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Original article
Does the width of the surgical margin of safety or premalignant dermatoses at the negative surgical margin affect outcome in surgically treated penile cancer?
  1. Sven Gunia1,
  2. Stefan Koch2,
  3. Anjun Jain2,
  4. Matthias May3
  1. 1Institutes of Pathology at the Johanniter Hospital Stendal, Stendal, Germany
  2. 2HELIOS Clinic Bad Saarow, Charité-University Medicine Academic Teaching Hospital, Bad Saarow, Germany
  3. 3Department of Urology, St. Elisabeth Clinic Straubing, Straubing, Germany
  1. Correspondence to Sven Gunia, Institute of Pathology at the Johanniter Hospital Stendal, Straße der Demokratie 1, Stendal 39576, Germany; sven-gunia{at}gmx.de

Abstract

Aims To evaluate the prognostic impact of the width of negative surgical margins (NSM) and associated and preinvasive lesions at the NSM in patients with penile squamous cell cancer (PSC).

Methods Enrolling 87 patients with NSM who underwent surgery for PSC, the archived margin slides and entirely wax-embedded surgical margins were retrieved from the pathology files. After step sections were cut, margins were stained with antibodies against CK5/6, p16, p53 and Ki-67 and subjected to in situ hybridisation for high-risk human papillomavirus (HPV). All NSM were histologically examined for squamous hyperplasia (SH), lichen sclerosus (LS) and subtypes of penile intraepithelial neoplasia (PeIN). Then, histological findings were correlated with cancer-specific mortality (CSM, median follow-up 34 months; IQR 6–70).

Results All NSM were negative for high-risk HPV and exhibited SH (p16 and p53 negative, Ki-67 variably positive), LS (p16 negative, variable p53 and Ki-67 positivity) and differentiated PeIN (dPeIN; p16 negative, Ki-67 positive, variable p53 positivity) in 28 (32%), 30 (34%) and 22 (25%) cases, respectively, whereas PeIN subtypes other then dPeIN did not occur. Pathological tumour stage was the only independent predictive parameter with respect to CSM in the multivariable analysis (p=0.001).

Conclusions SH, LS and dPeIN are frequent histological findings at the NSM of surgically treated PSC. However, neither the width of the NSM nor dPeIN, LS or SH at the NSM influences prognostic outcome.

  • HISTOPATHOLOGY
  • CARCINOMA
  • PENIS

https://doi.org/10.1136/jclinpath-2013-201911

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    Introduction

    The incidence of penile squamous cell cancer (PSC) greatly varies from country to country with the highest incidence having been reported for countries in Africa, South America and Asia and the lowest in the USA and in Europe.1

    PSC develops from histomorphologically clearly defined preinvasive lesions referred to as penile intraepithelial neoplasia (PeIN) which can be further classified as differentiated (simplex) PeIN (dPeIN), basaloid PeIN (bPeIN), warty PeIN (wPeIN) and warty-basaloid PeIN (w-bPeIN) and is frequently observed in penectomy specimens resected for PSC.2 ,3 Moreover, histopathologically, PSC might be accompanied by associated lesions such as squamous hyperplasia (SH) of the overlying epidermis or by penile lichen sclerosus (LS) which might also be seen in coexistence with PeIN.4

    At present, surgery aiming at negative surgical margins (NSM) represents the ‘gold standard’ in the treatment of patients with PSC because it is pivotal to remove all invasive cancer in order to achieve adequate oncological control.5

    In the present study, we aimed to evaluate the prognostic role of the width of the NSM as well as the so far neglected prognostic impact of associated and preinvasive lesions encountered upon histological examination of the NSM in penectomy specimens with respect to their association with postsurgical outcome in patients who underwent surgery for PSC.

    Methods

    Study patients

    Our recently described cohort encompasses 92 consecutive patients surgically treated for PSC between January 1993 and December 2010 in six different German hospitals.6 All patients were clinically staged to be without metastasis (cM0) before surgery using CT, MRI or chest x-ray.

    Among the 92 PSC patients, 87 patients had histologically confirmed NSM and comprised the study group of the present investigation. Briefly, 61 patients (70.1%) underwent partial and 26 patients (29.9%) total penectomy.

    Since the earlier part of our study cohort predated European Association of Urology (EAU) guidelines on penile cancer, clinical management of regional lymph nodes was performed according to the decision of the treating surgeon. Briefly, an inguinal lymph node dissection (ILND) was performed in 25 patients (28.7%). The remaining 62 patients were prevented from ILND owing to clinically unremarkable inguinal nodes (cN0) or based upon individual decision-making. None of the 92 study patients developed local recurrence of his PSC during postsurgical follow-up.

