Microsatellite instability in penile cancer: comparative analysis of primary tumors and metastases

1 Introduction

Penile cancer (PeCA) is a rare disease, yet, in developing countries, the incidence increases, and in Brazil, the incidence varies between 2.9 - 6.8/100,000 inhabitants (1) and 3–4 per 100,000 inhabitants in Uganda (2). The GLOBOCAN estimate for 2025, 39885 new cases, leading to 14485 deaths (3). The etiological factors associated with PeCA are phimosis, genital warts, HPV and HIV infection, the presence of lichen sclerosus and UV light to treat psoriasis (4–8). Epidermoid carcinoma is the most prevalent histology in PeCA (95%). In 2022, the World Health Organization (WHO) published the new international classification of PeCA: they are currently divided into HPV-associated and HPV-independent (9).

PeCA spreads via the lymphatic route, initially through the inguinal lymph nodes, followed by the pelvic and retroperitoneal lymph nodes (10). The presence of lymph node metastasis is therefore the main factor in poor prognosis (11). Another well-described factor that corroborates histopathological stratification is the presence of HPV DNA, represented by the expression of p16, and is correlated with a 54% reduction in the risk of mortality and a 37% reduction in the risk of disease recurrence, compared to the absence of HPV DNA (12). The combined prevalence of HPV DNA is 50.8% among all patients with PeCA; the predominant oncogenic HPV type was HPV16 with 68.3%, followed by HPV6 and HPV18 with 8.1% and 6.7%, respectively (13).

Although cancer treatment is increasingly guided by molecular biomarkers, their concordance between primary penile tumors and corresponding lymph node metastases remains poorly understood in PeCA. Immunotherapy offers new treatment opportunities for many cancer subtypes (14). Microsatellite instability-high (MSI-H) is a predictive biomarker of response to immunotherapy. The mismatch repair (MMR) system corrects potential mutations that occur during DNA replication. DNA microsatellite regions are subject to replication errors, and in the event of a deficiency in the repair system (dMMR), these errors can lead to instability in these regions (15, 16). The mutational tumor burden (TMB) is also used as a surrogate for the use of immunotherapy, and the correlation between TMB and MSI-H has been demonstrated in a real-world study, regardless of tumor type or treatment line (17). A study of 72 PeCA patients using immunohistochemistry revealed one patient with dMMR (deficient MMR0 and 12 with pMMR (proficient MMR). Of the 13 cases tested, one was MSI-H, with no correlation observed with the dMMR case (18). Another PeCA study with a larger sample size (n = 397) showed that 15% of cases were classified as high TMB (10 mut/Mb or more) (19).

Herein, we aimed to compare MSI status between primary tumors and corresponding lymph node metastases in a representative cohort of patients with PeCA. We also assessed HPV status using p16 as a surrogate marker and correlated these findings with clinicopathological characteristics.

2 Patients and methods

2.1 Study population and selection of cases

We retrospectively included consecutive patients diagnosed with penile squamous cell carcinoma between 2010 and 2023 at Barretos Cancer Hospital who underwent surgical treatment and had available formalin-fixed paraffin-embedded (FFPE) tissue from the primary tumor and lymph node metastasis. Patients lacking sufficient biological material for molecular or immunohistochemical analyses were excluded. Clinical-pathological features were retrieved from the medical records and collected using REDCap electronic data capture tool (20). Among the 116 cases, 114 patients had samples available for molecular and immunohistochemistry analysis; two patients were excluded due to a lack of biological material. All patients underwent inguinal or pelvic lymphadenectomy, and the decision to perform surgery was in accordance with the protocols of the local urology department. The analysis was made comparatively between the primary tumor and the lymph node metastases. This study was approved by the local IRB under Protocol No. CAAE 53660821.0.0005437.

