Factors Predicting Nodal Metastasis in Penile Cancer: Analysis from a Tertiary Center

Penile cancer is relatively uncommon malignancy affecting Indian men with a crude incidence rate of 1.18 per 100,000 population with a relatively higher incidence rate of 2.9 per 100,000 in South India [1]. Surgical management remains the mainstay of treatment for carcinoma penis. Inguinal lymphadenectomy remains an integral part of management for staging and therapeutic purpose; however, a subgroup of patients including stage Tis, Ta, and T1 can be closely observed as per the current guidelines [2]. We attempted to identify various pathological factors of the primary tumor that are associated with increased rate of nodal metastasis.

After approval from institutional ethical and regulatory board (Cancer Institute [WIA] Institutional Ethics Committee Approval No. IEC/2021/July 07), we performed a retrospective analysis on all patients with biopsy-proven penile cancer who underwent surgical treatment at our institute from January 2011 to March 2014. After histological confirmation, all patients underwent ultrasound of the abdomen and pelvis and chest X-ray as staging investigations. In nonmetastatic patients, management of primary disease was done by circumcision, partial amputation, or total amputation based on the extent of disease. All groins were initially staged by clinical examination and US-guided fine needle aspiration cytology. We performed cytology in all patients due to high incidence of palpable inguinal nodes seen in our country, with 1 series reporting an incidence of 96% as compared to 20% incidence in the West, probably due to barefoot walking [3]. In our study period of 3 ½ years, we changed our institutional policy for clinically and cytologically node negative patients, from observation initially to sentinel node biopsy or superficial inguinal block dissection. In the early years, cytologically negative patients were kept under observation, if the primary biopsy represented T1 and grade 1 disease. For higher stage or grade, a superficial inguinal block dissection was performed. In the subsequent years, after performing a validation study, a dynamic sentinel node biopsy using radiocolloid and blue dye was performed for cytologically negative patients, unless contraindicated [4]. In the event of histological node positivity or nodal recurrence, patients were subjected to completion ilioinguinal block dissection. Patients with cytologically node positive disease underwent ilioinguinal block dissection. For the purpose of the study, nodal status of patients in the observation group was based on uneventful follow-up of at least 24 months. As seen in the literature, nearly 86% of the recurrences tend to occur in the initial 2 years of follow-up; hence, this cutoff was decided [5]. Thus, patients who did not develop nodal recurrence after 2 years were assumed to be node negative. Patients on observation with shorter follow-up duration were excluded from the study.

The paraffin blocks of the specimen were retrieved and a single pathologist reviewed the blocks for the histological variables. Histological grading was done based on the categories specified by World Health Organization for penile cancer, including cytological atypia, keratinization, intercellular bridges, mitotic activity, and tumor margin. Based on the above criteria, they were graded into grade 1, well differentiated; grade 2, moderately differentiated; and grade 3, poorly differentiated. Patient who had received neoadjuvant therapy or who underwent surgical management elsewhere were not included in the analysis. The depth of invasion was measured from the basement membrane of the squamous epithelium to the deepest invasive carcinoma cell.

Immunohistochemistry for Ki67 was done after antigen retrieval in Tris-EDTA buffer at 100^o C for 30 min. After peroxidase blocking for 10 min, the slides were stained with the monoclonal mouse antibody for 15–30 min. After washing with buffer solution, Dako REAL EnVision Detection System was used to label the antigen.

All statistical analyses were performed using SPSS software version 17.0 (SPSS Inc., IBM, Chicago, IL, USA). Histological node positivity was the primary end point in the study. Student’s t test was used for nonparametric variables and the χ² test was used for categorical variables with the level of significance set at p < 0.05. The odds ratio was computed in univariate and multivariate settings using logistic regression analysis.

A total of 97 carcinoma penis patients underwent surgical management at our institute during the abovementioned period. The mean follow-up period of the patients was 23 months (1 month–47 months) and 2 patients were lost to follow-up. The clinicopathological characteristics of the patients are presented in Table 1.

Logistic regression was applied to the above data and grade III tumor, presence of lymphovascular invasion (LVI), tumor thickness >10 mm, perineural invasion (PNI) and Ki67 >50% were significantly associated with nodal metastasis (Table 2). On multivariate analysis (Table 3), only presence of PNI was found to be significant (odds ratio: 6.82) (95% confidence interval: 1.72–27.03) (p = 0.006).

Lymph node involvement is one the most important prognostic factors in penile cancer. The 5 year survival drops to 51% with the involvement of inguinal nodes, and it is dismal with involvement of pelvic nodes [6]. The incidence of occult metastasis in a clinically negative node is around 10–20% [7]. It becomes imperative to identify occult nodal metastasis as early lymphadenectomy has been shown to improve cancer-specific survival and can be curative for histologically node positive patients [8]. Though various noninvasive methods have been attempted to identify inguinal node involvement, none have been proven effective. Historically, clinical examination has been associated with false positive rate of 50%. Ultrasound-guided fine needle aspiration cytology has false negative rate is 15%. CT or MRI is not recommended for detecting occult metastasis [7]. Hence, in a clinically negative groin, invasive methods have been used to identify occult metastasis. However, this leads to nearly 60% negative inguinal nodal dissections and unnecessary morbidity of skin edge necrosis, scrotal, or limb edema associated with it [9]. Currently, the guidelines recommend invasive staging in clinically negative groin based on primary tumor stage, grade of the tumor, and presence of LVI. We conducted a retrospective review to identify factors in the primary tumor which predicts lymph node metastasis.