    During surgical pathology dissection, the maximum tumour diameter as well as the width of the NSM was grossly measured prior to fixation of the resected tissues. Then, all surgical margins were entirely wax-embedded for subsequent histological examination.

    IRB approval for evaluating cancer-specific mortality (CSM) based upon the retrospective evaluation of death certificates was obtained by the Medical Ethics Committee (MEC) of the federal state Brandenburg (LÄKB; MEC.-No.: 27608).

    IHC, ISH and central histopathological review of surgical margins

    The archived original conventionally H&E-stained slides performed from the entirely wax-embedded surgical margins as well as the archivated paraffin blocks were retrieved from the pathology files. Then, several 4 µm thick step sections were cut from each block for subsequent histological examination and immunohistochemistry (IHC).

    Immunostaining using an automated staining system (Ventana BenchMark) was performed in order to exclude foci of stromal microinvasion by PSC at the NSM as well as to facilitate the histopathological classification of associated (SH and LS) and preinvasive (PeIN) lesions in conventionally challenging cases (eg, SH vs dPeIN). Briefly, following deparaffinisation, rehydration and heat-induced epitope retrieval (CCl mild, Ventana BenchMark), margin sections were incubated with antibodies directed against CK5/6 (Dako, monoclonal, 1:50 dilution), mutant p53 (Ventana, monoclonal, dilution 1:50) and Ki-67 (Ventana; monoclonal) as well as monoclonal anti-p16INK4a (NeoMarkers; clone 16P04; dilution 1:50) for 30 min. Subsequently, sections were washed with phosphate-buffered saline, incubated with rabbit antimouse IgG 1:50, and then mouse peroxidase–antiperoxidase conjugate 1:200.

    All study margins were subsequently subjected to in situ hybridisation (ISH) for high-risk human papillomavirus (HPV) employing the INFORM HPV III Family 16 Probe (b) according to the manufacturer`s instructions. Adequate positive and negative controls were run for all IHC protocols as well as for ISH.

    All study slides were read by one clinical pathologist (SG) blinded to patient ID and clinical outcome. During this central histopathological review, in all cases in which the gross distance between PSC and the surgical margin was deemed to be 6 mm or less during surgical pathology dissection, the distance between the advancing tumour edge and the nearest skin and the deep excision margin was determined histologically by using a Vernier scale according to a previously described methodology.7

    During the central histopathological review, all NSM were classified as follows based upon the criteria recently published elsewhere: non-specific histological findings, SH, LS and PeIN. The last was subdivided into dPeIN, bPeIN, wPeIN and w-bPeIN based upon the criteria reported elsewhere.3 ,4 ,8 Briefly, SH showed hyperkeratosis with orthokeratosis, acanthosis with >10 cell layers, retained cell maturation and lack of nuclear atypia, whereas dPeIN exhibited parakeratosis with or without hyperkeratosis, acanthosis, abnormal epithelial maturation, variable keratin pearl formation and atypical cells at the bottom layer. For bPeIN to be diagnosed, the entire epithelium was required to be replaced by a monotonous population of small-to-intermediate-sized cells with basophilic cytoplasm, indistinct cell borders and a high mitotic rate and apoptotic bodies. For a diagnosis of wPeIN to be established, the surface was required to be undulating and to display atypical parakeratosis with at least a few cells featuring some degree of cytoplasmic keratinisation, nuclear pleomorphism and koilocytosis with multinucleation, perinuclear halo and dyskeratosis.

    Immunostaining evaluation employed the criteria recently reported.3 Briefly, for p16INK4a, only strong and diffuse full-thickness nuclear and/or cytoplasmic positivity in all epithelial cells was considered as positive.3 By contrast, for p53 and Ki-67 staining evaluation, patchy or diffuse nuclear positivity above the basal layer was considered as positive.3 Immunostaining with anti-CK5/6 served to exclude foci of invasive cancer underneath the basement membrane of the entirely wax-embedded surgical margins.

    Follow-up and statistical analysis

    Briefly, follow-up was performed in keeping with the EAU guideline.9 Among the five a priori excluded patients with positive surgical margins, four patients experienced local recurrence during follow-up.

