Supplementary Information
Control for cross-contamination
Prevention of, and control for cross-contamination during tissue analysis is the
prerequisite to obtain valid HPV DNA and RNA data. To monitor cross-contamination
during sectioning on a microtome, the FFPE rat liver control (RLC) was sectioned
after each 5th FFPE block. To monitor cross-contamination during sectioning on a
cryotome, DFT biopsy of NOM obtained by uvulopalatopharyngoplasty from a healthy
(cancer free) individual was sectioned after each 10th DFT biopsy. Both the RLC and
NOM were pretested for HPV negativity. Both the RLC (N=32) and NOM (N=5) as a
sectioning controls yielded negative HPV DNA results only when cleaning was
performed as described (Halec et al, 2012) and gloves were changed accordingly.
One vial of lyses buffer was used per each 11 biopsy samples in one batch to control
for cross-contamination during DNA and RNA extraction, respectively. Water
samples (one per 14 tissues) and PCR master mixes without template (one per 14
tissues) were included to control for reagents contamination and potential crosscontamination in (RT)-PCR experiments. Each hybridization plate contained
hybridization buffer without (RT)-PCR product as a negative control in hybridization.
Five separate laboratories were used for sectioning, and pre- and post-PCR
procedures.
HPV DNA, E6*I mRNA and IHC data per biopsy and per patient
Detailed overview with all the HPV DNA, HPV E6*I mRNA and IHC results per L-SCC
biopsy and per patient is displayed in Supplementary Table S1.
Definition of final IHC result per biopsy and per patient
The TMA´s with L-SCC (N=154) and NOM (N=6) tissue cores were stained both
manually and on DAKO autostainer using following antibodies: p16INK4a (Clone No.:
E6H4,
MTM
Laboratories,
Heidelberg,
Germany),
pRb
(Clone
No.:
1F8,
Visionbiosystems Novocastra, Newcastle upon Tyne, United Kingdom), cyclin D1
(Clone No.: DCS-6, DAKO, Denmark), and p53 (Clone No.: DO.7, DAKO, Denmark)
primary antibodies as described in (Halec et al, 2012; Holzinger D, Article in press,
2013). Due to presence of cartilage in laryngeal tissue, 48/154 (31%) L-SCC tissue
cores were lost from TMA´s during IHC procedure. For these cases, new whole
sections were made from FFPE biopsies and stained both manually and on DAKO
autostainer. All stained slides were scanned at a 40-fold magnification (0.23Lm/pixel)
and had an averaged file size of 12GB and were digitalized using Hamamatsu’s
NanoZoomer 2.0-HT scansystem (Hamamatsu Photonics, Japan). Visualization of
digitalized slides was then carried out using Hamamatsu’s NDP Viewer software
(version 1.1.27). At least two of the four different evaluators scored protein
expression in both manually and DAKO autostainer stained TMA´s and whole tissue
slides, and data were compared. To define protein expression as high or low in tumor
sections, expression of cellular proteins in NOM was used as guidance. Only two
protein expression categories were applied here, low and high. Normal protein
expression was in category low expression. Cutoff´s were defined and applied as
described in Table 1 of Result section.
For 34 patients multiple FFPE biopsies were available (Table S1) and 30 patients
had IHC data available from at least two biopsies. Among these 30 patients, for 26
(87%), heterogeneous expression of at least one protein was observed among
multiple biopsies (proteinlow in one, and proteinhigh in another biopsy from the same
tumor, Table S1). To define final protein expression per patient, the same protein
pattern needed to be seen in ≥50% of observations. If 50% of biopsies were
proteinlow, and 50% were proteinhigh, 2 evaluators examined the biopsies one more
time and consensus was reached.
References:
Halec G, Schmitt M, Dondog B, Sharkhuu E, Wentzensen N, Gheit T, Tommasino M, Kommoss F,
Bosch FX, Franceschi S, Clifford G, Gissmann L, Pawlita M (2012) Biological activity of
probable/possible high-risk human papillomavirus types in cervical cancer. Int J Cancer 132(1): 63-71
Holzinger D FC, Henfling N, Kaden I, Grabe N, Lahrmann B et al. (Article in press, 2013) Identification
of Oropharyngeal Squamous Cell Carcinomas with Active HPV16 Involvement by
Immunohistochemical Analysis of the Retinoblastoma Protein Pathway. Int J Cancer