Among the various types of cancer affecting the male population, penile cancer (PeCa) stands out due to its relatively high mortality rate. PeCa is a rare malignancy that arises on the skin or within the tissues of the penis. The most common histological subtype is squamous cell carcinoma, which accounts for over 90% of cases and originates in the epithelial cells covering the penile surface [1].

Incidence rates of PeCa vary globally and are strongly associated with socio-economic factors. The disease is more prevalent in Latin America, Africa, and Asia [2]. In Brazil, data from the National Cancer Institute (INCA) reported 408 and 444 deaths from PeCa in 2016 and 2017, respectively [3]. According to the International Agency for Research on Cancer (IARC), there were an estimated 36,230 new cases and 15,938 related deaths worldwide in 2020 [4].

Several risk factors have been implicated in the pathogenesis of PeCa. These include chronic infection with high-risk human papillomavirus (HPV) types—especially HPV-16 and HPV-18, along with poor genital hygiene, phimosis, smoking, and absence of circumcision. Additionally, chronic inflammatory and pre-malignant conditions of the penis, such as balanoposthitis, lichen sclerosus et atrophicus, and xerotic balanitis obliterans, have been associated with increased oncogenic potential [5].

The management of PeCa is largely guided by the stage of the disease, with particular emphasis on lymph node status and the existence of metastases. For patients with localized, low-grade T1 tumors, treatment options may include conservative approaches such as local excision, partial penectomy, laser ablation, or radiotherapy. In contrast, individuals with more advanced lesions (T2 or higher) often require more aggressive interventions, including partial or total penectomy, potentially combined with radiotherapy and/or chemotherapy. In cases of non-palpable inguinal lymph nodes and low-risk primary tumors, active surveillance may be appropriate. However, in patients with intermediate- to high-risk disease (e.g., T1b or higher), non-palpable lymph nodes may necessitate evaluation through bilateral dynamic sentinel lymph node biopsy (DSNB) [6] or even bilateral inguinal lymph node dissection (ILND), followed by close follow-up [7, 8]. Palpable inguinal or pelvic lymphadenopathy typically requires confirmation via percutaneous biopsy and may be managed with pelvic lymph node dissection (PLND), radiotherapy, systemic chemotherapy, or neoadjuvant chemotherapy regimens [9, 10].

With recent advances in next-generation sequencing technologies, cancer genomics has entered a new era of discovery. The comprehensive characterization of somatic mutations in tumors has enabled the identification of novel biomarkers with potential diagnostic, prognostic, and therapeutic value. Although the oncogenic role of HPV in PeCa is still under investigation, current evidence suggests that the E6 and E7 viral oncogenes contribute to malignant transformation by inactivating key tumor suppressor genes, including TP53 and RB1, thereby promoting uncontrolled cellular proliferation [11]. Nonetheless, HPV is not universally present in all PeCa cases, indicating that other molecular mechanisms also play a role in carcinogenesis [12].

The present study aimed to perform whole-exome sequencing of PeCa tissue from a Brazilian cohort. The objective was to identify relevant somatic mutations associated with tumor initiation or progression and to contribute genomic insights that may guide future clinical decision-making.

This study analyzed DNA samples extracted from the penile tissue of 18 individuals, comprising 12 patients with histologically confirmed squamous cell carcinoma of the penis and six control individuals without any clinical or histopathological evidence of PeCa, genetic disorders, or venereal disease, who underwent postectomy for aesthetic or functional reasons.

Our analysis identified exclusive somatic mutations and potential molecular biomarkers, thereby contributing to the growing body of genomic data in this rare malignancy and laying the groundwork for future studies investigating genotype-phenotype correlations and therapeutic targets.

The age at diagnosis among the cancer patients ranged from 33 to 81 years, with both the mean and median age being 57.5 years. Ethnic self-identification among these patients was as follows: 58.3% White (n = 7), 25.0% Black (n = 3), and 16.7% Mulatto (n = 2). A history of tobacco use was reported by 75% of patients, and 50% reported regular alcohol consumption (Table 2).

Clinically, microscopic lesions suggestive of HPV infection were observed in two patients. However, molecular testing confirmed HPV-16 DNA in only one of these cases, and in one additional patient who did not exhibit visible lesions. No samples tested positive for HPV-18.

Tumor staging revealed four cases classified as pT1, three as pT2, three as pT3, and two with staging information unavailable. Among the 12 cancer patients, 4 (33.3%) presented with metastases, initially affecting regional lymph nodes. Two of these patients subsequently developed distant metastases to bone and lung and succumbed to the disease. All patients underwent surgical treatment via penectomy; nine underwent partial penectomy with tumor excision and adequate safety margins, while three underwent total penectomy.

Whole-exome sequencing and subsequent variant analysis using the COSMIC database identified potentially pathogenic variants in 55 genes. Among these, the most frequently mutated genes were MUC16 (present in 11/12 patients, 91.7%) and PABPC1 (8/12 patients, 66.7%). Although mutations in PABPC1 were also detected in all control individuals, the mutation types differed: frameshift mutations were found in controls, while non-synonymous mutations were predominant in cancer patients.

The mutation profile among patients with PeCa predominantly included non-synonymous single-nucleotide variants, followed by frameshift mutations and insertions. Other genes with notable variants include SIRPA, NOTCH1, USP6, TP53, NOTCH2, KMT2D, ATM, HLA-A, JAK2, KRAS, and several others. While some of these genes have been previously implicated in cancer, many have not yet been described in the context of penile carcinoma. A summary of the identified mutations is provided in Figure 1.

