A 24-year old female patient that never smoked presented with an advanced laryngeal carcinoma. The patient showed a long history of RRP. At the age of three, she had undergone a first microlaryngoscopy with removal of laryngeal masses, which were diagnosed as papillomatosis in the histopathologic investigation. Several hundred revision surgeries had been necessary to control recurrences of the disease. In 2008, a further microlaryngoscopy and histopathologic examination revealed a malignant laryngeal squamous cell carcinoma of stage IV. Total laryngectomy with bilateral neck dissection was performed. Adjuvant chemoradiation with 5-FU and carboplatin to a total dose of 70 Gy was administered postoperatively. However, the patient developed inoperable paratracheal recurrence of the primary tumor and eventually died 21 months after the diagnosis of recurrence. The patient had never been treated with Cidofovir.
We analyzed formalin-fixed, paraffin-embedded tissue that had been archived at the Institute of Pathology at the University of Cologne Medical Center from previous resections (1985 and 1989) of the laryngeal papilloma, as well as fresh frozen tumor tissue from the laryngeal carcinoma and lymph node metastasis (2008). Furthermore, we analyzed 9 additional, randomly chosen fresh frozen tissue samples from our tumor tissue archive, i. e. three cases of laryngeal papilloma (two HPV6- and one HPV11-positive), three HPV16-associated oropharyngeal squamous cell carcinomas (OSCC) and three HPV-negative oropharyngeal squamous cell carcinomas. These samples served as controls for immunohistochemical stainings and Western blotting.
The ethics committee of the University of Cologne medical faculty approved this study. Written, informed consent had been obtained from the hereby reported patient and all other patients whose material was used for control. All control samples were anonymized prior to analysis.
Genomic DNA was extracted from five 10 microm-thick formaldehyde-fixed, paraffin-embedded tissue sections and of snap frozen tissue sections, respectively, using the Gentra puregene tissue kit with proteinase K treatment (Qiagen, Hilden, Germany) according to the manufacturer's protocol.
Paraffin was removed by graded xylol and ethanol baths before extraction of the DNA. Both a general primer GP5+/6+ PCR (150 base pairs [bp] product) and a nested PCR with degenerate primers A5/A10 (527 bp product) and A6/A8 (268 bp product) for HPV detection were applied. Five microl of these PCR products were separated on a 1.5% agarose gel and visualized by ethidium bromide staining. Direct sequence analysis of purified PCR products (QIAquick PCR purification kit, Qiagen, Hilden, Germany) was carried out with an ABI Prism 377 DNA sequencer using the Taq FS Big-Dye-Terminator cycle sequencing method (PE Applied Biosystems, Weiterstadt, Germany). Human papillomavirus typing was performed by comparison of the obtained sequences with database entries using NCBI Blast search. Additionally, HPV-typing was performed by bead-based multiplex hybridization of A6/A8 PCR products with 38 type-specific probes as previously described. HPV-types covered by the assay were HPV6, 11, 16, 18, 26, 30, 31, 33-35, 39, 40, 42-45, 51-59, 61, 66-68, 70-73, 81-84, 89.
beta-globin gene PCR served as a positive control for sufficient DNA of adequate quality, and to show that samples were free of PCR inhibitory substances (268 bp PCO4/GH20 PCR product).
The following primary antibodies were used: monoclonal mouse anti-human p16INK4A antibody, clone G175-405 (BD Biosciences, Heidelberg, Germany); monoclonal mouse anti-human AKR1C3 antibody, clone NP6-G6.A6 (does not cross react with AKR1C1, AKR1C2, and AKR1C4) (Sigma-Aldrich, Munich, Germany), monoclonal mouse anti-human p53 antibody, clone DO-7 (BioLogo, Kronshagen, Germany), and monoclonal mouse anti-human pRb antibody, clone J146-35 (BD Biosciences, Heidelberg, Germany).
Immunohistochemical staining on 5 microm-thick formaldehyde-fixed, paraffin-embedded tissue sections and cryostat sections, and slide evaluation was performed as previously described. Primary antibodies were detected using the avidin-biotinylated peroxidase complex (ABC) procedure (Vectastain-Elite-ABC kit; Vector Laboratories, Burlingame, USA) and peroxidase activity was visualized using diaminobenzidine/H2O2 (BD Biosciences, Heidelberg, Germany). Sections were counterstained with hematoxylin and mounted in Histofluid (Marienfeld, Lauda-Koenigshofen, Germany). Each analysis included negative and positive controls. Analysis was performed by two independent researchers (CUH and SFP) and consensus was achieved. Strong nuclear and cytoplasmic staining was considered positive for AKR1C3 expression.
