Novocastra p16 Antibody Demonstrated an Agreement of 98% with the Roche p16 Antibody

Leica Biosystems are pleased to welcome you to today's webinar, through which we will be presenting a clinical study utilizing our newly launched p16 IVD Ready-To-Use (RTU) antibody.

Within today's webinar, we will be presenting the design, execution, and subsequent results of the Leica Biosystems clinical study comparing immunohistochemistry assays for the detection of p16 protein in oropharyngeal cancers.

For this study, we were pleased to engage with two key opinion leaders within head and neck pathology. Firstly, Dr. Selvam Thavaraj, a senior lecturer in oral and maxillofacial pathology of King's College London, and also an honorary consultant in head and neck pathology working out of Guy's and St Thomas' Hospital Trust.

Alongside Dr. Thavaraj, Leica Biosystems were pleased to work with Dr. Max Robinson, again, jointly appointed as a senior lecturer in oral pathology at Newcastle University and as an honorary consultant in oral pathology at Newcastle upon Tyne NHS Trust.

With today's webinar being pre-recorded, there won't be an opportunity for a live question and answer session. However, should you have any questions for either our speaker or regarding the comparative study in question, please feel free to contact us and we will make sure to respond. There will be another reminder of the contact information towards the end of the webinar.

As speaker for today's webinar, Leica Biosystems are very pleased to welcome Dr. Selvam Thavaraj, a co-principal investigator for the comparison study. As mentioned, Dr. Thavaraj is a senior lecturer out of King's College London and an honorary consultant in head and neck pathology at Guy's and St Thomas' Hospital Trust, through which he is part of the histopathology service of one of the UK's largest head and neck cancer treatment centers. His research interests include biomarkers and the role of microbes in head and neck premalignancy and cancer, and more specifically, the diagnostic and prognostic aspects of HPV within these cancer types. I'll now hand over to Dr. Selvam Thavaraj, who can walk us through the specifics of the p16 comparison study.

Head and Neck Cancer

Thank you, Andrew, for that kind introduction. I'd like to start off by setting the scene in order to provide some context to the clinical importance of this study. Head and neck cancer, globally, is the sixth most prevalent cancer worldwide, and rather sadly, it is estimated to continue to rise at least over the next two to three decades. It is important to realize that there are subsite differences within head and neck cancer in terms of its epidemiology. The rather exponential rise in head and neck cancer is due largely to rise in the incidence and prevalence of oropharyngeal cancers, namely those cancers affecting the palatine tonsil and base of the tongue. This lies in contrast to cancers of the larynx, which in recent years has seen a decline in incidence and prevalence. These differences can be explained by varying etiological factors. The vast majority of oropharyngeal cancers are attributable to HPV infection, whereas those of the larynx are related to conventional risk factors of smoking and tobacco.

HPV in Oropharyngeal Cancer

The key drivers of high-risk HPV mediated oncogenesis are the viral oncoproteins, E6 and E7. One of the ways that E7 causes oncogenesis is through its inactivation of the retinoblastoma tumor suppressor protein. E7 binds to Rb and thereby releases the transcription factor E2F, which causes progression through the cell cycle.

p16 in Head and Neck Cancer

In the normal uninfected cell, release of E2F from Rb is under the negative control of p16. One of the functions of E2F is to cause transcription of p16, and therefore a negative feedback loop is set up between E2F and p16. In the infected state, E7 permanently binds Rb, causing disruption of this negative feedback loop, resulting in accumulation and overexpression of p16. Therefore, p16 acts as a surrogate marker for high-risk HPV infection.

Against the alarming epidemiological trends, it is also important to note that HPV associated oropharyngeal squamous cell carcinomas indicate a much more favorable prognosis when compared to site and stage matched HPV negative cancers.

The evidence for improved prognosis is so robust that recent staging systems have applied different algorithms to oropharyngeal squamous cell carcinomas based on p16 status. We now therefore stage oropharynx cancers according to p16 status. The importance of p16 in oropharyngeal squamous cell carcinomas is such that national guidelines in several countries stipulate that all oropharyngeal squamous cell carcinomas should undergo p16 testing.

