Received: 30 July 2020 | Accepted: 4 August 2020

DOI: 10.1111/jop.13103

ORIGINAL ARTICLE

RNA in situ hybridization for human papillomavirus testing in

oropharyngeal squamous cell carcinoma on a routine clinical
diagnostic platform

Rhonda Henley-Smith1,2,3 | Alice Santambrogio4 | Cynthia L. Andoniadou4 |

Edward Odell2,3 | Selvam Thavaraj2,5

1
Head and Neck Cancer Biobank, Guy's and
St Thomas' NHS Foundation Trust, London, Background: The current diagnostic standard for detection of high-risk human papil-
UK lomavirus (HPV) in oropharyngeal squamous cell carcinoma is via a two-stage algo-
2
Head and Neck Pathology, Guy's and St
rithm, namely p16 immunohistochemistry followed by HPV DNA in situ hybridization
Thomas' NHS Foundation Trust, London, UK
3
Centre for Host Microbiome Interactions,
in p16 positive cases. This study evaluated the feasibility of automated RNA in situ
Faculty of Dental, Oral and Craniofacial hybridization on a clinical platform as a single-step alternative to the two-stage algo-
Science, King's College London, London, UK
4
rithm within a routine diagnostic histopathology setting.
Centre for Craniofacial and Regenerative
Biology, Faculty of Dentistry, Oral & Methods: Thirty-eight cases positive for both p16 and DNA in situ hybridization, 42
Craniofacial Sciences, King's College p16 negative cases and 20 cases positive for p16 but negative for DNA in situ hybridi-
London, London, UK
5 zation were randomly selected. High-risk HPV RNA in situ hybridization was under-
Centre for Clinical, Oral and Translational
Science, Faculty of Dental, Oral and taken on all cases on an automated clinical platform. Manufacturer-recommended
Craniofacial Science, King's College London,
and on-slide additional p16/HPV positive and negative controls were used. Test qual-
London, UK
ity assurance and diagnostic RNA in situ hybridization were independently assessed
Correspondence
by two observers. A consensus diagnosis was reached in the presence of a third ob-
Selvam Thavaraj, Head and Neck Pathology,
Guy's and St Thomas' NHS Foundation Trust, server on discordant cases. All RNA in situ hybridization results were then correlated
London, UK.
against p16 and DNA ISH status.
Email: selvam.thavaraj@kcl.ac.uk
Results: Inter-slide RNA in situ hybridization staining variation was observed in con-
trol sections. RNA in situ hybridization demonstrated a high inter-observer agree-
ment rate (κ = .897, P < .001). Following consensus review, there was full concordance
between RNA in situ hybridization and the current standard.
Conclusion: Human papillomavirus testing by standalone automated RNA in situ hy-
bridization on a clinical diagnostic platform currently available in routine diagnostic
histopathology laboratories is a feasible alternative to the two-step algorithm of p16
and DNA in situ hybridization. Control tissue staining procedures need to be adapted
to achieve the most accurate results.

KEYWORDS

human papillomavirus, oropharyngeal carcinoma, RNA in situ hybridization, RNAScope

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2020 The Authors. Journal of Oral Pathology & Medicine published by John Wiley & Sons Ltd