Fluorescence in situ hybridization (FISH) for the detection of HPV6 on 5 microm thick formaldehyde-fixed, paraffin-embedded tissue sections, and evaluation of nuclear FISH signals was performed as previously described. In brief, sections were deparaffinized, treated with 85% Formic acid/0.3% H202 and dehydrated in an ethanol series containing 0.01 M HCl (acidic dehydration). The digoxigenin-labeled HPV6 probe (Panpath, Budel, The Netherlands) was applied under a coverslip at a concentration of 1 ng/microl in 50% formamide, 2x SSC pH 7.0, 10% dextran sulphate, and a 50x excess of carrier DNA (salmon sperm DNA) followed by hybridization over night. Detection was carried out with peroxidase-conjugated sheep anti-digoxigenin Fab fragments (Roche, Basel, Switzerland; 1:100 diluted in 4xSSC containing 5% nonfat dry milk), followed by a tyramide signal amplification (TSA) reaction using rhodamine-labeled tyramide. After dehydration in an ascending ethanol series, slides were mounted in Vectashield (Vector Laboratories, Burlingame, USA) containing 0.2 microg/ml 4,6-diamidino-2-phenyl indole (DAPI; Sigma, Germany). Slides were evaluated under a Leica DM-RE fluorescence microscope equipped with DAPI and rhodamine filters and images were recorded with the Metasystems Image Pro System (black and white CCD camera; Sandhausen, Germany).
Total RNA was extracted from five 10 microm-thick snap frozen tissue sections using the RNeasy mini kit (Qiagen) according to the manufacturer's instructions, which included DNase treatment. RNA concentration and quality were determined by RNA standard sense chips on a BioRad Experion system (BioRad, Munich, Germany). 500 ng RNA was reverse transcribed in 20 microl using oligo-dT primer (25 microM), dNTPs (10 mM each), DTT (0.1 M), 5x RT-buffer and 40 U SuperScript reverse transcriptase (Invitrogen, Karlsruhe, Germany). Quality of generated cDNA was determined by a standard GAPDH gene PCR (forward primer 5'-AATGGAAATCCCATCACC-3', reverse primer 5'-CAGCCTTGGCAGCGCCAG-3'; 441 bp product).
For detection of the physical status of HPV6 (episomal and/or integrated virus), a 3'-RACE APOT assay (amplification of papillomavirus oncogene transcripts) was used which was based on Klaes et al. and modified for HPV6 detection. After reverse transcription of RNA, a nested PCR with a set of newly designed 5'-Primers (1st 5'-primer: 5'-GGACGGACAAGATTCACAACC-3'; 2nd 5'-primer: 5'-CCTGTTGCTGTGGATGTGACAGC-3') both located in the E7 open reading frame of HPV6 and a 3'-Frohman primer (for both nested PCR-setups) was used. PCR products were separated on a 1.2% agarose gel. Candidate bands for viral integration different from episomal ones were cut out of the gel, purified (Gel extraction kit, QIAGEN) and sequenced. Sequences were compared with NCBI and UCSC database entries to determine virus-human fusion points indicating viral integration.
Sequence analysis of TP53 was performed by PCR amplification followed by direct sequencing based on the IACR protocol. Exons 4-8 were amplified from cDNA, Exons 2, 3 and 9-11 were amplified from DNA. Sequences were compared with reference sequence NC_000017.9.
High-resolution oligo-nucleotide aCGH was performed using a 105 K microarray (99,000 human sequence probes) (Agilent Technologies, Boeblingen, Germany) according to the manufacturer's protocol. In brief, 1.5 microg genomic tumor and reference DNA (G1521 female DNA, Promega, Mannheim, Germany) were digested with AluI and RsaI, labeled and processed according to the manufacturer's protocol and scanned using an Agilent G2567AA Scanner. Images were extracted using Feature Extraction 9.5 software and visualized using Genomic Workbench 6.5 software (both Agilent Technologies). Aberrations were calculated by the z-score algorithm with a threshold of 2.5. Primary array CGH data have been made publicly available at EMBL-EBI (Accession No. E-MEXP-3330) for use in subsequent analysis.
Total protein extracts were derived from five 10 microm-thick snap frozen tissue sections. Extracts were normalized after protein concentrations were determined by bradford assay according to the manufacturer's protocol (Roti-Nanoquant, Carl Roth, Karlsruhe, Germany) and equal amounts were resolved via SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Blotting of proteins on nitrocellulose membranes was carried out according to standard protocols in a semi-dry blotter using Towbin buffer (0.025 M Tris-HCl pH 8.0; 0.192 M Glycin; 20% Methanol). After blocking with 4% non-fat milk powder in TBS-T buffer (0.1% Tween 20 in Tris-buffered saline), monoclonal anti-AKR1C3 antibody (clone NP6-G6.A6, Sigma-Aldrich, Munich, Germany) followed by secondary anti-mouse IgG peroxidase conjugated antibody (Sigma-Aldrich A 9044) was applied to the membrane. Dilution of antibodies and subsequent washing was done in TBS-T buffer. For visualization, Pierce solution (Biozym, Hessisch Oldendorf, Germany) was applied to the nitrocellulose membrane. Chemiluminescence signals were documented with an Alpha Innotech Fluorchem FC2 system (Biozym). Blots were stripped and re-probed with monoclonal beta-actin antibody (Clone AC-74, Sigma-Aldrich) as control.