Presence of high-risk human papillomavirus DNA in penile carcinoma predicts favorable outcome in survival

Authors


Abstract

There is evidence that a subset of penile carcinomas is caused by infection with high-risk human papillomavirus (HPV). However, extensive studies on the possible influence of HPV infection on clinical outcome of penile cancer are lacking. This investigation is aimed to examine the prevalence of high-risk HPV in a large series of penile squamous-cell carcinomas (SCCs) and to determine the relationship between HPV and survival. Formalin-fixed, paraffin-embedded tumor specimens of 171 patients with penile carcinoma were tested for high-risk HPV DNA presence by GP5+/6+-PCR. The clinical course of the patients and the histopathological characteristics of the primary tumors were reviewed. High-risk HPV DNA was detected in 29% of the tumors, with HPV 16 being the predominant type, accounting for 76% of high-risk HPV containing SCCs. Disease-specific 5-year survival in the high-risk HPV-negative group and high-risk HPV-positive group was 78% and 93%, respectively (log rank test p = 0.03). In multivariate analysis, the HPV status was an independent predictor for disease-specific mortality (p = 0.01) with a hazard ratio of 0.14 (95% CI: 0.03–0.63). Our results indicate that the presence of high-risk HPV (29%) confers a survival advantage in patients with penile carcinoma. © 2006 Wiley-Liss, Inc.

Squamous-cell carcinoma (SCC) of the penis is uncommon and accounts for less than 1% of adult male cancers in developed countries. The incidence in Europe is 1 per 100,000 per year.1 The highest incidence occurs in the seventh decade. The development is most likely a stepwise chain of events over a period of years from preneoplastic lesions to SCC. The etiology of penile SCC appears to be multifactorial, with a history of smoking, phimosis and poor hygiene are commonly associated with this tumor.2, 3 Recent data have also provided corroborative evidence for a role of high-risk human papillomavirus (HPV) in the pathogenesis of a subset of penile SCC.4, 5 These high-risk HPVs are known to possess transforming capacity and are a necessary but insufficient cause of cervical SCC.6, 7 More recently, also a role of these viruses has been implicated in the development of other SCCs, such as those of the head and neck and vulva, next to penile SCCs. In contrast to cervical cancers, almost all of which contain high-risk HPV, the reported high-risk HPV prevalence in the other mentioned SCCs is much lower. This suggests that the other SCCs are etiologically heterogeneous, with only a subset of cases linked to high-risk HPV. However, the exact proportion of these noncervical cancers that can be attributed to high-risk HPV is still a matter of debate since reported prevalence rates are highly variable, possibly as a result of the different testing methods that have been applied. Moreover, for penile carcinomas the histological subtype seems to be a determinant for HPV presence. Nevertheless, keratinizing or not-otherwise-specified penile SCCs, which represent the most common penile SCCs, generally show a rather consistent HPV prevalence rate of about 35%.4, 5

Another complication is that various studies performed on head and neck and vulvar SCCs revealed a survival benefit for patients with a HPV-positive tumor,8, 9, 10, 11, 12 whereas others failed to do so.13, 14, 15 Also for penile SCC, 2 studies have been performed in which survival was correlated with the HPV-status of the primary tumor. These studies did not show survival differences between HPV-positive and HPV-negative tumors.16, 17

One of the possible explanations for the apparent discrepancies both in HPV prevalence rates and survival deals with the fact that sole HPV DNA detection might not be sufficient evidence of causation. Extensive analysis of HPV DNA containing head and neck carcinomas revealed that only about the half of these tumors showed transcriptional activity of the viral E6/E7 oncogene region and may be the direct consequence of HPV infection.13, 18, 19, 20 The latter is consistent with the notion that not all high-risk HPV infections represent clinically relevant infections.21

Also, a recent comprehensive study on penile SCCs showed evidence for distinct carcinogenic pathways in tumors with and without transcriptionally active HPV.5 However, unlike head and neck carcinomas, the number of cases that tested positive for high-risk HPV DNA by the consensus primer GP5+/6+-PCR method, but negative for E6/E7 transcripts, was rather low. Hence, HPV DNA detection by GP5+/6+ PCR better reflects causality for this tumor type than for head and neck SCCs.

Here, we determined high-risk HPV DNA presence by GP5+/6+-PCR on a large series of formalin fixed, paraffin-embedded SCC specimens of the penis and assessed the relevance of high-risk HPV-DNA presence on the primary tumor, regarding other local pathological parameters, incidence of lymph node metastases and disease-specific survival.