J Oral Pathol Med. 2020;00:1–8.  wileyonlinelibrary.com/journal/jop | 1

2 |
1 | I NTRO D U C TI O N
The recent increased worldwide incidence of oropharyngeal squa-
mous cell carcinoma (OpSCC) in several parts of the world is at-
tributable to high-risk (HR) types of human papillomavirus (HPV).1
Alongside these observations, there is now robust evidence that
patients with HPV-associated OpSCC have significantly improved
overall and disease-specific survival compared to site and stage-
matched HPV negative tumours. 2 The improved survival of HPV
positive OpSCC has led to several recent international develop-
ments: (a) the World Health Organisation have recognized these
3
tumours as a separate entity, (b) international staging systems for
OpSCC are now defined according to p16 immunohistochemistry,
4,5
a surrogate marker for HR HPV positivity and (c) several ongoing
clinical trials to evaluate treatment de-intensification for HPV pos-
itive OpSCC.6
The UK National Comprehensive Cancer Network, the National
Institute of Health and Care Excellence (NICE), the Royal College
of Pathologists and the College of American Pathologists have rec-
ommended that HPV testing be undertaken on all OpSCC.7-10 Their
guidelines stipulate p16 immunohistochemistry (IHC) as a minimum
for all OpSCCs. However, although p16 demonstrates excellent sen-
sitivity as a surrogate for direct assessment of HR HPV, a recent
meta-analysis indicates a somewhat suboptimal specificity of 83%.11
This lack of specificity is clinically problematic since a proportion
of patients will be inaccurately staged and may be inappropriately
selected for de-escalated treatment regimens. In view of the poten-
tial for false positivity, some guidelines suggest that all p16 positive
OpSCCs should also be subjected to HR HPV-specific testing.7,8,12
The current HR HPV-specific testing modality of choice is DNA
in situ hybridization (ISH), but this is not widely available. Moreover,
DNA ISH is known to have poor sensitivity necessitating prior
“screening” with p16 IHC with resulting cost and turnaround time
12-14
implications. Against this background, RNA ISH has emerged
as a potential alternative to two-stage HPV testing with acceptable
sensitivity and specificity rates.15-20
To date, most reports evaluating RNA ISH for HPV detection in
OpSCC have been limited to utilization of manual methods and re-
search platforms only thereby limiting its use in routine clinical prac-
tice. Therefore, we sought to determine the feasibility of automated
HPV testing by RNA ISH on a routine clinical platform within the
setting of a diagnostic histopathology laboratory and compared it
with current guidelines, namely p16 IHC, and where positive, DNA
ISH. We also undertook a comparison between automated RNA ISH
against manual testing.
2 | M E TH O DS
2.1 | Case selection
Eighty formalin-fixed paraffin-embedded (FFPE) OpSCC samples
taken between 2005 and 2016 were selected randomly from the
HENLEY-SMITH et al.
pathology archive. Of these, there were 38 cases that had been as-
sessed as positive for p16 and DNA ISH (p16+DNA+) and 42 cases
negative for p16 (p16−). In addition, 20 p16 positive but DNA ISH
negative (p16+DNA−) cases (of which 15 had previously undergone
manual RNA ISH testing as part of a prior study) were included. 21
2.2 | p16 IHC and DNA ISH
p16 and DNA ISH were performed as part of the routine diagnos-
tic work up as previously described.14 p16 IHC (clone E6H4, CINtec;
Roche mtm Laboratories AG) was performed on an automated plat-
form (Benchmark Ultra; Ventana Medical Systems) according to
manufacturer's instructions. OpSCCs showing strong and diffuse
nuclear and cytoplasmic positivity in >70% of tumour cells were
then subjected to HR HPV DNA ISH (INFORM Family III; Roche) on
an automated platform (Benchmark Ultra; Ventana Medical Systems)
according to the manufacturer's instructions. An OpSCC with high
p16 expression and manufacturer-provided FFPE material for high,
moderate and low copy number of HR HPV DNA (Ventana Medical
Systems) were used as positive controls for p16 and DNA ISH,
respectively.
2.3 | Manual RNA ISH
RNA ISH was carried out on 4µm FFPE sections using the RNAscope
2.5 HD Kit-BROWN Manual Assay (ACDBio, Bio-Techne). Timings for
retrieval were assessed using probe-HPV-HR7 (ACDBio, Bio-Techne)
detecting HPV 16, 18, 31, 33, 35, 52 and 58, E6/E7 mRNA, posi-
tive control Hs-UBC (ACDBio, Bio-Techne), negative control dapB
(ACDBio, Bio-Techne). Analyte control sections containing HPV neg-
ative (MCF-7), human breast adenocarcinoma), low copy number HR
HPV positive (SiHa) and high copy number HR HPV positive (CaSki
and HeLa) FFPE cell lines (HCL003, Histocyte Laboratories) were
placed on the same slide as the test sections.
The assay was performed following manufacturer protocol.
Slides were baked at 60°C for 1 hour, followed by deparaffinization
(2 × 5 minutes Xylene, 2 × 1 minute 100% Ethanol). Slides were left
to air dry and subsequently treated with hydrogen peroxide solution
for 10 minutes at room temperature. Sections were washed with
deionized water, and target retrieval was performed at 100°C for
12 minutes. After washing with deionized water, slides were treated
with Protease Plus at 40°C for 30 minutes. Sections were washed
with deionized water and incubated at 40°C for 2 hours for probe
hybridization. Amplification steps were performed at 40°C (1-4)
and room temperature (5-6) as follows: AMP1 30 minutes, AMP2
