DNA Replication Licensing Factors and Aneuploidy Are Linked to Tumor Cell Cycle State and Clinical Outcome in Penile Carcinoma Free

Penile carcinoma is a rare cancer in the Western world with an incidence of 0.1 to 0.9 per 100,000 males. However, there is notable geographic variation with much higher incidences in Africa, Asia, and South America (1). Patients with penile squamous cell carcinoma (PeScc) represent the largest subgroup (98% of all cases) and typically present with primary lesions on the glans, foreskin, or shaft of the penis (2). Locoregional lymph node status is currently the most powerful prognostic indicator identified in penile cancer. Early radical inguinal lymphadenectomy has been shown to convey a distinct survival benefit (3–6), but this surgical technique is limited by its associated high rates of morbidity and mortality (7) Furthermore, controversy exists over patient selection for both radical surgical and chemotherapeutic interventions. Treatment selection is currently based on the patient's age and health status and clinical lymph node stage, together with conventional prognostic factors including histologic grade, tumor-node-metastasis stage, and depth of invasion (8, 9). However, a proportion of men will be incorrectly staged using these algorithms and potentially inappropriately treated (10). Therefore, it is imperative to isolate novel biomarkers in patients with poor prognostic characteristics to identify high-risk patients and facilitate treatment stratification.

Tumors acquire a growth advantage over normal tissues through a variety of mechanisms, including acquisition of aneuploidy and dysregulation of the mechanisms that control cellular proliferation. The DNA replication licensing pathway has emerged as a powerful downstream mechanism for controlling the proliferative state of cells and ensures that DNA is replicated once and only once per cell cycle, thus maintaining genomic stability (11, 12). During late mitosis and early G₁ phase, the replication licensing factors (RLF) ORC, Cdc6, Cdt1, and Mcm2-7 assemble into prereplicative complexes, which render replication origins “licensed” for DNA synthesis. During S phase, Cdc7 kinase and cyclin-dependent kinases induce a conformational change in the prereplicative complex, resulting in recruitment of additional initiator proteins that collectively promote DNA unwinding and recruitment of DNA polymerases (13, 14). During S-G₂-M phases, the presence of the licensing repressor protein geminin prevents inappropriate reinitiation events at origins that have already been activated (15, 16). Recent studies suggest that dysregulation of replication licensing in early tumorigenesis may arise as a consequence of oncogene-induced cell proliferation, which can cause either underreplication or overreplication of chromosomal DNA and therefore contribute to the development of aneuploidy commonly seen during multistep tumor progression to an aggressive cancer phenotype (17).

Mcm2-7 (MCM) proteins are expressed throughout the cell cycle (G₁-S-G₂-M) but are tightly downregulated during exit into out-of-cycle quiescent (G₀), differentiated, or senescent states (11, 12, 14, 18–21). Thus, the MCM proteins represent novel biomarkers of growth and have been confirmed as powerful markers for cancer detection and prognostication in a wide range of tumor types (11, 17, 22). Moreover, expression profiling of MCM together with Ki67 (standard proliferation marker) and geminin (biomarker of S-G₂-M progression) allows cells in out-of-cycle states to be distinguished from those residing in cycle and can assign cells to G₁ and S-G₂-M phase (23, 24). Mcm2-7 protein expression also identifies noncycling cells with proliferative potential. The Mcm2/Ki67 ratio therefore defines the proportion of cells that are licensed to proliferate. Consequently, the higher the Mcm2/Ki67 ratio, the greater the proportion of cells that reside in a licensed noncycling state (11, 23–27). Because Ki67 is present throughout the cell cycle in proliferating cells, the “geminin/Ki67” ratio may be used as an indicator of the relative length of G₁ phase and the rate of cell cycle progression (11, 23–27). Similarly, the “Ki67-geminin” labeling index (LI) can be used to identify the numbers of cells transiting G₁ phase (27–29). This information is valuable for determining the cell cycle kinetics of dynamic tumor cell populations and is of prognostic significance (11, 23–27).

