Microsatellite Instability Proves Rare but Stable Biomarker in Penile Cancer Study

Penile cancer remains one of the most under-researched malignancies in oncology, and a new study from Brazil now offers the first paired molecular comparison between primary penile tumors and their lymph node metastases. Researchers at Barretos Cancer Hospital set out to determine whether microsatellite instability, a well-established predictor of response to immunotherapy, is preserved as tumor cells spread from the penis to regional lymph nodes. Their findings, published in Frontiers in Urology, reveal that this biomarker is rare in penile cancer but remarkably stable once present, and that it travels in lockstep with a signature of human papillomavirus-associated disease.

The scientific premise rests on the biology of DNA mismatch repair. During every round of cell division, the mismatch repair system acts as a molecular proofreader, correcting replication errors that accumulate in repetitive DNA sequences known as microsatellites. When genes such as MLH1, PMS2, MSH2, or MSH6 are lost or silenced, these short repeats begin to lengthen or shorten unpredictably, producing a state called microsatellite instability-high, or MSI-H. Tumors with this defect accumulate mutations rapidly, display high tumor mutational burden, and are among the most responsive cancers to immune checkpoint blockade. Because MSI-H status can be tested from archived tissue and predicts benefit from drugs like pembrolizumab, oncologists routinely measure it in colorectal, endometrial, and many other cancers. Whether the same biomarker behaves consistently in penile squamous cell carcinoma had never been systematically tested.

To answer that question, the team assembled a cohort of 116 patients with penile squamous cell carcinoma treated between 2010 and 2023, all of whom underwent inguinal or pelvic lymphadenectomy. The researchers obtained formalin-fixed paraffin-embedded tissue from both the primary tumor and the corresponding lymph node metastasis whenever available. They assessed microsatellite instability using a hexa-plex panel of six quasi-monomorphic mononucleotide repeat markers, including BAT25, BAT26, and HSP110, analyzed by polymerase chain reaction followed by capillary fragment analysis on an automated genetic sequencer. Cases with two or more markers shifting outside the quasi-monomorphic variation range were classified as MSI-H. In parallel, immunohistochemistry for all four mismatch repair proteins provided an independent validation layer, and p16 staining served as the World Health Organization-endorsed surrogate for HPV-associated carcinogenesis.

The molecular results were striking in their rarity and consistency. Of 99 evaluable primary tumors, only two were MSI-H, while three of 69 analyzed lymph node metastases showed instability, yielding an overall frequency of 2.6 percent across the cohort. Critically, every patient whose metastasis was MSI-H also carried the biomarker in the primary tumor, and the two evaluable primary tumors confirmed by molecular testing showed complete concordance with their matched metastases. Immunohistochemistry corroborated the molecular findings: all MSI-H samples, whether primary or metastatic, exhibited loss of the MLH1/PMS2 heterodimer while retaining proficient MSH2 and MSH6 staining. This pattern mirrors what has been documented in colorectal cancer, where MSI status is generally preserved between primary tumors and metastatic sites, and a recent meta-analysis identified MSI as one of the most stable immunotherapy predictive biomarkers across tumor pairs.

Perhaps the most provocative observation was that all three MSI-H patients were also p16-positive, indicating HPV-associated carcinogenesis. In the broader cohort, 22.8 percent of patients expressed p16 by immunohistochemistry, and this subgroup experienced a 55 percent relative reduction in the risk of death in multivariable analysis. Median overall survival had not been reached for p16-positive patients at the time of analysis, whereas p16-negative patients survived a median of 3.5 years. The convergence of HPV-driven transformation and mismatch repair deficiency in the same tumors suggests a potential biological interaction between viral oncogenesis and genomic instability, though the authors caution that the small number of MSI-H cases precludes any definitive conclusion about causality or mechanism.