There are also several de-escalation clinical trials in progress which stratify patients according to p16 status, and therefore accurate p16 testing is of utmost importance.

p16 Immunohistochemistry

Up until now, automated diagnostic p16 testing has been restricted to Roche Tissue Diagnostics Syntech p16 histology in vitro diagnostic assay. This product is intended for the qualitative detection of p16 on formalin-fixed paraffin-embedded tissue prepared from cervical biopsies in the context of a medical pathology laboratory to provide adjunctive information following established diagnostic methods.

Following the expiry of the patent held by Roche Tissue Diagnostics, Leica Biosystems has developed and commercialized a p16 clone 6H12 IVD assay. This product is intended for the qualitative identification by light microscopy of human p16 protein in formalin-fixed paraffin-embedded tissue using the automated BOND system.

Its recommendation is for the detection of p16 protein in normal and neoplastic tissues as an adjunct to conventional histopathology by non-immunological methods. It is therefore not restricted to any specific indication.

Study Objectives

Since users had become accustomed to a single diagnostic assay, it was necessary to evaluate the diagnostic utility of the new product against what was previously available. The primary objective, therefore, of this current study was to compare the immunohistochemical agreement rates of the Leica Biosystem against the Roche Tissue Diagnostic system. A secondary objective was to establish interobserver agreement, in this case, between two specialist head and neck pathologists for both the LBS and RTD assays. A third, but exploratory objective was to investigate agreement rates between the two systems against molecular testing, namely HPV DNA in situ hybridization and PCR.

Study Flow

Here's an overview of the study flow. Following sample sourcing, the study and comparative device staining were carried out at Leica Biosystems' Newcastle Clinical Laboratory and the Royal Victoria Infirmary in Newcastle, respectively. Samples were then read independently in succession by two pathologists, followed by final data analysis.

Sample Enrollment and Processing

The sample source was based on a previously research ethics committee-approved cohort published by Shakir and colleagues in 2016. This cohort was composed of 170 oropharyngeal squamous cell carcinomas diagnosed between 2002 to 2011. Tissue microarrays were constructed from tissue samples of this cohort, resulting in triplicate one-millimeter cores of a maximum of 170 unique specimens. There were 510 individual cores. Serial sections were cut at four microns, and specimens were de-identified and coded by study identification number.

All specimens were previously characterized for HPV status using molecular methods as part of the previous study. This was done using PCR with the OptiPlex HPV Genotyping Kit, in addition to high-risk HPV DNA in situ hybridization on the Roche platform using the Roche Inform Family 16 cocktail of probes. As part of the current study, DNA in ISH was further validated using the Leica Biosystem probes on the Leica BOND-III platform.

Staining Methodology

For the current study, the test antibody, namely the Leica p16 clone 6H12, was undertaken on the BOND-III platform, whereas the comparator device, the Roche Syntech p16 antibody, was performed on the Benchmark XT platform.

Scoring Criteria

Immunoreactivity was independently quantified by two pathologists using the Histoscore or H-score. Now, the H-score is defined as the sum of staining intensity ranging from zero to three, multiplied by the percentage of tumor cells at 5% increments at each intensity grade. This results in a quantity H-score ranging for each core between zero to 300.

Here's an example of how one particular core was scored. This tumor core showed 25% of tumor cells to have an intensity of one, 50% of tumor cells to have a moderate intensity staining, and a strong staining or score three was present in 25% of cells. This particular core resulted in an H-score of 200, which was arrived at by the sum of 25 times one, plus 50 times two, plus 25 times three. Each patient sample is presented in triplicate, and the final H-score for each tumor was arrived at by the average H-score of all interpretable cores.

For purposes of clinical diagnostic utility, oropharynx cases need to be either categorized as being p16 positive or p16 negative. The current guidance states that a tumor should be considered p16 positive if there is strong and diffuse nuclear and cytoplasmic staining in greater than 70% of tumor cells. Therefore, for the purposes of this current study, a negative p16 H-score was defined as being less than 140, whereas a positive score was defined as being equal to or greater than 140.

Staining Images

Here are some representative photomicrographs taken at high power of some of the study cases. The top row is the result from the Roche system, whereas the bottom row, that of the Leica system. Columns A and D were cases that were deemed p16 positive because these scores contributed to a final H-score of equal to or greater than 140. By contrast, column B was deemed p16 negative because as clearly can be seen here, it resulted in a H-score of less than 140. Column C, on the other hand, the core represented in the top panel using the Roche system contributed to a final H-score of 140, which is right on the cusp of the cutoff. Whereas using the Leica system, this core contributed to a final H-score of 232, which put it well into the positive category. Interestingly, this case, when assessed using molecular methods, was positive for HPV DNA.