Complex signaling pathways interlinked with redundant growth-regulatory mechanisms contribute to the diverse and heterogeneous effects of oncogenic mutations observed in diverse tumor types (30). Attempts to formulate improved biomarkers for cancer detection and progression alongside the development of novel chemotherapeutic agents against these new molecular targets have met with limited success to date (31). Targeting the DNA replication licensing pathway, which acts as an integration point for upstream mitogenic signaling pathways, is an attractive alternative approach to the identification of new prognostic and predictive markers (11). In light of the biological, prognostic, and therapeutic implications of these cell cycle regulators in tumorigenesis, we have investigated their role in the progression of penile carcinoma. We have used multiparameter analysis of Mcm2, geminin, and Ki67 to study the cell cycle kinetics of this tumor type in vivo and how deregulation of the replication licensing pathway is linked to acquisition of aneuploidy and clinical outcome. Our findings provide new insights into the biological mechanisms involved in tumor progression of penile carcinoma and how these novel biomarkers of growth might be exploited to predict the in vivo behavior of this rare tumor type.

From January 1988 to January 2007, 141 patients were diagnosed with carcinoma in situ or invasive squamous cell carcinoma of the penis. All patients had been treated within the North London Cancer Network and histologic specimens were reviewed by a uro-oncology pathologist at diagnosis. Paraffin wax–embedded tissue specimens were retrieved from the pathology archives for all patients and clinical information was sourced from hospital medical records. Local research ethics committee approval for the study was obtained from the joint University College London/University College London Hospitals Committees on the Ethics of Human Research. Excised tumors were histologically staged using the revised tumor-node-metastasis system criteria 2002 (32). Pathologic variables of the primary tumor included grade, local stage, subtype, extent (unifocal/multifocal), tumor size, depth of invasion, and lymphovascular invasion. All pathologic parameters were recorded by a specialist uro-oncology pathologist and independently reviewed by a second pathologist. Tumor grade was defined using Broders' classification (33): well differentiated (grade 1), moderately differentiated (grade 2), and poorly differentiated (grade 3). Tumor size was defined as the maximal dimension and depth of invasion measured from adjacent normal epithelium to the deepest invasive point. Lymphovascular invasion was determined microscopically and confirmed using antibodies against endothelial markers CD33 and CD34. Lymph node status was confirmed following pathologic review of inguinal and pelvic lymph node specimens attained through prophylactic or delayed lymphadenectomy. Patients who entered into surveillance programs without lymph node surgery were classified as negative after 2 y without disease presentation. Twelve patients with carcinoma in situ were removed from most analyses and 11 patients were lost to follow-up. Therefore, 118 patients were included in the long-term follow-up survival study. The median follow-up time was 20 mo (range, 0.8-162.4 mo). Table 1 summarizes the clinicopathologic characteristics of the patients. The mean age of all patients at the time of diagnosis was 62 y (range, 27-87 y).

Affinity-purified rabbit polyclonal antibody against full-length human geminin was previously generated and validated (14, 34). Ki67 monoclonal antibody (clone MIB-1) was obtained from DAKO and Mcm2 monoclonal antibody (clone 46) was from BD Transduction Laboratories. The specificity of Ki67 and Mcm2 monoclonal antibody has been extensively studied and validated in previous studies (24, 25, 27).

Consecutive serial sections were cut from each paraffin-embedded tissue block representative of the tumor. Three-micrometer sections were cut onto Superfrost Plus slides (Leica Microsystems), dewaxed in xylene, and rehydrated through graded alcohol to water. For antigen retrieval, slides were pressure cooked in 0.1 mol/L citrate buffer (pH 6.0) at 103 kPa for 2.5 min. Tissue sections were immunostained using the Bond Polymer Define Detection kit and Bond-X automated system (Leica) according to the manufacturer's instructions. Primary antibodies were applied at the following dilutions: Ki67 (1:70), Mcm2 (1:1,000), and geminin (1:500). Coverslips were applied with Pertex mounting medium (CellPath). Incubation without the primary antibody was used as a negative control and colonic epithelial sections were used as positive controls.