    Data are summarised as the median and IQR for continuous variables as well as by means of frequency tables for categorical variables. CSM was defined as to represent the end point of the study. Study patients identified as having died of PSC by means of death certificates had widely disseminated and often clinically symptomatic metastasis at the time of death.

    Univariable and multivariable Cox proportional-hazards regression models were used to determine the impact of clinical and pathological parameters including the histologically confirmed width of the NSM as well as associated and preinvasive lesions histologically observed at the NSM on CSM. Furthermore, assessment of area under curve (AUC) values for estimation of the predictive accuracy of the multivariable Cox model according to Harrell was performed (c-indices).10

    In all cases, a two-tailed p value <0.05 was required for statistical significance. Statistical analyses were performed using SPSS V.19.0 (SPSS Inc., Chicago, Illinois, USA).

    Results

    The median follow-up of the 87 study patients with NSM was 34 months (IQR 6–70 months). Pertinent study patient characteristics are presented in table 1. Out of the 25 patients in whom an ILND was performed, seven patients (28%) had histologically confirmed lymph node metastases from their PSC (pN+) while the other 18 patients had histologically negative lymph nodes (pN0).

    View this table:
    Table 1

    Pertinent characteristics of the 87 study patients surgically treated with (partial) penectomy for penile squamous cell cancer

    The median width of the NSM in our study group was 18 mm. Ten study patients (12%) had a width <6 mm (table 1).

    All 87 study margins histologically examined in the present study were negative for high-risk HPV by ISH and IHC.

    Complete serial sectioning and immunostaining of the entirely wax-embedded surgical margins did not reveal invasive carcinoma in any of the study margins examined.

    Briefly, the study margins exhibited SH, LS and dPeIN in 28 (32%), 30 (34%) and 22 (24%) cases, respectively. Conversely, bPeIN, wPeIN and w-bPeIN did not occur at any of the study margins examined.

    IHC was particularly helpful for segregating SH (p16 and p53 negative with variable Ki-67 positivity) from dPeIN (p16 negative and Ki67-positive with variable p53 staining).

    None of the 87 study patients developed local recurrence during postsurgical follow-up. Cancer-specific survival rates of the whole study group after 1, 3 and 5 years were 92%, 73% and 68%, respectively.

    The univariable analysis of CSM identified pT stage, pN status, histopathological grade, the width of the NSM as well as the type of surgery as prognostic parameters, whereas pT stage was the only independent predictive parameter in terms of CSM according to the multivariable analysis (HR 3.76, p=0.001; table 2). By contrast, the width of the NSM as well as SH, LS and dPeIN histologically confirmed at the NSM failed to represent independent predictive parameters in terms of CSM. The AUC value (predictive accuracy) of the multivariable model for CSM was 0.85 (95% CI 0.77 to 0.94).

    View this table:
    Table 2

    Univariable and multivariable analyses using Cox proportional-hazards models for predicting cancer-specific mortality

    Discussion

    The achievement of a histologically confirmed NSM is the mainstay of surgical treatment for PSC.5 ,11 However, since penile surgery is often accompanied by poor cosmetic and functional results as well as significant psychological morbidity, unnecessary radical surgery should be avoided.12–14 Therefore, the traditional historical teaching proposing a 2-cm NSM has been abandoned and, instead, the recent EAU guideline recommends to achieve NSM without further stipulating how broad the margin of safety should be.9

    In the recent literature, to our knowledge, only two advanced studies conducted by the same group of investigators addressed the issue of what surgical margins are required to achieve oncological control in men surgically treated for PSC, and both studies reported that more conservative techniques involving margins of only a few millimetres are adequate.7 ,15 However, both studies enrolled patients (n=51 and n=179) who were treated with organ-sparing surgery at a tertiary referral centre. Due to this previous patient selection, the results presented by those studies might differ from observations deduced from a study cohort surgically treated in general hospitals not affiliated to specialised referral centres. Moreover, both studies failed to examine the prognostic impact of associated and preinvasive lesions histologically confirmed at the NSM.7 ,15