2.2 p16 Immunohistochemistry

Immunohistochemistry (IHC) was performed on paraffin-embedded tissue from the patients’ tumors, in sections up to 4 μm thick, using the BenchMark^® Ventana Ultra automated staining system (Roche Diagnostics, Switzerland). p16 expression was assessed using the Cintecp16 Histology 50 Kit (CE) from Roche Diagnostics for automated immunohistochemistry, when not expressed, they are classified by the WHO as HPV-independent, and when positive, HPV-associated. Samples with strong and diffuse nuclear and cytoplasmic staining in more than 75% of the tumor cells were considered positive for p16 (21, 22).

2.3 Mismatch repair Immunohistochemistry

For cases in which MSI-H was detected by PCR, immunohistochemistry for four MMR proteins (MLH1, PMS2, MSH2, and MSH6) was performed. The antibody clones used were MLH1 (clone ES05, Agilent Technologies Canada Inc.), MSH2 (clone G219-1129, Becton Dickinson Biosciences, Canada), MSH6 (clone EPR3945, Abcam, Canada), and PMS2 (clone A16-4, BD Biosciences). Normal adjacent tissue from each sample served as a positive control. MMR protein loss was defined by the absence of IHC staining in the tumor cell nucleus, while normal cells remained stained, ensuring the technical validity of the experiment. The immunohistochemical staining results were evaluated according to the scoring system reported in the literature: (i) MMR proficient (pMMR): cases with all four MMR stains positive; (ii) dMMR: cases with loss of one of the two heterodimers, including MLH1/PMS2 or MSH2/MSH6 (23).

2.4 DNA isolation and microsatellite instability (MSI) assay

Tumor DNA was isolated from FFPE sections using QIAamp DNA Micro Kit (Qiagen, Germany), accordantly manufacturer’s guideline. The MSI assay was performed using HT-MSI+ kit (Cellco, São Carlos, Brazil) composed of six quasi-monomorphic mononucleotide repeat markers (NR27, NR21, NR24, BAT25, BAT26, and HSP110), following the manufacturer’s protocol (24, 25). The analysis was performed using 3500 Genetic Analyzer automated sequencer (Applied Biosystems, USA) and visualized by the GeneMapper software version 4 (Applied Biosystems, USA), according to manufacturer’s recommendations. Cases with the presence of two or more markers out of the quasimonomorphic variation range (QMVR) were classified as MSI-H (presence of microsatellite instability), and cases with one or without markers out of QMVR were classified as MSS (absence of microsatellite instability).

2.5 Statistical analysis

The comparative analysis methodology was done using the kappa index and the chi-squared test. Survival curves were plotted using the Kaplan–Meier method and the event of interest (death) was considered for the outcome of overall survival (OS). Alive patients and those lost to follow-up were censored. Univariate and multivariate analysis were performed using Cox regression method. Statistical analyses were performed using IBM SPSS Statistics for Mac OS, Version 25 (IBM). P values < 0.05 were considered statistically significant.

3 Results

3.1 Clinical–pathological characterization of PeCA patients

One hundred and sixteen cases were selected according to the inclusion and exclusion criteria (Table 1), two patients did not have enough samples from either the primary or the metastatic lymph nodes. All patients were squamous cell carcinomas (SCCs). The median age was 55 years. All patients underwent lymphadenectomy, either inguinal (91.4%), pelvic (0.8%) or both (7.8%). More than three quarters of the patients had less than an elementary school education. Of samples, 13.8% of them were pN1, 19.0% pN2 and 33.6% pN3. (Table 1).

Table 1. Clinical-pathological characteristics of patients with PeCA.

PeCA, penile cancer.

According to the WHO 2022, 71,0% of cases were the usual squamous cell carcinoma cell carcinoma histological subtype, 5.3% were verrucous, 0.8% sarcomatoid and 22,8% basaloid subtype (Figure 1).

Of note, 39 cases underwent lymphadenectomy and were classified as pN0, and therefore, metastasis analysis was carried out in 77 cases. Approximately 30% of patients received neo or adjuvant chemotherapy, and almost 20% received first-line chemotherapy. Patients received predominantly platinum-based combination chemotherapy according to institutional protocols. (Table 1).