Protein expression analysis was done by determining the LI of the markers in each tumor, as previously described (23–27, 35). Consecutive serial sections cut from the same formalin-fixed, paraffin-embedded tissue block were used to stain for the markers. Slides were evaluated at ×100 magnification to select the advancing edge of the tumor. Selected areas at the advancing edge plus three to five adjacent fields perpendicular to the advancing front moving progressively toward the center of the tumor were image captured at ×400 magnification with a charge-coupled device camera and AnalySIS image analysis software (SIS). Images were subsequently printed for quantitative analysis, which was undertaken with the observer unaware of the clinicopathologic variables. Both positive and negative cells within the field were counted and any stromal or inflammatory cells were excluded. A minimum of 500 cells was counted for each case. The LI was calculated using the following formula: LI = number of positive cells/total number of cells × 100. Normal foreskin and colon specimens were used as external controls. Normal adjacent epithelium acted as an additional internal control. Reassessment of 10 randomly selected cases by an independent assessor showed high levels of agreement.

For each case, one 40-μm section of formalin-fixed, paraffin-embedded tissue, obtained from the same block as that assessed for immunohistochemistry, was used to prepare a suspension of nuclei. Sections were deparaffinized in xylene, rehydrated through decreasing alcohol gradient, and washed twice in PBS. Rehydrated tissue sections were incubated at 37°C in a shaker water bath for 2 h in the presence of 20 mg/mL bacterial proteinase type XXIV (Sigma). Chilled PBS was added to stop enzymatic digestion and the suspension of nuclei was filtered through a nylon mesh filter and centrifuged at 1500 rpm for 5 min. The supernatant was then discarded and the pellet was resuspended in 3 mL of fresh PBS. A volume of 100 μL of the suspension was cytospun at 1500 rpm for 5 min to prepare a monolayer on a Superfrost Plus slide (Visions Biosystems). The density of the nuclear preparation on the slide was checked under a light microscope and an adjusted volume of the suspension was cytospun if correction of the density was required. The monolayer preparations were air dried and fixed overnight in 4% formaldehyde. After washing in distilled water, slides were incubated in 5 mol/L HCl for 1 h at room temperature for hydrolysis. Slides were then rinsed in distilled water and incubated in Feulgen-Schiff's solution for 2 h in the dark. Finally, the slides were washed in running tap water for 10 min, dehydrated in increasing alcohol gradient, cleared in xylene, and coverslipped.

The Fairfield DNA Ploidy System (Fairfield Imaging) was used for image processing, analysis, and classification. This consisted of a Zeiss Axioplan microscope equipped with a 40/0.75 objective lens (Zeiss), a 546-nm green filter, and a black and white high-resolution digital camera (C4742-95; Hamamatsu Photonics K.K.) with 1024 × 1024 pixels at 10 bits per pixel. The integrated absorbance of each nucleus was calculated based on measurements of absorbance and area. Background absorbance was measured and corrected for each nucleus. At least 1,000 nuclei were scanned for each case and stored in galleries, which were then edited to discard spliced, overlapping, and pyknotic nuclei. A minimum of 300 tumor nuclei per case was used by Histogram Draftsman 1.4 (Fairfield Imaging) to create the histograms. Lymphocytes and plasma cells were included as diploid internal controls and sections of high-grade bladder cancer and normal foreskin tissue as external controls for aneuploid and diploid populations, respectively.

Ploidy histograms were constructed for 121 cases, with 9 cases not linked to survival data. Twenty specimens could not be fully processed due to insufficient or poor-quality tissue material. Histograms were classified according to the following previously published criteria (36): The tumor was classified as diploid if only one G₀-G₁ peak (2c) was present, the number of nuclei in the G₂ (4c) peak did not exceed 10% of the total number of nuclei, and the number of nuclei with a DNA content exceeding 5c did not exceed 1%. A tumor was defined as tetraploid when a peak in the 4c position was present together with a peak in the 8c position or the fraction of nuclei in the 4c region exceeded 10% of the total number of nuclei. A tumor was defined as polyploid when a peak in the 8c position was present together with a peak in the 16c position. The tumor was defined as aneuploid when noneuploid peaks were present or the number of nuclei with a DNA content exceeding 5c/9c, not representing euploid populations, exceeded 1%. The histograms were classified by two independent assessors with a high level of agreement and without knowledge of the clinicopathologic variables. For the purposes of statistical analysis, tetraploid and polyploid tumors were grouped together with aneuploid tumors.