    We present the first multi-centre study looking at the prognostic implications of the width of the NSM as well as associated and preinvasive lesions histologically confirmed at the NSM in a study cohort enrolling 87 patients who underwent surgery for PSC. Briefly, our data are in line with the recent EAU guideline and support the contention that the width of the NSM does not represent an independent prognostic parameter by itself in terms of CSM. Moreover, our present investigation is the first to suggest that dPeIN, SH and LS histologically confirmed at the NSM do not represent independent prognostic parameters. From a practical standpoint, keeping in mind that PeIN shows multifocality in approximately 15% of the cases, it is tempting to suggest that surgeons should employ more conservative techniques producing margins of only a few millimetres rather than aiming at removal of all surrounding dPeIN.8 A clinically important implication of our so far unreported observation is that the histological finding of dPeIN, SH or LS upon examination of surgical margins in the setting of intraoperative frozen sections does not warrant any further surgical interventions as long as the surgical margin is not infiltrated by PSC. However, since only 22 study patients of our cohort showed dPeIN at the NSM and none of the study margins examined exhibited bPeIN, wPeIN or w-bPeIN by histological examination, further clinical studies are clearly needed in order to validate the prognostic implications of various PeIN subtypes histologically confirmed at the NSM.

    Further limitations of the present study encompass its retrospective multi-centre design and the fact that ILND was performed in a minority of the study patients enrolled. Moreover, all study margins assessed were negative for high-risk HPV by ISH and IHC. Since a subset of PSCs are pathogenically linked to high-risk HPV subtypes, further studies are needed to clarify the prognostic implications of high-risk HPV persisting at the NSM epithelium. Additionally, the length of follow-up available in our study might be too short to be certain that the premalignant dermatoses evaluated do not have any prognostic impact. Therefore, until our findings are validated by advanced studies in the future, surgically treated PSC patients should either participate in regular clinical surveillance or be trained to self-examination.

    In conclusion, our present data are in line with the current EAU guideline by indicating that the width of the NSM does not represent an independent prognostic parameter in patients with surgically treated PSC. Moreover, our preliminary data suggest that dPeIN, SH and LS histologically confirmed at the NSM do not seem to represent independent prognostic parameters and, therefore, do not warrant any further surgical intervention if encountered during intraoperative frozen sections performed from surgical margins. Therefore, at present, more conservative techniques seem to offer adequate oncological control in PSC patients as long as the surgical margin is not infiltrated by PSC. However, advanced clinical studies are clearly needed in order to validate the prognostic implications of various PeIN subtypes as well as persistent high-risk HPV subtypes histologically confirmed at the NSM.

    Take-home messages

    • At present, a histologically negative surgical margin represents the ‘gold standard’ in the surgical treatment of penile squamous cell cancer.

    • Since the width of the negative surgical margin does not seem to represent an independent prognostic parameter in patients with negative surgical margins, unnecessary radical surgery should be replaced by more conservative surgical techniques in the treatment of penile cancer.

    • Our preliminary data suggest that differentiated penile intraepithelial neoplasia, squamous hyperplasia and lichen sclerosus histologically confirmed at the negative surgical margin are frequent and do not represent independent prognostic parameters by themselves in terms of cancer-specific mortality in surgically treated penile cancer patients.

    • These so far unreported data might have important clinical implications since differentiated penile intraepithelial neoplasia, squamous hyperplasia and lichen sclerosus histologically encountered during intraoperative frozen sections performed from the surgical margins do not warrant any further surgical interventions which might result in poor cosmetic and functional results as well as significant psychological morbidity.

    • Further studies are clearly needed to validate the prognostic implications of various subtypes of penile intraepithelial neoplasia (differentiated, basaloid, warty and warty-basaloid) as well as of persistent high-risk human papillomavirus infection histologically confirmed at the negative surgical margin.

    Acknowledgments

    In alphabetical order, we would like to express our thanks to the following department directors who kindly supported the present study by providing archival slides and wax-embedded tissues for investigations: V Henn, MD (department of pathology, Ruppiner Clinics GmbH, Germany), J Jander, MD (department of pathology, Rhön Clinic Frankfurt/Oder, Germany), H Lobeck, MD (department of pathology, Ernst-von-Bergmann Clinic Potsdam, Germany), R Pauli, MD (department of pathology, Health Care Centre Brandenburg an der Havel GmbH, Germany) and M Tuffaha (Carl-Thiem Clinic Cottbus, Germany).

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    Footnotes

    • Contributors Each author contributed significantly to the research of the present study (SG: study concept and design, acquisition, analysis and interpretation of the data, drafting and critical revision of the manuscript, statistical analysis; SK: supervision and revision of the manuscript; AJ: acquisition of the data; MM: analysis and interpretation of the data as well as statistical analysis).

    • Competing interests None.

    • Ethics approval Medical Ethics Committee of the federal state Brandenburg, Germany (Landesärztekammer Brandenburg, LÄKB; MEC-No.: 27608).

    • Provenance and peer review Not commissioned; externally peer reviewed.