Materials and methods

Patients

Investigations were carried out on 176 patients with SCC of the penis treated at our institute, between 1963 and 2001. The study represents the patients of whom paraffin-embedded tumor material from the primary tumor and clinicopathological data were available. Of 55 patients analyzed in this study, a corresponding frozen tissue specimen was tested during the course of a previous study.5 The age of the 176 patients ranged from 27 to 94 years (mean age: 64 years).

Surgical procedures

Small (T1) tumors were treated by local excision, and more extended tumors (T2–3) were mainly treated by (partial) penectomy. Furthermore, all patients with lymph node metastases underwent inguinal lymph node dissection. Inguinal lymphadenectomy consisted of the removal of all lymphatic tissue between the inguinal ligament cranially, the adductor longus muscle medially, the sartorius muscle laterally, the crossing point of the sartorius and adductor longus muscles caudally, and the pectineus muscle and the superficial femoral artery and vein dorsally. Additional ipsilateral pelvic (external iliac and obturator) lymph node dissection was performed if 2 or more inguinal lymph node were involved. Patients only underwent adjuvant regional radiotherapy if histopathological examination showed extracapsular involvement or when at least 2 lymph nodes were invaded by tumor.

Histopathological features

A pathologist (M.P.W.G.) reviewed all resection specimens of the 176 patients without knowledge of clinical outcome. Primary tumors were staged retrospectively according to the 1987 TNM system of the International Union Against Cancer and pathologically graded according to Broders as grade I, well differentiated; grade II, moderately differentiated; or grade III, poorly differentiated.22 Vascular invasion was defined as tumor cells within endothelium lined spaces. As tumor diameter, the macroscopically largest distance between 2 opposite tumor borders was taken. Also the presence of a sclerosing morpheic growth pattern was judged. This growth pattern is characterized by small strands of tumor cells emerging from the tumor mass growing deeply in the penile tissue between sclerosing collagen bundles.

Sample preparation and high-risk HPV-DNA detection and typing

Of each specimen, a series of consecutive 5-μM sections were cut, the first and last of which were haematoxylin–eosin stained for histomorphological confirmation of tumor presence, whereas the in-between sections were collected in a reaction vessel for processing for PCR, as described previously.23 Various measures were taken during sample preparation to avoid contamination. Gloves were worn throughout the cutting and preparation procedures; new blades were used for cutting new specimens, and the microtome was cleaned each time in between different specimens. All sections were handled with disposable tips, and in between the different tumor specimens blocks, with only paraffin were cut; the resulting sections were treated similar to those of the tumor samples and checked for contamination by PCR.

To assess the quality of the DNA, β-globin PCR analysis was performed on crude extracts of formalin-fixed, paraffin-embedded penile carcinoma specimens, using the primers BGPCO3 and BGPCO5 that amplify a DNA fragment of about 200 bp.23 Samples of 5 patients were negative in the β-globin PCR and excluded from further HPV DNA analysis. The latter included 2 of the 55 patients of whom frozen tissue was previously analyzed, which scored β-globin PCR negative as well.5 Detection of HPV DNA in the formalin-fixed, paraffin-embedded SCC samples of the remaining 171 patients was performed by general primer GP5+/GP6+- PCR enzyme immunoassay (PCR-EIA), essentially as described before.5 Subsequent genotyping for 14 high-risk HPV types (i.e., 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) was performed by reverse line blot analysis, as previously described.23, 24 The HPV detection and typing data were in complete agreement with those of the frozen tissue of the 53 cases for which frozen material of adequate quality was tested before.5

Follow-up and statistical methods

Patients were followed regularly at The Netherlands Cancer Institute. Data were collected retrospectively, and these data were analyzed immediately after collection. Mann-Whitney U test, Chi-square test and Fischer exact test were used for comparison of patient and tumor characteristics. Multiple logistic regression analyses was used to determine pathological variables related to HPV status. Disease-specific survival plots were made using the Kaplan-Meier method and were compared with the log rank test. Patients with clinical signs of disease at the time of death or with unknown death cause were considered as death due to disease. Cox proportional hazards analysis was applied to assess the independent effects of several prognostic factors on survival. All variables of the univariate analysis were included in the multivariate analysis. Effects were characterized with 95% confidence intervals on the hazard ratio scale. The significance level was set at .05. Analyses were performed with the Statistical Package for the Social Sciences software (SPSS, Chicago, IL).