Relationships between biomarker expression and other factors were assessed using the Mann-Whitney U, Kruskal-Wallis, and Jonckheere-Terpstra tests. Data were summarized as the median value and interquartile range of LIs observed across the cohort. Multivariable analyses for lymph node status were carried out in three steps using logistic regression: (a) all factors were assessed separately and those with P < 0.05 were retained, (b) remaining pathologic and biomarker factors were entered into two separate models and backward elimination was applied with P = 0.05, and (c) remaining factors were entered into a single model and backward elimination was applied to produce a final model. Multivariable analyses for overall survival were carried out in a similar fashion using Cox's proportional hazards model. The discriminatory ability of this model was quantified using Harrell's c-index (37), which is analogous to the receiver operating characteristic area and gives the probability that two randomly selected patients have concordant predictions and survival times. The c-index takes values between 0.5 (random predictions) and 1 (perfect concordance). Patients were divided into tertile model-based risk groups and labeled as low, medium, and high risk for disease progression. Patients with incomplete data were excluded from multivariable analyses. All tests were two sided and used a significance level of 0.05 with 95% confidence intervals (95% CI), and no allowances were made for multiple hypothesis testing. All analyses were done using Stata 10 for Windows (StataCorp).

Protein expression profiles for Mcm2, Ki67, and geminin were determined in benign, dysplastic, and malignant lesions of the penis (Fig. 1) using previously characterized monospecific antibodies against Mcm2, geminin, and Ki67 (14, 25, 34). In normal penile squamous epithelium, Mcm2, geminin, and Ki67 expression was restricted to the basal and suprabasal layers forming the transit-amplifying compartment. Cells in the superficial layers showed a fully differentiated phenotype with flattened morphology and showed low Mcm2, Ki67, and geminin LIs (<4%). In striking contrast, dysplastic lesions showed very high replication Mcm2, geminin, and Ki67 expression throughout the full thickness of the epithelium, reflecting an expanded proliferative compartment and arrested differentiation (Fig. 1). As expected, penile cancers showed high levels of Mcm2, geminin, and Ki67 expression, indicative of a hyperproliferative state (Fig. 1).

Mcm2, geminin, and Ki67 LIs were highly significantly associated with tumor grade, with more poorly differentiated tumors showing a higher LI (all P < 0.0001; Table 2). Median Mcm2 expression was greater than median Ki67 expression, with both biomarkers mapped over a broad range within each tumor grade (Fig. 2). Mcm2 and Ki67 levels were higher than geminin expression in these tumors, reflecting the lower growth fraction identified by geminin, which is only present during S-G₂-M (14). There was strong correlation between all biomarkers tested, highlighted by the high concordance between Mcm2 and Ki67 LIs (Pearson coefficient ρ = 0.87), consistent with their linkage to the cell division cycle. The Ki67-geminin score was associated with an increase in tumor grade (P < 0.0001), indicative of an increase in the number of cells transiting G₁ phase (11, 27). Thus, the proportion of tumor cells actively cycling increases with increasing grade. There was little evidence, however, of an increase in the geminin/Ki67 ratio with increasing grade. This ratio is an indicator of the relative length of G₁ phase, and the results suggest that increased recruitment of cells into the cell division cycle was not linked to accelerated cell cycle progression as seen in other tumor types (e.g., epithelial ovarian cancer; ref. 25). There was evidence of a trend for decreasing “Mcm2/Ki67” ratio with increasing grade (P = 0.09), reflecting a shift in the proportion of nonproliferating cells that are licensed for DNA replication in well-differentiated tumors to a population of actively cycling cells in poorly differentiated tumors (25–27, 34).