15 minutes, AMP3 30 minutes, AMP4 15 minutes, AMP5 30 min-
utes and AMP6 15 minutes. Washes in between amplification steps
were carried out at room temperature with Washing Buffer. Sections
were incubated with DAB solution at room temperature for 10 min-
utes, washed in deionized water and nuclei were counterstained
with Vector Hematoxylin QS (Vector Laboratories H-3404). Slides
HENLEY-SMITH et al.
were dehydrated and mounted in VectaMount Permanent Mounting
Medium (Vector Laboratories H-5000).
2.4 | Automated RNA ISH
RNA ISH was performed on 4µm formalin-fixed paraffin-embedded
(FFPE) tissue sections on the Leica Bond III automated platform
(Leica Biosystems) according to manufacturer's instructions. On-
slide analyte controls sections were used as detailed above.
Sections were baked onto slides at 60°C for 30 minutes and
deparaffinized at 72°C for 30 minutes. Epitope retrieval was un-
dertaken using ER2 at 95°C for 15 minutes followed by enzyme
denaturation at 40°C for 15 minutes. Target probe (RNAscope 2.5
LS probe-HPV-HR18, ACDBio, Bio-Techne) was applied and allowed
to hybridize at 42°C for 2 hours. Detection kit (RNAscope 2.5 LS
Assay-Brown, Leica Biosystems) was applied as follows: amplifica-
tion steps 1-4 were performed at 42°C (AMP1 DAB 30 minutes,
AMP2 DAB 15 minutes, AMP3 DAB 30 minutes and AMP4 DAB
15 minutes) followed by steps 5-6 at room temperature (AMP5 DAB
30 minutes and AMP6 DAB 15 minutes). Sections were then incu-
bated with diaminobenzidine (DAB) for 20 minutes, counterstained
with haematoxylin for 5 minutes and blued for 2 minutes, all at room
temperature. All wash steps between reagents were performed on
the automated platform at room temperature then slides were re-
moved and placed into deionized water, dehydrated, cleared and
mounted. For each case, additional FFPE sections on separate slides
were probed for diaminopimelate B (RNAscope 2.5 LS Negative
Control Probe_dapB, ACDBio, Bio-Techne) as a negative control
and ubiquitin C (RNAscope 2.5 LS Positive Control Probe-Hs-UBC,
ACDBio, Bio-Techne) as a positive control, the latter to confirm the
presence of amplifiable mRNA. Brown punctate signals co-localizing
with nuclei and/or the cytoplasm of tumour cells were designated
as positive.
2.5 | Statistical analysis
Inter-observer agreement analysis was calculated with SPSS for
Windows version 25 (IBM).
3 | R E S U LT S
3.1 | Quality assessment
Of the 100 cases which underwent automated RNA ISH testing, 12
required retesting for the following reasons: false positive on HPV
negative cell line control (MCF-7,Figure 1A,B), very low to undetect-
able staining on the low HPV copy number cell line control (CaSki,
Figure 1C,D), lack of diffuse UBC control staining (Figure 1E,F) and
failure of any of the positive cell line controls to stain (Figure 1G,H).
All 12 cases were deemed acceptable following retesting. RNA
| 3
integrity was maintained in all archival samples as demonstrated by
the presence of UBC staining.
3.2 | Inter-observer correlation
Initial independent assessment by 2 observers resulted in 16 discord-
ant cases. Following assessor calibration, all 16 initially discordant
cases and 12 randomly selected concordant cases were indepen-
dently reassessed. Of the initial 16 discordant cases, 5 remained
discordant following reassessment (κ = .897, P < .001). All 5 discord-
ant cases had very low or sparse RNA copy signal (Figure 2). These
5 cases were resolved at a consensus review in the presence of a
third observer. Subtle non-specific staining was occasionally present
within the on-slide HPV negative cell line control (MCF-7) and was
therefore used as a referent when resolving discordant cases.
3.3 | Correlation with diagnostic standard
All p16+DNA+ and p16− cases were positive and negative for RNA
ISH, respectively (100% concordance). Of the 20 p16+DNA− cases,
18 were positive and 2 negative for RNA ISH (Figure 3).
3.4 | Comparison with manual testing
Of the 20 p16+DNA− cases, 15 had previously undergone manual
RNA ISH and there were 14 concordant cases with automated test-
ing (93%, κ = .815, P ≤ .001). One case previously assessed as HPV
negative by manual staining was deemed positive by automated
testing (Figure 4).
4 | D I S CU S S I O N
The increased incidence of HPV OpSCC necessitates accurate and
efficient HPV testing for treatment planning, prognostication and
stratification in clinical trials. The current minimum standard for HPV
testing in OpSCC is p16 IHC alone, but this yields a suboptimal speci-
ficity with insufficient diagnostic accuracy to fulfil these roles.11 To
increase specificity, most centres utilize high-risk HPV DNA ISH as
a second-tier test in p16 positive cases but, this two-tier testing is
not available in all centres, lacks optimal reproducibility and slows
turnaround time. Therefore, an accurate and easily implementable
single-tier HPV test for OpSCC is required. 22 We evaluated the fea-
sibility of RNA ISH on an automated clinical platform (Bond III, Leica
Biosystems) as a single-step HPV test in a diagnostic laboratory
setting. In our study, RNA ISH alone demonstrated 100% concord-
ance with the two-tier algorithm of p16 IHC followed by DNA ISH in
p16+ cases and proved to be a viable standalone alternative.
OpSCC that is p16+ but DNA ISH− causes difficulty in prog-
nostication and stratification in clinical trial. Such cases may arise
4 | HENLEY-SMITH et al.

