The study was approved by the ethical board in Lund with diary number 2015/907. Inclusion criteria were men over 18 years, having penile cancer surgery at Skane University Hospital (SUH) in Sweden between 22nd of June 2015 and 10th of August 2021. SUH is one of two national referral centers for penile cancer. Excised tumor tissues were transported fresh to the Department of Pathology, SUH and a 3 mm single-use punch biopsy was taken from the tumor and submerged in 1 mL RNAlater (Ambion). Another 3 mm single-use punch biopsy was taken 10 mm outside the macroscopic margins of the tumor and submerged in 1 mL RNAlater (Ambion). In two samples 10 mm adjacent to the tumor, the HPV analysis was not adequate, due to no detectable human DNA in the sample. In one sample 10 mm adjacent to the tumor, the analysis was not performed.
Controls comprised age-matched men over 18 years, circumcised for non-malignant reasons at urological departments in Skane. At surgery, a biopsy of approximately 5 mm was taken from excised penile skin, and submerged in 1 mL RNAlater (Ambion) as described (22).
The biopsies were transported to the Department of Microbiology where they were transferred to 1 mL GITS-solution (4M guanidinium thiocyanate, 22mM NaCitrate and 5% Sarcosyl (N-Lauroylsarcosine sodium salt) and 1% mercaptoethanol) and incubated at room temperature overnight. Then DNA was extracted with the Total NA-kit (Roche, Stockholm, Sweden) using MagNA Pure LC (200 µL input and 100 µL output). Sample adequacy was assessed by testing 5 µL of the sample for the human beta globin gene with a real-time PCR (23). Simultaneous identification of 40 genital HPV types was carried out by MGP-PCR in 25 µL, containing 5 µL of extracted material and subsequent Luminex analysis (24, 25), including probes for 40 HPV types: 6, 11, 16, 18, 26, 30, 31, 33, 35, 39, 40, 42, 43, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 67, 68 (a and b), 69, 70, 73, 74, 81, 82, 83, 85, 86, 87, 89, 90, 91 and 114.
The integration of HPV-DNA into the human genome most frequently disrupts the E2 gene (26). As a surrogate marker for the physical status of HPV16, the quantity of HPV16 E2 gene was determined as described by Letsolo et al. (27). We used the mean log10 values of E2 and E7 copy numbers from each sample and calculated the ratio of E2/E7 gene copy numbers of HPV16 to investigate the presence of integrated, mixed and episomal forms of HPV16. HPV16 was classified as follows: ‘integrated’ when no E2 copy numbers could be detected and E7 copy numbers were present; ‘mixed status’ when E2/E7 ratios were 0.1–0.8; and ‘episomal status’ when the presence of E2/E7 copy number ratio was > 0.8. The samples were analyzed in duplicate. In addition, another aliquot (200 µL) of the GITS-lysate was used for mRNA extraction using the Oligotex Direct mRNA Mini Kit (Qiagen). The extraction was performed according to the manufacturer’s protocol for isolation of PolyA mRNA from animal tissues. The mRNA was eluted by adding 45 µL of Oligotex elution buffer (70°C) to the column and centrifuging for 1 minute at maximum speed. Purified mRNA was stored at -80°C until use. Quantitative PCR of HPV16 E7 mRNA was analyzed in triplicate and performed as previously described (27). To compensate for the smaller elution volume of the mRNA extraction (45 µL) compared to that of the DNA extraction (100 µL), the HPV16 mRNA copy numbers were divided by 2.5. The HPV16 mRNA expression level was given as HPV16 mRNA copy numbers per HPV16-DNA copy.
Histopathological diagnosis and classification of the subtype of penile cancer was retrospectively reviewed by two experienced pathologists subspecialized in uropathology and participating in the national multidisciplinary team conference of penile cancer in Sweden. A recent study showed that pathologists who have experience of penile cancer diagnostics have a good concordance in identifying HPV-related and non-HPV-related histological subtypes of penile cancer (28).
The glass slides were retrieved from the pathology archive, and one representative slide of the tumor was chosen for each case in hematoxylin-eosin (H&E) stain. Assessment of histological subtype and histological grade was carried out using glass slides (106 cases) and high-resolution digital slides (40 cases). The slides were scanned using NanoZoomer S360 (S60 for large histologic sections) by Hamamatsu, and Sectra IDS7 software. The pathologists were blinded to the results of the HPV detection by PCR in the tumor material to avoid any bias in tumor assessment. The histological subtypes were determined according to the WHO criteria in “Classification of tumors of the urinary system” and the International Society of Urological Pathology recommendations (2016) (4, 29). Following the separate assessment of all cases, the results were compared to identify eventual discrepancies, which were found in six cases where the diagnosis was difficult to make without HPV status. p16INK status was used in five cases and HPV result in one case and thereby the pathologists agreed upon the final diagnosis.
All cases and controls completed a questionnaire, regarding medical history, medication, smoking habits, number of sexual partners and former symptoms and procedures performed on the penis (Supplementary 1). Controls completed the questionnaire in connection with the circumcision, while penile cancer cases completed the questionnaire after surgery. If the questionnaire was not returned, a new letter was sent to the patient and if not returned, the patient was informed by phone and if necessary, a further questionnaire was sent.
The study was approved by the ethical board in Lund with diary number 2015/907.
The statistical analysis was performed using SPSS, version 26, IBM Statistics, IBM Corp., Armonk, NY, USA. Correlation was calculated with Chi-square tests and Fisher’s exact test in small numbers. When p < 0.2, odds ratios were calculated with multiple logistic regression adjusted for > 10 sexual lifetime partners, smoking, former smoking, former skin disease, former phimosis, former penile biopsy, former penile surgery and former genital warts.