% CI: 4.7–125, P<0.001).Conclusions:We demonstrate the successful application of qMSP to a large series of historical Ca ex PSA samples and report on a panel of TSGs with significant differences in their methylation profiles between benign and malignant variants of pleomorphic salivary adenoma. qMSP analysis could be developed as a useful clinical tool to differentiate between Ca ex PSA and its benign precursor.

Predictors of swallowing outcome in patients treated with surgery and radiotherapy for advanced oral and oropharyngeal cancer.

Retaining effective swallowing is a key element when optimising outcomes in the management of head and neck cancer. We report the functional swallowing outcomes for a cohort of 31 individuals with advanced oral and oropharyngeal cancer who underwent free or pedicled flap reconstruction of surgical defects. Swallowing was assessed pre and immediately post surgery and at four months post treatment. Swallowing assessments were related to site, size and volume of defect and composition of flap reconstruction. The effect of radiotherapy on swallowing was assessed among 17 of the 31 individuals who were submitted to radiotherapy after surgery. The proportion of patients on a total oral diet four months post treatment varied significantly by site of defect (Fishers exact test p=0.006), from 100% (7/7) of patients with a lateral defect to only 22% (2/9) of patients with a central defect. The proportion of patients on a total oral diet at the final assessment did not vary by flap reconstruction or radiotherapy.

Mandibular reconstruction with vascularised bone flaps: a systematic review over 25 years

To explore the techniques for mandibular reconstruction with composite free flaps and their outcomes, we systematically reviewed reports published between 1990 and 2015. A total of 9499 mandibular defects were reconstructed with 6178 fibular, 1380 iliac crest, 1127 composite radial, 709 scapular, 63 serratus anterior and rib, 32 metatarsal, and 10 lateral arm flaps including humerus. The failure rate was higher for the iliac crest (6.2%, 66/1059) than for fibular, radial, and scapular flaps combined (3.4%, 202/6018) (p<0.001). We evaluated rates of osteotomy, non-union, and fistulas. Implant-retained prostheses were used most often for rehabilitation after reconstruction with iliac crest (44%, 100/229 compared with 26%, 605/2295 if another flap was used) (p<0.001). There were no apparent changes in the choice of flap or in the complications reported. Although we were able to show some significant differences relating to the types of flap used, we were disappointed to find that fundamental outcomes such as the need for osteotomy, and rates of non-union and fistulas were under-reported. This review shows the need for more comprehensive and consistent reporting of outcomes to enable the comparison of different techniques for similar defects.

Human Papillomavirus Testing in Head and Neck Squamous Cell Carcinoma: Best Practice for Diagnosis

Human papillomavirus (HPV)-associated oropharyngeal carcinoma is now recognized as a clinically distinct subtype of head and neck cancer with important prognostic implications. HPV testing is recommended as the standard of care for all patients diagnosed with oropharyngeal squamous cell carcinoma. Here, we describe a practical approach to HPV testing within the setting of the diagnostic histopathology laboratory.