We observed an association between both high geminin LI and an increase in the geminin/Ki67 ratio with advanced tumor growth. An increase in these indices is indicative of accelerated cell cycle transit and was linked to increasing tumor stage (geminin, P = 0.05; geminin/Ki67, P = 0.02) and depth of invasion (geminin, P = 0.02; geminin/Ki67, P = 0.03; Table 3). Furthermore, we noted a significant association between increased biomarker expression and geminin/Ki67 ratio with increasing tumor size (Mcm2, P < 0.01; Ki67, P = 0.02; geminin, P < 0.001; geminin/Ki67, P < 0.01; Table 3). Thus, larger tumors contain a greater proportion of cycling cells and this increased growth fraction is also coupled to accelerated cell cycle transit. Interestingly, a strong association was found for increased Mcm2, Ki67, and geminin expression with aggressive tumor subtypes (Table 3). Previous research has shown that verrucous and warty tumors behave in a biologically indolent fashion with low risk for disease progression compared with more aggressive variants, such as papillary and basaloid tumors, which are associated with poorer clinical outcomes (38). We observed that low-risk tumors were significantly associated with lower Ki67 and RLF expression, with the lower geminin and Ki67-geminin scores indicating smaller numbers of cells transiting S-G₂-M and G₁ phase, respectively, when compared with aggressive papillary and basaloid tumors. The subtypes associated with poorer clinical outcome therefore displayed a more aggressive cell cycle phenotype.

We next sought to investigate the relationship between RLF expression, anaplasia (arrested differentiation), and tumor DNA ploidy in our study cohort. Tumors showing increasing anaplasia also strongly exhibited aneuploidy as determined by image cytometry (P < 0.0001; Supplementary Table S1; Supplementary Fig. S1), suggesting that arrested differentiation and aneuploidy are linked in PeScc. Moreover, the subset of aneuploid tumors was significantly linked to increased Mcm2, geminin, and Ki67 expression (P ≤ 0.01); a decreased Mcm2/Ki67 ratio (P = 0.03); and increased Ki67-geminin score (P = 0.01; Supplementary Table S2), indicating an increased proportion of actively cycling tumor cells in aneuploid tumors compared with diploid tumors. Interestingly, further analysis revealed a significant association between aneuploidy and the development of distant metastases (P = 0.04).

To investigate the correlation of RLF expression and locoregional disease progression, protein expression profiles for each biomarker were compared with lymph node status (positive/negative) using logistic regression. Ninety-six men with recorded outcomes for these variables were analyzed, with 37 men (38.5%) identified with positive locoregional disease. In univariate analysis, Mcm2 (P = 0.02), geminin (P = 0.02), and Ki67 (P = 0.03) expression were all significant predictors of nodal status. Tumor grade (P = 0.02) and stage (P < 0.01), presence of vascular invasion (P = 0.04), and ploidy status (P = 0.03) were also predictive of nodal status, with poorly differentiated tumors [odds ratio (OR), 8.76; 95% CI, 1.80-42.73], high-stage disease (OR, 4.80; 95% CI, 1.81-12.74), presence of vascular invasion (OR, 2.66; 95% CI, 1.04-6.75), and aneuploidy (OR, 2.96; 95% CI, 1.09-8.01) closely linked to advanced disease with regional lymph node involvement. Multivariate analyses were used to construct a final model using these factors. After backward elimination, biomarker information (Mcm2, Ki67, or geminin LI), vascular invasion, and ploidy status were excluded from the final analysis. Tumor grade (P = 0.006) and local stage (P = 0.003) were independent predictors of positive lymph node disease (Supplementary Table S3). The area under the receiver operating characteristic curve for this model was 0.78 (95% CI, 0.69-0.87). Although all biomarkers were predictive markers in univariate analysis, no single biomarker was a significant predictor after adjustment for grade and stage. This is due in part to the significant associations between biomarker LI and tumor grade and stage, making it difficult to separate their independent effects.

