Patients diagnosed with VSCC in the northern region of Sweden between 1990 and 2013 were identified through the Swedish Cancer Registry. Out of 258 correctly classified patients treated at the University Hospital in Umeå, 34 declined to participate, 81 were excluded due to missing clinical data and in 31 cases material could not be obtained. Finally, this study was based on 112 patients with an age range of 37–94 years. Patients' records were retrieved from the Department of Oncology at the University Hospital in Umeå and clinical data were collected. Formalin-fixed, paraffin-embedded (FFPE) specimens from diagnostic biopsies or resection material from the time of diagnosis were retrieved from the biobank at Västerbotten County Council (Umeå, Sweden). FFPE-material was handled and stored in room temperature. All patients alive at the start of the study signed informed consent for the use of their tissues. This research was approved by the Regional Ethical Review Board at Umeå University.

DNA was extracted from 25–30 µm FFPE sections using the QIAamp DNA Tissue FFPE Tissue kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's instructions. To check for possible contamination, a negative control consisting of an empty test tube was included between every fifth sample and treated likewise. DNA-samples were stored at 20°C.

HPV-PCR analysis was carried out using 125 ng of extracted DNA with the general primer pair GP5+/6+, amplifying a fragment of the conserved HPV L1 gene (23). Primer sequences were as follows: Forward, 5′-TTTGTTACTGTGGTAGATACTAC-3′ and reverse, 5′-GAAAAATAAACTGTAAATCATATTC-3′. The 50 µl PCR mixture consisted of 5 µl GeneAmp 10X PCR Gold buffer, 3.5 mM MgCl₂ (both from Applied Biosystems Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 200 µM of each dNTP (GeneAmp dNTP mix) (Thermo Fisher Scientific, Inc.), 25 pmol of each primer and 1 unit of AmpliTaq Gold DNA Polymerase (Applied Biosystems Life Technologies; Thermo Fisher Scientific, Inc.). Amplification was performed in a Biometra professional thermocycler (Thermo Fisher Scientific, Inc.) or a T100 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA). The reaction was initiated with denaturation for 4 min at 94°C, followed by 40 amplification cycles of denaturation at 94°C for 1 min, annealing at 44°C for 1 min and elongation at 72°C for 2 min. The final cycle ended with a prolonged elongation step at 72°C for 10 min. PCR products were run on a 2% agarose gel in 50 mM Tris/37 mM Borate/1.3 mM EDTA and stained with 0.5X GelRed (Biotium, Inc., Hayward, CA, USA). Gels were visualized under UV-light. Fragments of 130–150 bp were considered HPV-positive.

To identify samples that were incorrectly classified as HPV-negative when using the GP5+/6+ primer pair due to disruption of the L1 gene, GP5+/6+ -negative samples were re-analyzed using the general primer pair CpI/IIG (24). Primer sequences were as follows: Forward, 5′-TTATCWTATGCCCAYTGTACCAT-3′ and reverse, 5′-ATGTTAATWSAGCCWCCAAAATT-3′. The 50-µl PCR mixture consisted of 5 µl GeneAmp 10X PCR Gold buffer, 200 µM of each dNTP (GeneAmp dNTP mix), 3 mM MgCl₂, 17 pmol CpI, 26 pmol CpIIG, and 1 unit of AmpliTaq Gold DNA Polymerase. The amplification consisted of denaturation for 5 min at 94°C, followed by 40 amplification cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min and elongation at 72°C for 2 min. The final cycle ended with a prolonged elongation step at 72°C for 4 min. Samples with products of ~188 bp were considered positive.

To assess the quality of the DNA-preparations, samples negative for both primer pairs were run on PCR using the s14 sense/antisense primers (25). Primer sequences were as follows: Forward, 5′-TCGAAAGGGGAAGGAAAAGA-3′ and reverse, 5′-CAGTGACATGGACAAAAGTG-3′. The 50-µl PCR mixture consisted of 5 µl GeneAmp 10X PCR Gold buffer, 200 µM of each dNTP (GeneAmp dNTP mix), 1.5 mM MgCl₂, 15 pmol of each primer and 1 unit of AmpliTaq Gold DNA Polymerase. The amplification consisted of denaturation for 1 min at 94°C, followed by 40 amplification cycles of denaturation at 94°C for 30 sec, annealing at 50°C for 30 sec and elongation at 72°C for 45 sec. The final cycle ended with a prolonged elongation step at 72°C for 5 min. Samples with products of ~127 bp were considered to contain amplifiable DNA.