3.2 p16 expression and histological analysis

As above mentioned, 26 cases (22.8%) had positive p16 expression (Figure 2), being considered HPV-positive. This evaluation was performed on primary tumors in 103 cases, except when material was unavailable, in which case it was performed on metastases in 11 cases. The survival rate (Figure 3) of patients who expressed p16 did not reach the median, while patients who were p16 negative had a survival rate of 3.5 years (p 0.027). Univariate Cox regression analysis identified extracapsular extension, advanced nodal stage, AJCC stage III–IV, and receipt of chemotherapy as significant predictors of worse overall survival (Table 2). Although p16 expression was not significantly associated with survival in the univariate analysis, it showed a trend toward significance and was therefore included in the multivariable model. In the multivariable analysis, AJCC stage III–IV remained independently associated with an increased risk of death (HR 5.9), whereas positive p16 expression was independently associated with a reduced risk of death (HR 0.45) (Table 2).

Table 2. Univariate and multivariate analysis of clinicopathologic characteristics and survival in patients with PeCA.

HR, hazard ratio; CI, confidence interval; PeCA, penile cancer.Bold values means statistically significant.

3.3 Microsatellite instability analysis

MSI analysis of primary tumors was performed on 99 samples, of which 2 were MSI-H, 70 were MSS, and 27 were inconclusive due to DNA quality issues related to DNA degradation associated with formalin-fixed paraffin-embedded (FFPE) tissue in older archival specimens. The analysis of lymph node metastasis, 69 samples were analyzed, of which 3 were MSI-H, 55 MSS, and 11 were inconclusive.

In this regard, we obtained 2.6% patients being MSI-H. In the primary tumor, one patient was inconclusive for MSI and was pMMR in IHC, the two others were MSI-H and showed dMMR in IHC (Figure 4). In the metastases samples of these same three patients, all were MSI-H by molecular analysis and with loss of heterodimer MLH1/PMS2 by IHC (Table 3).

Table 3. Description of the 03 patients who had MSI and IHC results.

AJCC: American Joint Committee on Cancer; CR: chemo-radiation therapy; SCC: squamous cell carcinoma; IHC: immunohistochemistry; OS overall survival; * Expression by immunohistochemistry.

The median survival between MSI-H and MSS was not significant (p 0.421), with worse outcomes for MSI-H 0.9 years vs. 11.2 years.

4 Discussion

Brazil has one of the highest incidences of penile cancer worldwide. In the present study, we report for the first time the concordance of MSI status between paired primary penile tumors and their corresponding lymph node metastases, demonstrating complete concordance in all MSI-H cases. We also observed that all MSI-H tumors were p16-positive, suggesting a possible association with HPV-related carcinogenesis.

Our cohort is representative of patients with penile squamous cell carcinoma treated in Brazilian referral centers, with a median age of 55 years, a predominance of uncircumcised men, advanced-stage disease, and clinicopathological characteristics comparable to those reported in other national series (26). Consistent with previous Brazilian studies, overall survival was similar to published data, and nodal involvement remained the strongest prognostic factor, reinforcing its central role in disease progression and survival (26, 27). These findings support the representativeness of our cohort and provide an appropriate clinical context for the interpretation of the molecular analyses.

All patients in our study underwent lymphadenectomy, either inguinal or pelvic, with a noteworthy pN0 frequency of 33.6%. This finding highlights the well-known clinical dilemma regarding the indication of lymphadenectomy, since only half of clinically positive lymph nodes harbor pathologically confirmed metastases (28). Conversely, other data suggest that up to 36.9% of cN0 cases may represent false positives (29). Although the prognostic value of nodal disease (N+) is well established, clinical evaluation alone does not appear accurate enough to determine surgical management, as corroborated by our findings.