(A) (B)

(C) (D)

(E) (F)

(G) (H)
HENLEY-SMITH et al. | 5

F I G U R E 1 Quality assessment. Examples of cases requiring retesting following initial quality assessment. A and B, Sparse punctate
chromogen signal (black arrows, A, medium power magnification, B, high-power magnification) visible on human papillomavirus (HPV)
negative cell line (MCF-7) control material. C and D, Lack of chromogen signal on high copy number HPV positive cell line (CaSki) control
material (C, initial test, D, re-test). E and F, Absence of chromogen signal on associated control section probed for UBC (E, initial test, F, re-
test, inset medium power magnification). G and H, Failure of any of the on-slide cell line control material to stain (G, initial test, H, re-test)

either by HPV-independent p16 overexpression or from the subop-
timal sensitivity of DNA ISH causing a false-negative result.13,16,19
Therefore, the HPV status of these OpSCCs remains ambigu-
ous. We, and others, have shown previously that p16+DNAISH−
OpSCCs have poorer clinical outcomes than those in which DNA
ISH is positive. 21,23-25 Manual RNA ISH testing of HPV DNA ISH
negative cases has been shown to identify these false-negative re-
sults. In the current study, we validated a novel automated clini-
cal diagnostic platform against a sub-cohort previously subjected
to manual RNA ISH testing and showed a strong correlation be-
21
tween automated and manual methods (κ = .815). The platform
utilized in our study is already in routine use within the diagnos-
tic pathology laboratory setting. Therefore, automated RNA ISH is
likely to be easily implementable, either as a standalone HPV test,
a second HPV-specific test after p16 IHC or to resolve p16+DNA
ISH− results.
Our data further confirm the greater inter-observer reliability of
RNA ISH compared with DNA ISH.14,19 In keeping with the observa-
tion by Rooper et al,19 all discordant cases in our study were restricted
to cases with low signal intensity and/or transcript number indicating
the necessity for observer training and calibration in RNA ISH inter-
pretation. Reference to negative controls is necessary to distinguish
low HPV transcript signals from non-specific punctate staining.26-28
We show that such control tissue must be stained on the same slide
as the test tissue, since in this study we observed heterogeneity in
staining patterns of serial sections placed on different slides during
the same test run. Our study mitigated against this potential source
of error by including on-slide HPV negative cell line control material.