Histological grade has been consistently seen as an important predictor of lymph nodal metastasis. The frequency of nodal metastasis increases from 9–47.5% to 67–85% as grade increases from I to III [7]. In a previous study from our institute, histological grade was the most significant factor for predicting nodal metastasis [10]. Aita et al. [11] demonstrated a better survival for low-grade cancers. An important caveat noted with grade determination is the interobserver variation and reproducibility [12]. Although at our place, the incidence of penile carcinoma is high and the pathologist was experienced, it may be a challenge for centers where these cases are scarcely encountered.

On evaluating the National Cancer Database, Winters et al. [13] found LVI as the strongest independent predictor of occult lymph node metastasis. A similar suggestion has been made by other authors also [14, 15]. Grade and presence of LVI have also been included in the EAU risk stratification for clinically node negative disease. However, as noted by Graafland et al. [16], 77% of the EAU high-risk patients would undergo a negative bilateral lymphadenectomy, leading to additional morbidity [16]. This underlines the search for additional predictive factors and a better risk stratification.

Perineurium forms the covering layer for nerve fascicles formed by flattened fibroblast and acts as a protective sheath. Since the perineural space is devoid of blood or lymphatics, cancer cells can theoretically breach this barrier at 2 anatomical locations, including distal nerve endings and entry points of epineural vessels. Once the barrier is breached, tumor cells can access the central nervous system [17]. The significance of PNI in penile cancer was demonstrated by Velazquez et al. [18] who defined it as tumor infiltrating the clear and open perineural space, and found it to be a significant predictor of nodal metastasis along with histological grade. They created a nomogram using these factors and found 80% probability of nodal metastasis for grade III tumors with PNI [18]. In another study, PNI was found to be predictive of local recurrence after glansectomy, along with grade and positive margins [19]. The presence of PNI has also been correlated with poor survival [20]. In our study also, PNI has proven to be a significant predictor of lymph node metastasis on multivariate analysis. A recent meta-analysis has shown an increased risk of inguinal lymph node metastasis and a higher cancer-specific mortality associated with presence of PNI especially in younger patients [21]. Even though various authors have questioned its role as an independent prognostic factor, it has been included in the AJCC TNM 8th edition staging manual [11, 22, 23].

Similar to that in malignant melanoma, Emerson et al. [24] suggested the role of depth of invasion in penile cancer and tumor progression. Ficarra et al. [14] included it in their nomogram for predicting prognosis; however, further studies have revealed its ambiguity as a prognostic factor with various authors suggesting different outcomes [25]. In our study, increasing depth of invasion was associated with increasing risk of nodal metastasis with depth >1 cm showing statistical significance in univariate analysis. Probably, a larger sample size would bring more clarity to its role.

The limitation of histological markers in predicting nodal metastasis has shifted the attention toward molecular markers. One of the markers under evaluation is Ki67, which is regarded as a marker of proliferation and its presence may signify aggressive disease as suggested by a correlation with tumor grade [26]. Zhu et al. [27] tried to determine its prognostic significance in penile cancer with a threshold for Ki67 set at 20%. They reported a significant correlation between expression of Ki67 and lymph node metastasis on univariate analysis but not on multivariate analysis [27]. A similar finding has been seen in our cohort also. In another study, where a cutoff of 40% was taken, there was strong correlation between Ki67 and tumor grade; however, no association was seen with lymph node metastasis or stage [26].

Advanced tumor stage and infiltrating pattern of invasion showed trend toward poorer prognosis, but were not significant on univariate analysis. Various authors have studied other molecular and biochemical markers, like p53, DNA binding protein inhibitor-1, superoxide dismutase 2 and squamous cell carcinoma antigen overexpression, matrix metalloproteinase-9, high e-cadherin, higher neutrophil to lymphocyte ratio, diffuse PD-L1 expression, and C-reactive protein >20, but further validation studies are required before incorporating them to clinical practice [15, 27, 28]. Our study is limited by its retrospective nature, small sample size and patients being treated at different time periods during which the understanding of the disease changed considerably leading to use of different modalities. Ours is a single-center experience thereby restricting its applicability. Currently a risk-adapted strategy is increasingly used by many centers which lead to unnecessary surgery. This strategy based on the characteristics of primary tumors needs further refinement, and addition of molecular markers will probably lead to better stratification. In our study, PNI appeared to be the strongest predictor; however, larger prospective studies are required to search for more accurate predictors of nodal metastasis.

Primary tumor characteristics, including high grade, LVI, PNI, tumor thickness >1 cm, and Ki67 >50% were significantly associated with nodal metastasis in penile cancer. PNI emerged to be the strongest independent predictor and its presence should suggest occult lymph node metastasis.

The study design complies with the Declaration of Helsinki ethical standards. The study protocol was approved by Cancer Institute (WIA) Institutional Ethics Committee (Reference No: IEC/2021/July 07) Written informed consent was obtained from all the participants.

The authors have no conflicts of interest to declare.

The authors have no financial support to declare.

A.R. and N.K. are responsible for conception and design, drafting the article, critical revision of the article, and final approval of the version to be published. K.M. and D.C. performed the data collection, analysis, and interpretation and drafted the article.

The data that support the findings of this study are available on request from the corresponding author.