The identification of p16 expression has been incorporated into WHO classifications as a validated surrogate biomarker of HPV-driven carcinogenesis. In our study, 22.8% of patients were p16-positive, and this subgroup demonstrated a 55% relative risk reduction of death. The Brazilian state of Maranhão, which has the world’s highest incidence of penile cancer, shows a frequency of 62% HPV-associated PeCA but also a high prevalence of other risk factors, such as poor genital hygiene (73%) and zoophilia practices (60%) (30). Olesen et al. evaluated 2,295 patients in a systematic review and meta-analysis and found 41.6% p16 positivity globally, though prevalence varies widely (13). Another systematic review and meta-analysis of 649 patients, 47% of whom were p16-positive, demonstrated better disease-specific survival among HPV- and p16-positive patients (31). Furthermore, a prospective phase II trial using atezolizumab with or without radiotherapy for PeCA reported that 43.8% of patients were p16-positive and experienced better survival outcomes compared to p16-negative patients (32). These data are aligned with our findings, underscoring the prognostic significance of HPV-associated PeCA in both chemotherapy and emerging immunotherapy contexts.

The low frequency of MSI-H observed in our cohort is consistent with findings in other squamous cell carcinomas, including vulvar SCC and head and neck SCC, where mismatch repair deficiency and MSI-H are uncommon molecular events (33, 34). MSI-H was identified in only 2.6% of cases, indicating that defective mismatch repair is a rare event in penile SCC. To our knowledge, this is the first study to compare MSI status between paired primary penile tumors and corresponding lymph node metastases; therefore, no previous studies in penile cancer are available for direct comparison. We observed complete concordance of MSI status between the primary tumors and matched metastases. Similar high concordance has been reported in colorectal cancer, where MSI/MMR status is generally preserved between primary tumors and metastatic sites (35, 36). A recent meta-analysis further demonstrated that MSI is among the most stable predictive biomarkers of immunotherapy across primary and metastatic tumors (37). Immunohistochemistry confirmed the molecular findings, with loss of the MLH1/PMS2 heterodimer in all MSI-H metastatic samples and in the two evaluable primary tumors, consistent with the expected MMR-deficient phenotype (23). All MSI-H tumors were also p16-positive. However, the small number of MSI-H cases precludes conclusions regarding an association between HPV-associated carcinogenesis and mismatch repair deficiency.

The association between genomic instability and tumor immunogenicity is well established, particularly in tumors with high TMB and MSI-H status, which derive greater benefit from immune checkpoint blockade. Although immunotherapy has recently been investigated in advanced penile cancer, MSI status has not been prospectively evaluated as a predictive biomarker in clinical trials (38). Necchi et al. reported that approximately 15% of penile SCCs harbor high TMB (19), while the HERCULES trial demonstrated substantially higher objective response rates among patients with TMB-high tumors treated with pembrolizumab plus platinum-based chemotherapy (75% vs. 36.4%) (38). Because MSI-H represents one of the biological mechanisms underlying high TMB, whereas other alterations such as POLE mutations may also generate hypermutated tumors (39), comprehensive molecular profiling may help identify the subset of patients most likely to benefit from immunotherapy. Although MSI-H appears to be rare in penile SCC, its established predictive value across solid tumors supports routine consideration of MSI testing, particularly in patients with advanced or treatment-refractory disease.

Our study comprises a relevant cohort considering the rarity of the PeCA, and it appears representative given the similarity of sample characteristics and clinical outcomes with the literature. We employed validated MSI-H testing methodology in the Brazilian population, and the paired analysis of primary tumors and nodal metastases provided novel insights into PeCA carcinogenesis and progression, as well as hypothesis-generating findings for future research.

Despite our findings, the study also has some limitations, including its relatively small sample size, single-institution data, and retrospective nature, which may introduce selection biases. Additionally, variability in HPV prevalence across Brazilian population may limit the generalizability of our findings, and HPV status was inferred solely by p16 immunohistochemistry, an accepted WHO surrogate marker that may misclassify HPV status in the absence of HPV DNA or RNA testing.

5 Conclusion

MSI-H is rare in penile cancers and is associated with p16-positive tumors. We observed a 55% reduced relative risk of death in p16 positive patients. More comprehensive, multicenter studies across different local cultures are needed to confirm our results.

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Footnotes

Edited by: Łukasz Zapała, Medical University of Warsaw, Poland

Reviewed by: Maira Du Plessis, St George’s University, Grenada

Mikhail Ermakov, Medical University of Graz, Austria