Genotyping was performed using the PapilloCheck^® HPV screening-kit and the CheckScanner™ laser scanner (Serial no. 700×0177) (Greiner Bio-One North America Inc., Monroe, NC, USA). In brief, this method detects 24 different HPV types through PCR amplification of a 350-bp fragment of the E1 gene and hybridization to specific DNA probes on a DNA chip. The HPV-types detected by the assay included eighteen high-risk types (16, 18, 45, 31, 33, 52, 58, 35, 59, 56, 51, 39, 68, 73, 82, 53, 66 and 70) and six low-risk types (6, 11, 40, 42, 43 and 44/55).

FFPE sections (4 µm) were deparaffinized, rehydrated, and rinsed in water. Immunohistochemistry was performed using Ventana standard procedure on a Ventana BenchMark ULTRA instrument (Ventana Medical Systems, Inc., Tucson, AZ, USA). Antigen retrieval was performed with a CC1 buffer (Ventana Medical Systems, Inc.). Antibodies used were as follows: Rabbit anti-LRIG1 (product. no. AS184165; AgriSera AB, Vännäs, Sweden), 22 µg/ml; rabbit anti-LRIG2-151 (12), 3 µg/ml; rabbit anti-LMO7-1250 (15), 24 µg/ml; rabbit anti-LMO7 (cat. no. HPA020923; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 2 µg/ml; mouse monoclonal anti-p16^INK4a (E6H4) (Ventana Medical Systems, Inc.), 1 µg/ml. For validation of anti-LRIG1 and anti-LMO7 antibodies, please refer to Figs. S1 and S2, respectively.

Evaluation of immunostainings was performed by a senior pathologist without knowledge of the disease outcome. Immunoreactivity was scored based on both staining intensity and percentage of immunoreactive epithelial cells within the tumor. Intensity was evaluated on a four-grade semi-quantitative scale; no staining, weak intensity, moderate intensity or strong intensity. For statistical purposes, intensity scores were grouped into no/weak and moderate/strong. The percentage of positive cells was grouped into ‘at or above median’ or ‘below median’ to obtain relatively even groups. For simplicity, at or above median is henceforth referred to as ‘high’ and below median as ‘low’ staining percentage. For p16^INK4a immunostaining, only cases with a strong nuclear and cytoplasmic expression in a continuous segment of at least 10–20 cells were considered positive.

Patient characteristics were analyzed with the independent sample t-test for comparison of means or the Pearson Chi-square test for ordinal variables. Two-sided P-values were reported. Disease-free survival (DFS) was defined from the date of diagnosis to the date of accession to the patients' records or the date of recurrence [according to pathological anatomical diagnosis (PAD) or the date of first recorded clinical progression]. Death without documented recurrence was censored at the date of death. Overall survival (OS) was defined as the time from the date of diagnosis to the date of death irrespective of cause. If a patient was still alive at the time of accessing the patients' records, the case was censored at that date. DFS and OS were illustrated in Kaplan-Meier graphs and a log-rank test was used for comparison of variables. In the figures presented, the x-axis was truncated at 120 months of follow-up. In the multivariate analysis, tumors were grouped into low and high percentage of positively-stained cells with a cut-off value at median. All parameters revealing a significant difference when comparing survival were included. The median age in each group was compared with the Mann-Whitney test. P-values <0.05 were considered to indicate a statistically significant difference. All statistical analyses were performed using the SPSS software (IBM SPSS Statistics for Windows, version 23; IBM Corp., Armonk, NY, USA). Due to failure of DNA preparation or immunohistochemical staining, occasional data were missing in various statistical analyses.

The 112 patients diagnosed with VSCC during the period of 1990 to 2013 from whom clinical data and representative material could be obtained were included in the study. The cases that were excluded due to lack of material exhibited no significant difference compared to those that were included regarding age, FIGO-stage or histopathological grade.

Mean age at diagnosis was 70 years. The mean age at diagnosis of patients with HPV-negative and HPV-positive tumors was 72 and 66 years, respectively. This difference was significant (P=0.008). HPV-negative and HPV-positive tumors differed significantly also regarding lichen sclerosus et atrophicus (LSA) in disease history (P=0.001) and rate of recurrence or persistence of disease (P=0.027). Within the HPV-negative group, LSA was more common, and recurrence was more likely to occur. Cases negative vs. positive for p16^INK4a also differed significantly, and in the same manner, regarding record of LSA in disease history (P<0.001) and rate of recurrence (P=0.027). Clinical characteristics in relation to HPV-status are summarized in Table I.