Statistical Analysis

In order to determine the primary objective of the study, which was the agreement between the Roche and Leica systems, the primary measurements were overall percent agreement, average positive percent agreement, and average negative percent agreement. The average negative percent agreement was arrived at using this formula: 100 times two of the number of concordant cases divided by the total number of discordant cases. The average positive percent agreement was arrived in a similar way, and the overall agreement was arrived using this formula: 100 times the total concordant cases divided by the total number of cases.

A two-sided 95% confidence interval for overall average positive and average negative agreement rates was arrived at using the nonparametric bootstrap statistical method. Percent agreement rates for secondary analysis, namely the concordance between pathologists' reads, were arrived in a similar way. When we look at the data comparing the Leica Biosystems and Roche Tissue Diagnostics agreement rates, there was a very high average positive agreement, average negative agreement, and overall agreement approaching 99%. It is a consistent finding for both pathologists, and it is important to note that the lower bound 95% confidence interval is in excess of 96%, indicating a true result. This indicates that the Leica Biosystems p16 antibody is a viable alternative to the Roche Tissue Diagnostics Syntech assay, as demonstrated within this oropharyngeal cancer cohort.

Of the three p16 discordant cases, two were the result of discrepancy scores in one of the pathologists, and one case was discordant for both pathologists. In all three discordant cases, p16 was positive using the Leica Biosystems assay, but negative for the Roche Tissue Diagnostics test.

In two of the Leica Biosystems positive but Roche Tissue Diagnostics negative cases, the tumor was positive for HPV DNA as assessed by in situ hybridization and PCR. In relation to the secondary objective, which was to establish interobserver agreement rates for each of the p16 assays, there was, once again, very strong average positive, average negative, and overall interobserver agreement rates for the Leica Biosystems assay approaching 99%.

Once again, these results are valid since the lower bound 95% confidence interval was greater than 96%. Interestingly, the interobserver agreement rates for the Leica Biosystems p16 were identical to those of the Roche Tissue Diagnostics assay. From a clinical diagnostic aspect, it is important to have strong interobserver correlation, especially since oropharyngeal cancers are staged and stratified in clinical trials according to p16 status.

As for our exploratory objective, which was to investigate agreement rates between Leica Biosystems p16 and molecular HPV testing, namely Leica Biosystems' high-risk HPV DNA in situ hybridization and PCR, the overall agreement rate ranged from 85.8 to 88.8%, and this is entirely in keeping with published reports ranging from 79 to 95%. This suggests that the Leica Biosystems p16 assay may be a reliable surrogate marker for high-risk HPV.

Study Conclusion

To draw the presentation of the study to a conclusion, our results show that there is a high agreement between the Leica Biosystems and Roche Tissue Diagnostic p16 assays, with interassay agreement rates exceeding 95%. Furthermore, the results show that the Leica Biosystems p16 assay demonstrated strong interobserver agreement rates exceeding 95% when read by two specialist head and neck pathologists. Therefore, the Leica Biosystems p16 assay is now shown to be a suitable alternative to the Roche Tissue Diagnostics Syntech p16 histology assay as demonstrated across a range of oropharyngeal cancer specimens.

Finally, here is a reference list of publications to provide context of p16 utility in head and neck cancer that were cited in this presentation.

Thank you very much, Selvam, both for your collaboration on this study and for the excellent presentation. Leica Biosystems are pleased to announce that the p16 IVD automated antibody, derived from proprietary NovaCastra clone 6H12, is now available for use and compatible with the clinical Leica BOND MAX and BOND-III staining platforms. This can be ordered under product code PA0016. The p16 antibody has been developed and validated for use with the BOND Polymer Refine Detection System, product code DS9800, with the associated staining protocol available for download now within the BDD software version 81 or higher. Please contact your local Leica Biosystems representative for availability within your region, or indeed, submit a query to the email address on the following slide. As a final reminder, should you have any questions regarding the p16 comparison study or for our guest speaker, Dr. Selvam Thavaraj, please feel free to submit them to andrew.margerrison@leicabiosystems.com. Thank you all very much for your attendance.