The therapeutic strategy for PeCa is primarily guided by disease stage, especially in cases with regional lymph node involvement or distant metastases. According to the National Comprehensive Cancer Network (NCCN) Guidelines (v1.2024), the preferred systemic treatment for patients with metastatic or recurrent disease is a regimen combining paclitaxel, ifosfamide, and cisplatin (TIP). An alternative first-line option includes cisplatin in combination with 5-fluorouracil (5-FU). In parallel, recent studies have increasingly focused on the molecular characteristics of advanced PeCa, exploring the potential of targeted therapies and immune checkpoint inhibitors based on underlying genomic alterations [10].

Through comprehensive exome sequencing, we identified several unique mutations potentially associated with tumor progression and adverse clinical outcomes.

Among the 12 patients, five exhibited distinguishing clinical characteristics: advanced local disease requiring total penectomy (P5, P7, P9), HPV positivity (P1, P9), and evidence of metastases (P1, P7, P9, P11), with two patients (P1, P7) ultimately succumbing to the disease. The remaining patients, who did not experience disease progression, served as a reference group for outlining a linear model of PeCa progression.

Patient P1, a 52-year-old Caucasian tobacco and alcohol user with HPV-16 infection, presented with advanced disease, experienced recurrence one-year post-surgery, and developed metastases to the bone and lung, ultimately leading to death. This patient harbored pathogenic mutations in PABPC1, MUC16, SIRPA, NOTCH1, RGPD3, DDX10, TSC2, RSPO3, and JAK2.

The tumor suppressor gene DDX10 has been associated with inhibition of tumor growth, and its reduced expression has been implicated in accelerated cancer progression [13]. TSC2, a critical regulator of the mTORC1 pathway, acts as a tumor suppressor. Loss-of-function mutations in TSC2 lead to mTORC1 hyperactivation, stimulating anabolic processes and inhibiting autophagy [14, 15]. Notably, HPV-16 E6 proteins can bind and degrade TSC2, further activating mTORC1 and enhancing tumor cell proliferation, metastasis, and resistance to apoptosis by inhibiting pro-apoptotic proteins such as Bad and Bax [16, 17].

The NOTCH1 gene, frequently implicated in squamous carcinomas, was also mutated in P1. Ali et al. [18] identified NOTCH1 mutations in 25% of PeCa samples and proposed it as a potential therapeutic target. Patient P7, diagnosed with metastatic PeCa at just 33 years of age, was a non-smoker and non-drinker who died two months after a total penectomy. This patient exhibited mutations in TP53, NOTCH2, ATM, and USP6. Although the role of USP6 in PeCa is not well-defined, Zhou et al. [19] previously reported its frequent mutation in PeCa cell lines.

The NOTCH2 mutation c.302G > T, a frameshift variant with a high CADD score, is predicted to be pathogenic. The NOTCH family genes encode transmembrane proteins involved in cellular survival and differentiation [20]. Although few studies have examined NOTCH2 in PeCa, frequent alterations have been noted, suggesting potential relevance [19]. The ATM gene mutation (c.8800A > G), a non-synonymous variant, is also predicted to be pathogenic. ATM plays a pivotal role in genomic stability and DNA damage repair. Previous studies have linked ATM mutations with poor outcomes in PeCa [21, 22]. The co-occurrence of ATM and TP53 mutations may disrupt DNA repair and apoptotic mechanisms, contributing to the aggressive course observed in this patient.

Patient P9, a 65-year-old Caucasian with a history of alcohol consumption, presented with HPV-16-positive, locally advanced PeCa. Mutations in AFF1, STRN, and EML4 were identified. The EML4/ALK fusion gene has been extensively studied in lung adenocarcinoma and squamous cell carcinoma and is associated with metastasis [23]. Similarly, STRN mutations can generate STRN/ALK fusion proteins, previously linked to metastatic progression in colorectal, breast, and renal cancers [24–26]. These findings suggest a potential role of these genes in the lymph node metastases observed in this patient.

Patient P11, a white male tobacco user without HPV infection, presented with unilateral lymph node metastasis. Mutations were found in ZNF521 and CBLC. ZNF521 encodes a transcriptional co-repressor involved in chromatin remodeling and has been implicated in the pathogenesis of breast, lung, and colorectal cancers through its role in genomic instability [26–31]. The CBLC gene, affected by a non-synonymous mutation (c.1174G > A), is known to influence tumor suppression, cell cycle regulation, proliferation, and DNA repair [32, 33]. Although CBLC has not been widely studied in cancer, its moderate-impact mutations merit further exploration.

Interestingly, patients who underwent partial penectomy with tumor-free margins experienced no recurrence or metastasis. This subgroup, including the oldest patients and those with the highest mutation counts, suggests that neither age nor the number of mutations independently predicts disease aggressiveness. Instead, specific “driver” mutations and gene interactions may play a more decisive role. These insights support the development of targeted gene panels for identifying mutations associated with carcinogenesis and aggressive clinical behavior in PeCa.

Including healthy controls with no history of tobacco or alcohol use, penile lesions, or venereal disease allowed for the identification of shared mutations (PABPC1, MUC16, and KAT6B) between groups. However, controls typically harbored frameshift or isolated mutations, while cancer patients displayed non-synonymous mutations with broader genomic involvement, suggesting distinct mutation profiles. Of note, RSPO3—classified as pathogenic, showed no known gene interactions and was absent from the dbSNP database, suggesting it may represent a novel finding worthy of future investigation.