Forty percent (44/109) of tumors were HPV-positive, 26% (26/99) were p16^INK4a-positive and 23% (22/97) were positive for both HPV and p16^INK4a. The latter category was referred to as HPV-driven in accordance with a recent hypothesis (4,5). Four tumors were HPV-negative and p16^INK4a-positive whereas 14 tumors were HPV-positive and p16^INK4a-negative. Fifty-seven cases were negative for both HPV and p16^INK4a. In three cases the DNA preparation was of insufficient quality and in 13 cases p16^INK4a data could not be retrieved. From a total of 44 HPV-positive samples, 41 were subjected to genotyping. Of these, 20 samples were successfully genotyped. Ten samples failed the internal control, probably due to insufficiency of material or quality of the sample. In eleven cases, internal control was satisfactory, however, the genotyping rendered a negative result. This may be explained by bad quality of the preparation or, alternatively, disruption of the E1 gene in the specific tumors. Among successfully typed tumors, HPV16 was the most prevalent type (13/20) and HPV33 the second most prevalent type (6/20). HPV42 was found in one of the samples positive for HPV33 and one sample contained HPV58 only (data not shown).

Both HPV- and p16^INK4a-positivity were associated with a lower rate of recurrence: 34.1% (15/44) of HPV-positive cases and 60% (39/65) of HPV-negative cases had a recurrence or persistent disease, 26.9% of p16^INK4a-positive cases and 57.5% of p16^INK4a-negative cases had a recurrence or persistent disease, and 22.7% of HPV-driven cases and 57.9% of not HPV-driven cases had a recurrence or persistent disease (data not shown). HPV-positive and p16^INK4a-positive patients also exhibited a longer DFS than HPV-negative and p16^INK4a-negative patients (P=0.006 and P=0.010, respectively; Fig. 1B and C). Similarly, HPV-driven cases revealed a longer DFS than not HPV-driven cases (P=0.005) (Fig. 1D). However, no significant association between HPV- (P=0.152; Fig. 1A) or p16^INK4a-status and OS was observed.

LRIG1 immunoreactivity was observed primarily in cell nuclei whereas LRIG2 immunoreactivity was primarily cytoplasmic and membraneous (Fig. 2A and B). When considering the percentage of immunoreactive tumor cells, there was a better clinical outcome for patients with a high proportion of LRIG-positive cells (Fig. 3). For LRIG1, this association was, however, neither significant when analyzing OS (Fig. 3A-D) nor DFS (data not shown). LRIG2 was significantly associated with a favorable OS but not DFS in the whole cohort (Fig. 3E; P=0.001 and P=0.051, respectively). Among the HPV-negative and not HPV-driven strata, LRIG2 was significantly associated with a favorable prognosis both regarding OS (P=0.008 and 0.001, respectively; Fig. 3F and G) and DFS (P=0.031 and 0.009, respectively). LRIG2 immunoreactivity was also an independent prognostic marker for OS in multivariate analysis (Table II).

When comparing intensity of LRIG1 divided into no/weak vs. moderate/high, no significant differences were observed between groups considering OS or DFS (data not shown). However, when stratified according to HPV-status, no/weak LRIG1 staining was associated with a better OS in HPV-negative cases (P=0.044) (data not shown). Stratifications into p16^INK4a-status, HPV-/not HPV-driven tumors or FIGO-stage provided no further significant findings. Intensity of LRIG2 computed in the same manner showed no significant differences between groups.

LMO7 immunostaining was evaluated using two different antibodies, one developed by our laboratory (LMO7-1250) and one commercially available (LMO7; Sigma-Aldrich; Merck KGaA). The former exhibited staining primarily in the nuclei whereas the latter stained predominantly around cell membranes and cytoplasmatically (Fig. 1C-E). High staining percentage with the LMO7-1250 antibody was significantly associated with a favorable OS, but not DFS (data not shown), in the whole cohort (OS: P=0.011; DFS: P=0.139; Fig. 3I). In the HPV-negative and HPV-driven strata, the association was significant regarding both OS (P=0.021 and P=0.008, respectively; Fig. 3J and K) and DFS (P=0.038 and P=0.042, respectively). Additionally, among tumors with FIGO stage III–IV at diagnosis, high LMO7 staining percentage was associated with a favorable prognosis (P=0.011; Fig. 3L). The LMO7 antibody also revealed association with a favorable prognosis but only regarding OS and in the HPV-negative, p16^INK4a-negative and HPV-driven strata (data not shown). Similar to LRIG immunoreactivity results, evaluation of staining intensity produced inconclusive results.