Supplementary Material Detailed Description of the Detection Methods

Supplementary material – Detailed description of the detection methods

p16 immunohistochemistry (p16 IHC)

p16 detection was performed on 4-μm thick whole tumor sections cut from formalin-fixed, paraffin-embedded tissue blocks using a mouse monoclonal antibody against p16 (CINtec® Histology Kit, clone E6H4™, ready-to-use, Roche mtm laboratories AG (Mannheim, Germany)) on a Ventana BenchMark ULTRA immunostainer (Ventana Medical Systems, Inc. (Tucson, AZ, USA)) according to the standardized protocol. The Ventana CC1, ethylendiaminetetraacetic acid-Tris, pH 8.0 solution was utilized for antigen retrieval and the OptiView DAB IHC Detection Kit with 3,3´-diaminobenzidine as chromogen was used for visualization of the antigen. Cases of HR-HPV-positive tonsilar SCC and of chronic tonsillitis were used as positive and negative controls, respectively.

All cases were independently assessed by two pathologists (J.L., A.R.), who were blinded to the results of other methods, and classified binarily as positive or negative. According to recent studies [31-34] and current College of American Pathologists (CAP) recommendations [35], a case was considered positive if ≥ 70 % of tumor cells showed strong nuclear and/or cytoplasmic staining. Discrepant cases were resolved by concensus review.

HPV DNA in situ hybridization (ISH)

Chromogenic ISH for detection of HR-HPV DNA was performed on 4-μm thick whole tumor sections cut from formalin-fixed, paraffin-embedded tissue blocks on a Ventana BenchMark ULTRA immunostainer (Ventana Medical Systems, Inc.) using the INFORM® HPV III Family 16 Probe (B) (Ventana Medical Systems, Inc.), a cocktail targeting HR-HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66. Briefly, tissue sections were deparaffinized, heat-treated and subjected to protease digestion. After denaturation for 12 min, the cocktail probe was added and hybridized for 120 min. Finally, the Ventana ISH iView Blue Plus Detection Kit (Ventana Medical Systems, Inc.) with nitro blue tetrazolium as chromogen was utilized. Control slides provided by the manufacturer were used on each run.

All cases were independently assessed by two pathologists (J.L., A.R.), who were blinded to the results of other methods, and classified binarily as positive or negative. Any definitive nuclear staining in the tumor cells observed as diffuse and/or dot-like navy-blue precipitate, was considered positive. Discrepant cases were resolved by concensus review.

HPV E6/E7 mRNA in situ hybridization (ISH)

ISH for detection of HR-HPV E6/E7 mRNA transcripts was performed manually on 4-μm thick whole tumor sections cut from formalin-fixed, paraffin-embedded tissue blocks using the RNAscope® Probe HPV-HR18 (Advanced Cell Diagnostics, Hayward, CA, USA), a cocktail targeting HR-HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82, and detection system RNAscope® 2.0 HD Reagent Kit (Brown) (Advanced Cell Diagnostics) as previously described [36]. In brief, tissue sections were baked for 1 h at 60ºC, followed by deparaffinization, incubation with pretreatment reagent 1 for 10 min at room temperature, pretreatment reagent 2 for 15 min at 100ºC, and pretreatment reagent 3 for 25 min at 40ºC. Tissue sections were then incubated with probe cocktail for 2 h at 40ºC. Detection of immobilized probes was performed according to the manufacturer´s instructions. 3,3´-diaminobenzidine was used as chromogen. Finally, the sections were counterstained with hematoxylin. Control probes for the bacterial gene DapB (negative control) and for the housekeeping gene ubiquitin C (positive control – evidence of adequate RNA) were used on each case.

All cases were independently assessed by two pathologists (J.L., A.R.), who were blinded to the results of other methods, and classified binarily as positive or negative. Any definitive nuclear and/or cytoplasmic staining in the tumor cells observed as brownish dots and/or clusters, was considered positive. Discrepant cases were resolved by concensus review.

HPV DNA polymerase chain reaction (PCR) and typing

The HPV DNA detection was performed by PCR as follows. The HPV DNA was extracted from formalin-fixed, paraffin-embedded tumor tissue after deparaffinization in xylen and rehydration in ethanol using the commercial DNA Sample Preparation Kit (Roche, Basel, Switzerland) according to the manufacturer’s protocol.

PCR amplification of β-globin sequences was performed to confirm sample fitness for PCR assay. Two products (length 110 bp and 268 bp) of real-time PCR were amplified using primers PC03 5‘-ACACAACTGTGTTCACTAGC-3‘, PC04 5‘-CAACTTCATCCACGTTCACC-3‘, GH20 5‘-GAAGAGCCAAGGACAGGTAC-3‘ [37] (Generi Biotech, Hradec Kralove, Czech Republic) and detected by measurement of fluorescence in LightCycler® 480 (Roche). The PCR reaction was performed in a volume of 20 μL, containing 7.5μL of KAPA SYBR Fast qPCR Kit Master Mix 2x (Kapa Biosystems, Inc., Boston, USA), 0.3μL of 10 μmol of each primer (PC03/PC04 or PC04/GH20), 6.9μL of H2O for PCR and 5 μL of HPV DNA at dilutions under 20 ng per reaction. The PCR protocol was then carriedout with initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 3 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s.