Results

High-risk HPV DNA presence

High-risk HPV DNA was detected in 29% (50 of 171) of the formalin-fixed, paraffin-embedded penile SCC specimens. Thirty-eight (76%) of HPV DNA positive tumors contained HPV-16, 3 (6%) HPV-18, 2 (4%) HPV-33, 3 (6%) HPV-45 (4%), 1 (2%) HPV-56, 1 (2%) HPV-58 and 1 (2%) HPV-59. Multiple infections with HPV-16 and HPV-18 were observed in 1 case (2%). When confining the analysis to the most common histological subtype of penile carcinoma, i.e., not-otherwise-specified SCC, the overall HPV prevalence was 31% (47/152).

Survival, clinical and pathological characteristics in the high-risk HPV-positive versus negative patients

Clinical and pathological variables of patients with high-risk HPV-positive versus high-risk HPV-negative tumors are presented in Table I. In univariate analysis, only presence of a sclerosing morphea-like growth pattern was significantly associated with HPV-negative tumors (chi-square: p = 0.04). There was no time trend for HPV prevalence in tumors (chi-square: p = 0.15). Multiple logistic regression analysis also revealed that morphea-like growth was the only factor related to HPV status: HPV-positive tumors showed less morphea-like growth (OR = 0.28; 95% CI = 0.08–1.0).

Table I. Clinical and Histopathological Characteristicsof 171 Penile Carcinoma Patients Divided in Patients with PV-DNA Negative Tumors (n = 121) and Patients with HPV-DNA Positive Tumors (n = 50).
 HPV-DNA
NegativePositivep-Value
  •  Values in parentheses indicate percentage values.

  • 1

    Chi-square test

  • 2

    Mann-Whitney test

  • 3

    Fisher's exact test

  • 4

    Lymph nodes were considered as tumor positive if tumor was found at presentation or within the first 2 years of follow-up.

No. of patients
 1963–199363 (76)20 (24)0.11
 1994–200158 (66)30 (34) 
Total121 (71)50 (29) 
Mean age63.763.70.992
pT stage
 T141 (65)22 (35)0.243
 T267 (72)26 (28) 
 T313 (87)2 (13) 
Tumor diameter
 <2.0 cm23 (66)12 (34)0.413
 ≥2.0 <5 cm89 (74)32 (26) 
 ≥5cm9 (60)6 (40) 
Grade
 I46 (75)15 (25)0.601
 II62 (69)28 (31) 
 III13 (65)7 (35) 
Vascular invasion
 Absent112 (93)45 (90)0.553
 Present9 (7)5 (10) 
Type of squamous-cell carcinoma
 Not otherwise specified105 (69)47 (31)0.213
 Basaloid1 (100)0 (0) 
 Verrucous10 (83)2 (17) 
 Warty3 (75)1 (25) 
 Sarcomatoid2 (100)0 (0) 
Sclerosing morphea-like growth
 Absent99 (68)47 (32)0.043
 Present22 (88)3 (12) 
Lymph node status4
 Tumor negative81 (70)34 (30)0.901
 Tumor positive40 (71)16 (29) 

Median follow-up was 95 months (range 12–492). The cumulative probabilities for disease-specific survival by high-risk HPV status are presented in Figure 1. There was a significant difference in 5-year disease-specific survival between the high-risk HPV-positive and negative group: 92 vs. 78% (p = 0.03, log-rank test). In the high-risk HPV-positive group, 3 (2 HPV-16 and 1 HPV-58) of the 50 patients died of penile cancer versus 24 of 121 patients with a high-risk HPV-negative tumor (Fisher's exact: p = 0.02).

Figure 1.

Disease-specific survival after primary tumor treatment; HPV-positive tumors (n = 50) appears to provide a significant survival benefit over HPV-negative tumors (n = 121, log rank test p = 0.03).

Table II shows univariate 5-year disease-specific survival rates according to clinical and pathological variables. The status of the lymph nodes was the strongest predictor followed by tumor stage and HPV status. Using a Cox proportional hazard model to control for the other prognostic variables, lymph node status again turned out to be the strongest independent predictive factor of disease-specific survival, followed by HPV status, pathological T classification and vascular invasion (Table III).

Table II. 5-Years Disease-Specific Survival Rates According to Clinical and Pathological Variables: Univariate Analysis
 No. of patients5 years (95%CI) disease-specific survival (%)p-Value(log-rank test)
  • 1

    Lymph node status considered as tumor positive if tumor positive lymph nodes were found at presentation or within the first 2 years of follow-up.