Next, we investigated the survival time for patients with PeScc using Cox's proportional hazards model. The study group for the analysis included 118 men with a recorded survival time. Of these, 26 patients were dead (22%) and 92 were alive (78%) at the time of analysis. Univariate analysis showed that Mcm2 (P = 0.02) and Ki67 (P = 0.04) expression and Ki67-geminin score (P = 0.03) were all significantly associated with overall survival. Age (P = 0.002), tumor stage (P = 0.02), depth of invasion (P < 0.0001), tumor multifocality (P = 0.002), vascular invasion (P = 0.04), and ploidy status (P = 0.008) were also predictive of overall survival outcomes. Advanced age [hazard ratio (HR), 1.05 per year; 95% CI, 1.02-1.1], higher-stage tumors (HR, 3.89; 95% CI, 1.52-9.99), multifocal tumors (HR, 3.69; 95% CI, 1.63-8.32), increased depth of invasion (HR, 1.045; 95% CI, 1.025-1.064), presence of vascular invasion (HR per mm, 2.23; 95% CI, 1.02-4.87), and aneuploidy (HR, 4.28; 95% CI, 1.46-12.58) were associated with a significantly shorter overall survival time. It was also noted that lymph node status (negative or positive) was an excellent prognostic factor on univariate analysis (HR, 11.07; 95% CI, 3.74-32.71), in keeping with previous reports (5, 6). Notably, tumor grade failed to predict outcome in this series (P = 0.28), emphasizing the limitations with current grading systems. Multivariate analyses were used to construct a final model involving 84 patients (Supplementary Tables S3-S7). Backward elimination resulted in the exclusion of biomarker information (Mcm2, Ki67, or geminin LI), vascular invasion, local tumor stage, and depth of invasion from the final model. However, age (P = 0.004), lymph node status (P < 0.001), tumor multifocality (P = 0.002), and aneuploidy (P = 0.03) were identified as independent predictors of overall survival (Supplementary Table S8). We quantified the discriminatory ability of this model using Harrell's c-index. In isolation, these predictors have c-index values of 0.77 (lymph node status), 0.68 (age), 0.65 (aneuploidy), and 0.64 (multifocality). The multivariable model has a c-index of 0.88. Kaplan-Meier curves (Fig. 3) show the survival advantages between patients with diploid versus aneuploid tumors and between tertile risk groups derived by splitting the patients into equal-sized groups based on their predicted risk from the model. Of the 26 deaths analyzed, 24 were cancer specific. We applied the same modeling methodology to cancer-specific survival, resulting in the same predictors having univariable associations that were significant at the 5% level. The same biomarker model was then selected (Mcm2 only) but ploidy status was omitted from the pathologic model (lymph node metastases, age, and extent were selected). The final model omitted Mcm2 (as before), leaving the predictors lymph node metastases, age, and extent (for HRs and P values for this model, see Supplementary Table S9).

In this study, we have assessed the utility of Mcm2 and geminin, alongside DNA ploidy status, as novel biological predictors of outcome in men with PeScc. Our results show that Mcm2 and geminin LIs and aneuploidy are prognostic indicators and predictors of locoregional metastasis. The inverse relationship between RLF expression and differentiation status recapitulates our findings in the in vitro HL60 monocyte/macrophage differentiation model system (18) and has been noted in several other malignancies (25–28, 34, 35, 39–46). This relationship reflects the mutually antagonistic circuits that control cell proliferation and differentiation in human cells and highlights the potential clinical utility of RLFs for improving current tumor grading systems (33). Our data show that analysis of RLFs and ploidy status in primary biopsy material from PeScc can provide important additional prognostic information and identify those tumors with an aggressive cell cycle phenotype, the latter characterized by an increased growth fraction [i.e., an increase in the numbers of cells traversing G₁ (Ki67-geminin score) and S-G₂-M phases (geminin LI)] and accelerated cell cycle transit (geminin/Ki67 LI; refs. 11, 25, 26, 28, 34). Notably, the aggressive tumor cell cycle phenotype was linked to increasing tumor size, stage, and depth of invasion and to morphologic subtypes associated with an adverse prognosis. The aggressive cell cycle phenotype was also linked to tumor ploidy status, suggesting that dysregulation of the DNA replication licensing pathway and cell cycle machinery is linked to the development of aneuploidy in PeScc. In addition, Mcm2, Ki67, and geminin LIs and aneuploidy were identified as significant predictors of locoregional metastasis, and aneuploidy was identified as a predictor of distant metastasis.