All samples were screened for the presence of HPV DNA by PCR amplification with EIA Kit HPV GP HR (Diassay, Rijswijk,Netherlands)with primers GP5+/GP6+ located within the HPV L1 gene. The sequences of the forward and reverse primers used were 5´-TTTGTTACTGTGGTAGATACTAC-3´ (GP5+) and 5´-GAAAAATAAACTGTAAATCATATT-3´ (GP6+). The PCR reaction was performed in a volume of 50 μL, containing 39.8μL of HPV GP PCR Master Mix, 0.2μL of polymerase and 10 μL of HPV DNA at various dilutions. The PCR protocol was then carriedout with initial denaturation at 94°C for 4 min, followed by 40 cycles of denaturation at 95°C for 20 s, annealing at 38°C for 30 s, extension at 71°C for 80 sand final extension at 71°C for 4 min. Detection was performed by EIA in a microplate according to the manufacturer’s protocol. This method detects HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 45, 51, 52, 53, 56, 58, 59, 61, 66, 67, 68 (and 68a), 69, 71, 72, 73, 81, and 82 (MM4 and IS39).

Samples showing HPV DNA presence by the above mentioned procedure and/or by ISH and samples showing any degree of immunohistochemical expression of p16 were all subsequently analyzed using the Linear Array HPV SPF10 Genotyping Test (LBP, Rijswijk, Netherlands).The test involves three steps: PCR amplification of target DNA, nucleic acid hybridization, and detection of 44 HPV types, specifically 3, 4, 5, 6, 7, 8, 11, 13, 16, 18, 26, 27, 30, 31, 32, 33, 34, 35,37, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, and 74. PCR amplification was performed using primers SPF10 located within the HPV L1 gene. The PCR reaction was performed in a volume of 50 μL, containing 40 μL of Master mix and 10 μL of HPV DNA at various dilutions. The PCR protocol was then carriedout with an initial denaturation at 94°C for 9 min, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 45 s, and extension at 72°C for 45 s. The PCR product was denaturated with denaturation solution and hybridized onto a strip containing specific probes for the 44 above mentioned HPV types and hybridization control lines. Detection was done using 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium as the chromogen. Positive reaction was visible as a blue line on the strip.

HPV E6/E7 mRNA reverse transcription and polymerase chain reaction (RT-PCR)

The HPV E6 and E7 viral protein detection was performed by RT-PCR as recently described [38]. HPV RNA was extracted from paraffin-embedded tissue after deparaffinization in xylen and rehydration in ethanol using the commercial RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol.

Reverse transcription of RNA was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA). The reverse transcription was performed in a volume of 20 μL, containing 2 µL of 10x RT Buffer, 0.8 µL of 25x 100 mM DTP Mix, 2 µL of 10x RT Random Primers, 1 µL of MultiScribe Reverse Transcriptase, 4.2 µL of Nuclease-free H2O and 10 µL of RNA per reaction. The reverse transcription protocol was then carriedout with an initial step at 25°C for 10 min, followed by 37 °C for 120 min, 85°C for 5 min, and cooling at 4°C for an unlimited time.

PCR amplification of β-globin sequences was performed to confirm sample fitness for PCR assay. The 110 bp product of RT-PCR was amplified using primers PC03 5‘-ACACAACTGTGTTCACTAGC-3‘, PC04 5‘-CAACTTCATCCACGTTCACC-3´ [37] (Generi Biotech, Hradec Kralove, Czech Republic). The PCR reaction was performed in a volume of 20 μL, containing 7.5μL of KAPA SYBR Fast qPCR Kit Master Mix 2x (Kapa Biosystems, Boston, USA), 0.3μL of 10 μmol of each primer (PC03/PC04 or PC04/GH20), 6.9μL of H2O for PCR and 5 μL of HPV cDNA at dilutions under 20 ng per reaction. The PCR protocol was then carriedout with initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 3 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s.

PCR amplification of E6 and E7 viral protein sequences was performed by RT-PCR. The PCR reaction was performed in a volume of 20 μL, containing 7.5μL of Power SYBR Green qPCR Master Mix (2x) (Applied Biosystems, Carlsbad, CA, USA), 0.3μL of 500 nmol of each primer according to the tested HPV type (for each HPV 16, 18, and 35: HPV E6-Forward, HPV E6-Reverse, HPV E7-Forward, HPV E7-Reverse) and 5 μL of HPV cDNA per reaction. PCR protocol was then carriedout with an initial denaturation at 95°C for 10min, followed by 50 cycles of denaturation at 95°C for 10 s, annealing at 58°C for 15 s, and extension at 60°C for 15 s.