HPV Status and Overall Survival of Patients with Oropharyngeal Squamous Cell Carcinoma – A Retrospective Study of a German Head and Neck Cancer Center

Materials and Methods

Study design. From 1988 to 2008, human tumor samples from patients with HNSCC were collected in the ENT Department of the J.W. Goethe University Hospital in Frankfurt am Main. The specimens were derived from biopsies of resected tumors and were stored at −80°C for further investigations. Patients with primary tumors of the oropharynx were included in this study. All patients had died at baseline. A total of 104 frozen tumor samples were obtained. All included cases had histologically confirmed diagnosis of squamous cell carcinoma. Medical records and our tumor documentation system ‘AdOnco’ were reviewed (18, 19). Clinical information, including patients' characteristics, were recorded for statistical analysis. Tumor stage was classified according to the current classification of malignant tumors (TNM) and Union internationale contre le cancer (UICC) staging classification (seventh edition) (20). For HPV detection, the DNA from each tumor specimen was purified first. Subsequently, the HPV status was determined by a two-step polymerase chain reaction (PCR) combined with p16 immunohistochemistry (p16 IHC) as described previously (13). Statistical analysis was performed with BiAS™ (Version 10.02^© 1989-2013 epsilon-Verlag, Hochheim, Darmstadt, Hesse, Germany) in cooperation with the Department of Biostatistic and Mathematic Modelling of the J.W. Goethe University Hospital in Frankfurt am Main. The study protocol was approved by the Ethics Committee of the Department of Medicine of the J.W. Goethe University Hospital in Frankfurt am Main.

DNA purification. HPV DNA was purified using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). A 25 mg piece of frozen tumor tissue was cut and 180 ml tissue lysis buffer (ATL) (Qiagen, Hilden, Germany) containing 0.5% Reagent DX (Qiagen) was added. The disruption was performed with steel beads (5 mm diameter) by Tissue Lyser LT (Qiagen) on 50 Hz for 40 sec. Proteinase K (20 μl) was added and digestion took three hours in a water-bath (56°C). The samples were shaken three times each hour. AL buffer (Qiagen, Hilden, Germany) (200 μl) was added and mixed samples were incubated for 10 min in a water bath at 70°C, ethanol was added and the mixture was transferred into QIAamp Mini columns. Following centrifugation (8000 rounds per minute (rpm) for 1 min), the remaining DNA was washed with AW1; 500 μl, 8000 rpm for one minute) and AW2 buffer (500 μl, 14,000 rpm for 3 min) The DNA was eluted with 200 μl AE buffer (Qiagen, Hilden, Germany) (8,000 rpm for 1 min). DNA concentration and purity was measured with Helios α spectrometer (Thermo Scientific, Dreieich, Hesse, Germany; wavelength: 260 nm, 280 nm).

HPV detection. HPV status was determined by a two-step PCR combined with p16 IHC (13, 21).

PCR. For PCR, 40 ng DNA was used. Firstly general HPV DNA was detected with the primers MY09 (5’-CGT CCM ARR GGA WAC TGA TC-3’) and MY11 (5’-GCM CAG GGW CAT AAY AAT GG-3’), which amplify a 450 bp fragmet of the HPV L1 gene (12). As an internal control, a fragment of the human ß-globin gene (110 bp) was amplified by the primers Pr03 (5’-ACA CAA CTG TGT TCA CTA GC-3’) and Pr04 (5’-CAA CTT CAT CCA CGT TCA CC-3’) (23). In a second step, the HPV DNA-positive cases underwent a type-specific PCR for analysis of the high-risk HPV subtypes HPV-16 and HPV-18. For this reason, the specific primers Pr1 (5’-CCG AGC ACG ACA GGA ACG ACT-3’), Pr2 (5’-TCG TTT TCT TCC TCT GAG TCG CTT-3’), Pr3 (5’-GTC AAA AGC CAC TGT GTC CT-3’) and Pr4 (5’-CCA TCC ATT ACA TCC CGT AC-3’) were used. The primers Pr1 and Pr2 amplify a 172 bp fragment of the HPV18 genome (E6/7) and primers Pr3 and Pr4 amplify a 499 bp fragment of the HPV16 genome (E7+6/1) (24). All primers were purchased from Invitrogen (Darmstadt, Germany). PCR was prepared with Platinum Blue PCR SuperMix (Invitrogen) and performed on an Eppendorf Mastercycler (Eppendorf AG, Hamburg, Germany). After 2 min heat-shock activation of Taq DNA polymerase, the PCR ran for 45 cycles: 95°C denaturation for 30 sec, annealing at 50°C for 30 sec and extension at 72°C for 1 min. Previously examined samples positive for HPV 16 and HPV 18 were used as controls. The PCR products were analyzed on a 2% agarose gel in 0.5 × TRIS-Borat-EDTA-Puffer (TBE) (Qiagen, Hilden, Germany). The Gel electrophoresis run 45 min at 150 V. The gel was stained in 3×GelRed (Biotrend, Cologne, Germany) for 30 min. The bands were detected by Kodak Image Station 44C by exposing for 30 sec.

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Figure 1.

Kaplan-Meier estimates of 5-year overall survival according to HPV-status.

p16 IHC. Detection of p16 protein by IHC was performed with CINTec Histology Kit (mtm laboratories AG, Heidelberg, Germany). The slices were cut by Cryotome (Reichert-Jung 2800 Frigocut-E, Depew, NY, USA), fixed in methanol for 10 min and stored at −20°C. For the staining procedure, the sections were rehydrated and blocked against peroxidase for five minutes. All incubation steps were performed in a moist chamber. After washing (5 min), the sections were incubated with antibody to p16^INK4a (mtm laboratories AG, Heidelberg, Germany) (or negative control) for 30 min. The Antibody solution was removed with wash buffer and sections were incubated with visualization reagent for 30 min. After a wash step, the sections were stained with substrate-chromogen solution, 3,3’-diaminobenzidine tetrahydrochloride (DAB) (DCS, Hamburg, Germany) for 10 min. The Reaction was stopped in water. Hematoxylin (AppliChem, Darmstadt, Hesse, Germany) counterstaing was performed for 30 sec followed by blueing for 2 min. The sections were then dehydrated by incubation in ascending ethanol series (70%, 95%, 100%, ProTaqs Clear (quartett)) and mounted with ProTaqs Paramount (quartett). The microscopy images were taken with an AxioCam ICc1 (Zeiss, Oberkochen, Germany).