The clinical context of the cohort underscores why these findings matter. Penile cancer is uncommon in high-income countries but carries a heavy burden in regions with limited access to genital hygiene and preventive care. Brazil reports incidence rates between 2.9 and 6.8 per 100,000 inhabitants, and the state of Maranhão holds the highest recorded incidence worldwide. Globally, GLOBOCAN estimates nearly 40,000 new cases and roughly 14,500 deaths in 2025. In the Barretos cohort, the median age was 55 years, more than three quarters of patients had less than an elementary school education, and over a third presented with advanced nodal disease classified as pN3. Nodal involvement remained the strongest prognostic factor, and extracapsular extension, advanced nodal stage, and AJCC stage III-IV disease each independently predicted worse survival.

The lymphadenectomy data also illuminate a persistent clinical dilemma. Although all 116 patients underwent lymph node dissection, 33.6 percent were ultimately classified as pN0, meaning no pathologically confirmed metastasis was found. Prior studies suggest that only about half of clinically positive nodes harbor true metastases, while up to 36.9 percent of clinically node-negative cases may in fact be false negatives. Clinical examination alone, the authors argue, is simply not accurate enough to guide surgical decisions, reinforcing the need for better preoperative risk stratification in this disease.

Where does immunotherapy fit into this picture? Immune checkpoint inhibitors have only recently been explored in advanced penile cancer, and MSI status has never been prospectively evaluated as a predictive biomarker in this setting. The HERCULES trial, a nonrandomized study of pembrolizumab plus platinum-based chemotherapy, demonstrated substantially higher objective response rates among patients with high tumor mutational burden, 75 percent versus 36.4 percent. Because MSI-H represents one biological route to a hypermutated phenotype, and other alterations such as POLE mutations can produce similar effects, comprehensive molecular profiling may be needed to identify which patients stand to benefit most. A separate analysis of nearly 400 penile cancer cases found that roughly 15 percent harbored high tumor mutational burden, suggesting that while MSI-H itself is rare, the broader hypermutated landscape may be more common than the 2.6 percent figure implies.

The study’s limitations are worth noting. It was conducted at a single institution, relied on retrospective data collection, and involved a relatively small sample, all of which introduce potential selection bias. HPV status was inferred solely from p16 immunohistochemistry, an accepted but imperfect surrogate that can misclassify cases in the absence of direct HPV DNA or RNA testing. DNA degradation in older archival specimens left 27 primary tumor samples and 11 metastasis samples inconclusive for molecular analysis, a common challenge when working with formalin-fixed tissue. Median survival between MSI-H and MSS patients did not differ significantly, though the raw numbers, 0.9 years versus 11.2 years, hint at worse outcomes for the small MSI-H group that larger cohorts may need to clarify.

Nevertheless, the study establishes an important foundation. It is the first to demonstrate complete concordance of MSI status between paired primary penile tumors and their lymph node metastases, providing reassurance that a biopsy of either site can reliably inform immunotherapy decisions. It also links the rare MSI-H phenotype to p16 positivity, raising the hypothesis that HPV-associated carcinogenesis and mismatch repair deficiency may intersect in penile cancer biology. The authors call for prospective, biomarker-driven trials and larger multicenter studies across different geographic and cultural settings to confirm these results. For a cancer that has long been neglected by the precision oncology revolution, even a 2.6 percent finding represents a meaningful step toward bringing penile cancer into the era of molecularly guided treatment.

Subject of Research: Microsatellite instability and HPV-associated biomarkers in primary penile cancer and lymph node metastases

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

Article References: Araujo, H. S., Teixeira, G. R., Laus, A. C., Berardinelli, G. N., Preto, D. D. A., Reis, R. M., & Cárcano, F. M. (2026). Microsatellite instability in penile cancer: comparative analysis of primary tumors and metastases. Frontiers in Urology, 6, Article 1908537. https://doi.org/10.3389/fruro.2026.1908537

Image Credits: AI Generated

DOI: 10.3389/fruro.2026.1908537

Keywords: penile cancer, microsatellite instability, mismatch repair, HPV, p16, immunotherapy, lymph node metastasis, biomarkers, MLH1, PMS2, squamous cell carcinoma, Barretos Cancer Hospital