(A) (B)

(C) (D)

F I G U R E 2 Inter-observer disagreement. A-D, Individual examples of inter-observer discordance. All cases demonstrating inter-observer
discordance contained sparse punctate chromogen signal co-localizing with tumour cell nuclei (black arrows). Each of these cases was p16
negative and were finally assessed as RNA ISH negative on consensus review
6 |
(A)
(C)
F I G U R E 3 p16 positive but DNA ISH negative case resolved by RNA ISH. An example of an OpSCC which was p16 positive, but human
papillomavirus (HPV) DNA ISH negative, clearly demonstrating HPV positivity by RNAISH. A, Haematoxylin and eosin (H&E), B, p16, C, DNA
ISH, D, RNA ISH (inset high magnification)
Only tumours containing signals of greater density and intensity than
that seen in the HPV negative control cells were considered positive,
thereby preventing potential over-interpretation of chromogen signals.
The inclusion of this additional quality control measure may also ex-
plain why there were no p16−RNAISH+ cases in our cohort, an obser-
vation reported in studies utilizing magnification-based interpretation
methods alone.19,20 Nevertheless, further work is required to define
low-end RNA ISH signal threshold cut-offs validated against qRT-PCR
29
reference standards and clinical outcomes.
Inter-slide variation also affects the UBC control. A diffuse
and strongly intense UBC signal on a separate slide does not
necessarily indicate the acceptability of the test section and
should be interpreted with caution; its presence only validates
the quality and presence of amplifiable mRNA within the FFPE
tissue block. We observed inadequate staining in on-slide HPV
positive cell line controls (CaSki and SiHa) despite acceptable
staining of the associated UBC control, thereby rendering the
test unreliable. This observation further highlights the necessity
for positive and negative controls on the same slide. Although
an UBC control for each test case is important for diagnostic
HENLEY-SMITH et al.
(B)
(D)
interpretation, DapB probing for each test case on separate
slides is not required if the HPV negative control material is on
the same slide as the test tissue. This reduction in control slides
would increase testing capacity.
An additional novel aspect of our study is the demonstration of
preserved RNA integrity in archived tissue samples. Interpretable
RNA ISH signals were present in tissue sections from formalin-fixed
specimens paraffin embedded up to 14 years prior to testing. These
findings support the utility of this technique in retrospective tis-
sue-based studies.
5 | CO N C LU S I O N
Human papillomavirus testing by standalone automated RNA ISH
on a clinical diagnostic platform currently available in routine di-
agnostic histopathology laboratories is a feasible alternative to
the two-step algorithm of p16 and DNA ISH. Control tissue stain-
ing procedures need to be adapted to achieve the most accurate
results.
HENLEY-SMITH et al. | 7

(A) (B)

(C) (D)

F I G U R E 4 Discordance between manual and automated RNA ISH. A single p16 positive but DNA ISH negative case demonstrated
discordance between manual (A, low-power magnification, B, high-power magnification) and automated (C, low-power magnification, D,
high-power magnification) RNA ISH, the latter demonstrating clear positivity

AC K N OW L E D G E M E N T S analysis; Funding acquisition; Investigation; Methodology; Project

The target and control mRNA probes (RNAscope 2.5 LS probe-HPV- administration; Supervision; Writing-original draft; Writing-review
HR18, RNAscope 2.5 LS Negative Control Probe_dapB, RNAscope & editing.
2.5 LS Positive Control Probe-Hs-UBC) were kindly provided by
Advanced Cell Diagnostics). The Leica Bond III and detection kit PEER REVIEW
(RNAscope 2.5 LS Assay-Brown) were kindly provided by Leica The peer review history for this article is available at https://publo​
Biosystems. ns.com/publo​n/10.1111/jop.13103.

C O N FL I C T O F I N T E R E S T ORCID
All authors declare no conflict of interest. Selvam Thavaraj https://orcid.org/0000-0001-5720-7422

AU T H O R C O N T R I B U T I O N S
Rhonda Henley-Smith: Data curation; Formal analysis; Investigation;
Methodology; Project administration; Validation; Writing-original
draft; Writing-review & editing. Alice Santambrogio: Data cura-
tion; Formal analysis; Investigation; Methodology; Writing-review
& editing. Cynthia Andoniadou: Investigation; Methodology;
Supervision; Writing-review & editing. Edward W Odell: Formal
analysis; Supervision; Writing-original draft; Writing-review & ed-
iting. Selvam Thavaraj: Conceptualization; Data curation; Formal
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How to cite this article: Henley-Smith R, Santambrogio A,
Andoniadou CL, Odell E, Thavaraj S. RNA in situ hybridization
for human papillomavirus testing in oropharyngeal squamous
cell carcinoma on a routine clinical diagnostic platform. J Oral
Pathol Med. 2020;00:1–8. https://doi.org/10.1111/jop.13103