Age
 ≤ 606578 (68–88)0.28
 > 6010685 (77–93) 
pT stage
 16395 (89–100)0.003
 2-310874 (64–84) 
Tumor diameter
 < 2.0 cm5189 (79–99)0.15
 ≥ 2.0 cm12079 (71–87) 
Grade
 1-215183 (77–89)0.18
 32074 (54–94) 
Vascular invasion
 Absent15883 (77–89)0.08
 Present1469 (43–95) 
Morphea-like growth
 Absent14684 (78–90)0.08
 Present2570 (60–80) 
Lymph nodes1
 Tumor negative11594 (90–98)<0.0001
 Tumor positive5660 (46–74) 
HPV DNA
 Absent12178 (70–86)0.03
 Present5092 (84–100) 
Table III. Cox Multivariate Regression Analysis: Independent Prognostic Factors for Disease-Specific Survival
FactorsAdjusted HR1 (95% CI)p-Value
  • 1

    Hazard ratio adjusted for all other variables as listed in Table II.

pT stage
 pT11.0 (reference)0.03
 pT2-34.0 (1.1-14.0) 
Vascular invasion
 Absent1.0 (reference)0.014
 Present4.5 (1.4-14.6) 
Lymph nodes
 Negative1.0 (reference)<0.001
 Positive7.0 (2.8-17.6) 
HPV-DNA
 Absent1.0 (reference)0.017
 Present0.21 (0.06-0.76) 

Discussion

To our knowledge, the present report describes the most extensive study on HPV DNA presence in penile SCC. We realize that the use of formalin-fixed material in this study may have resulted in a slight underestimation of the actual high-risk HPV prevalence. However, the predominant histotype of the penile SCC analyzed, namely not-otherwise-specified penile SCCs (sometimes also referred to as keratinizing SCC), revealed a high-risk HPV prevalence rate (i.e., 31%) that was in the same range as found in a previous study using a more sensitive PCR assay.4 This together with the fact that exactly the same high-risk HPV prevalence was observed in the paired frozen and formalin-fixed specimens of the 53 subjects of which both specimens have been tested by the same GP5+/6+-PCR assay5 suggests that the use of formalin-fixed specimens in this study has only a minor influence on the disease-specific survival data.

The most common viral type in penile carcinoma was HPV 16, which was detected in 76% of high-risk HPV-positive cancers. This result is more or less similar to those reported by other investigators, in which HPV 16 was found in 65–74% of HPV-positive tumors.2, 4, 25, 26 In this study, presence of a sclerosing morphea-like growth pattern of the primary tumor is significantly associated with HPV-negative tumors. This is an observation that has not been described before in the literature. Bezerra et al. found that HPV-positive penile carcinomas had less lymphatic embolization.16 Gregoire et al. observed that HPV DNA was detected significantly more often in penile carcinomas exhibiting a more vertical growth pattern and in histologically defined high-grade tumors.27

The current analysis suggests a survival benefit for penile cancer patients in whom high-risk HPV DNA was present in the primary tumor compared with patients in whom no high-risk HPV DNA could be detected. Although historical control analysis is not ideal to compare survival, because of potential time-related changes in various characteristics, the benefit could not be explained by differences in patient or primary tumor histopathological characteristics.

In contrast to the established role of HPV as a risk factor, little is known about its prognostic significance in penile SCC. To our knowledge, only 2 reports addressed the relation between HPV and prognosis of penile carcinoma. Both studies, comparing clinical outcome of patients with HPV-positive versus HPV-negative tumors, reported no survival differences.16, 17 These studies, however, were limited by relatively small numbers of patients (29 and 82, respectively). The explanation for the suggested increased risk of death among patients with HPV-negative tumors is unclear, but seems to be independent of currently understood risk factors. In penile carcinoma, the prognostic significance of lymph node invasion on survival has been well established.28, 29 Predictive histopathological features for lymph node metastasis include tumor grade, invasion depth and lymphatic embolization by neoplastic cells.30, 31, 32 Remarkably, HPV status was not related to the risk of lymph node metastases or vascular invasion (Table I). Therefore, the data in this study suggest that high-risk HPV-positive penile tumors comprise a distinct molecular and clinical entity and, although largely speculative, the survival benefit of patients with HPV-positive carcinomas may be owing to a lower degree of gross genetic alterations, as has been previously found in head and neck SCC.19 As an alternative, the presence of virus in penile carcinoma may confer an increased immune surveillance, thereby making the HPV-associated malignancy less aggressive than those without viral epitopes.

In conclusion, patients with penile carcinoma in which high-risk HPV DNA can be detected by GP5+/6+-PCR seem to have an increased disease-specific survival.

Acknowledgements

We thank P.M. van Diemen for her excellent technical assistance on HPV-testing.

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