We have previously shown in the HL60 monocyte/macrophage differentiation model system that loss of proliferative capacity and cell cycle withdrawal following engagement of the somatic differentiation program is tightly coupled to downregulation of core constituents of the DNA replication licensing machinery, including the Mcm2-7 proteins (11, 18). This coupling between loss of proliferative capacity, cell cycle withdrawal, downregulation of the Mcm2-7 helicase complex, and differentiation has been observed in anal, bladder, cervical, colonic, esophageal, oral, pancreatic, and prostatic epithelia (28, 35, 39, 41, 42, 45–50). In normal stratified squamous penile epithelium, we observed downregulation of RLFs as cells exit the cell cycle and engage the somatic differentiation program. This seems to be a ubiquitous mechanism for lowering the proliferative capacity of cells in stem-transit–differentiating self-renewing tissue systems (11, 14, 18–20). In contrast, block to the differentiation program (arrested differentiation) that characterizes dysplastic (preinvasive) lesions is associated with persistent expression of MCM proteins even in surface epithelial layers, indicative of cells failing to withdraw from the cell cycle (11, 14, 18, 20, 45). As previously observed for dysplastic lesions of the cervix, esophagus, bladder, oral, and anal mucosa, high-level MCM expression was detected in penile dysplasia (11, 28, 35, 41, 42, 45, 48, 49). Surface sampling of penile lesions followed by immunoexpression analysis for MCM proteins could therefore provide a rapid method for distinguishing benign hyperplastic lesions from dysplasia. This approach has already been exploited in screening for cervical cancer and detection of esophageal, lung, bladder, anal, and oral dysplasia (11, 42, 45).

Predictive nomograms for lymph node involvement and cancer-specific survival in PeScc using traditional histopathologic information alone have been developed but require prospective validation (51, 52). In our study, Mcm2 and Ki67 LIs, Ki67-geminin score, age, tumor stage, depth of invasion, tumor extent, vascular invasion, and nodal and ploidy status were all identified as predictors of overall survival, with lymph node status, tumor extent, and ploidy status identified as independent predictors of overall survival. Interestingly, we have shown that these parameters can be incorporated into a simple prediction model to stratify patients into high-, intermediate-, and low-risk groups for disease progression in PeScc. Notably, the multivariable model suggests that low-risk patients are at minimal risk of disease progression compared with men assigned to moderate- or high-risk groups. This is highlighted by overall survival rates at 3 years of 97%, 93%, and 27%, and 5-year survival figures of 97%, 72%, and 18%, for low-, moderate-, and high-risk men, respectively. This provides a potentially powerful approach for early treatment stratification of patients, with low-risk patients assigned to surveillance programs, and targeting of high-risk patients with radical surgical and adjuvant chemotherapeutic interventions. Further studies in additional patient cohorts are now warranted to confirm the predictive power of this model in the risk stratification of PeScc.

Inhibition of the DNA replication initiation machinery has been shown to provoke a cancer cell–specific apoptotic response as a result of the loss or impairment of a putative checkpoint for replication-competent origins during tumorigenesis (17, 53). Here, we have shown that increasing dysregulation of the DNA replication licensing pathway is linked to emergence of an aggressive cell cycle phenotype that affects the in vivo behavior of this tumor type. The DNA replication licensing pathway therefore seems to be also a potentially attractive therapeutic target in PeScc.

No potential conflicts of interest were disclosed.