Abstract
Penile squamous cell carcinoma (pSCC) is a rare genitourinary tumor associated with notable psychosexual distress and poor prognosis. Traditional prognostic factors for pSCC include TNM stage and histological grade, with lymph node metastases being a critical indicator of poor prognosis. This study aimed to evaluate the prognostic impact of the following immunohistochemistry markers routinely used in histopathology practice: GATA3, IMP3, HIF-1-α, CK7, CA-IX, HER2, and TTF-1. A retrospective cohort of 145 patients with pSCC was analyzed using tissue microarray and immunohistochemical techniques. Overall survival (OS) was correlated statistically with detected marker expression. Key findings include that low GATA3 expression is associated with significantly worse OS in univariate Cox regression truncated at 3 years of follow-up. Low GATA3 retained prognostic impact when adjusted for major clinicopathological variables: Age, pT and pN stage, grade, lymphatic, venous, and perineural invasion, lymphocytic infiltrate, and expression of p16, p53, and PD-L1. Low GATA3 expression was associated with shorter cancer-specific survival (CSS) at 10 years follow-up. IMP3, CK7, and CA-IX showed statistically insignificant trends towards poorer prognosis. CK7 and CA-IX were more frequently expressed in high grade pSCC and in p16/HPV-positive tumors. IMP3 and CA-IX were associated with regional lymph node metastases. All cases were negative in TTF-1 and HER2. This study suggests GATA3 as a potential prognostic marker in pSCC.
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Introduction
Penile squamous cell carcinoma (pSCC) is a relatively rare genitourinary tumor whose prognosis improvement has remained elusive, especially in advanced stages, over the past several decades1. Some studies on Western populations describe a slowly increasing incidence, particularly among young men, which is likely related to sexually transmitted human papillomavirus (HPV) infection2,3. In pSCC, two distinct types of lesions may arise: HPV-associated SCC, characterized by undifferentiated squamous intraepithelial lesions (SIL) and the expression of the oncoprotein p16, and HPV-independent SCC, associated with chronic inflammation and differentiated dysplasia/differentiated penile intraepithelial neoplasia (d-PeIN). d-PeIN is usually associated with older age, relatively preserved morphology of squamous epithelium, and a high risk of progression into an invasive pSCC. The traditional prognostic factors include TNM stage4, histological grade5, and histological subtype6. Lymph node metastases represent a late indicator of a poor prognosis7. There are controversies concerning the pT1b stage as it displays worse prognosis compared to pT2-48. Novel prognostic markers such as immune cell infiltration, tumor budding9, high tumor mutational burden (TMB)10, and several genomic alterations11 have been described, but more efficient markers are needed. Recently, there has been a global surge in scientific research concentrating on prognostic factors pertinent to pSCC.
Given the increasing recognition of certain markers as surrogate prognostic indicators or therapeutic targets in squamous and epithelial cancers, we aimed to explore the prognostic utility of a routinely available IHC panel, which could be related to modern anticancer therapy or used as specific markers for other tumors: GATA binding protein 3 (GATA3), Insulin-like growth factor II-messenger RNA-binding protein 3 (IMP3), Hypoxia-inducible factor 1-α (HIF-1-α), Cytokeratin 7 (CK7), carbonic anhydrase IX (CA-IX), Human epidermal growth factor receptor-2 (HER2), and Thyroid transcription factor-1 (TTF-1). The most important clinical-pathological variables (stage, histological subtype, grade, lymphatic/vascular/perineural invasion) and pivotal prognostic immunohistochemistry markers (i.e. p16, p53, PD-L1) have been studied in our previous works9,10. This study aims to analyze the prognostic impact of other important previously unexplored markers in pSCC.
Materials and methods
Cohort acquisition
A retrospective cohort of 156 patients with surgically removed or biopsied and histologically verified invasive pSCC with sufficient amount of representative formalin-fixed paraffin-embedded (FFPE) archival tumor tissue was analyzed. Invasive carcinomas of all stages were included, without regard to adjuvant therapy. No patient in the cohort received neoadjuvant therapy. Follow-up data were obtained from medical records or exported from the Czech National Oncological Registry. Overall survival (OS) was calculated from the date of surgery to the date of recorded death. The stage of the tumors was recorded based on medical records. Stage I-IV was assigned in accordance with TNM Classification4 and Union for International Cancer Control (UICC). All available haematoxylin and eosin (H&E) slides were reviewed by two experienced surgical pathologists (JH and ZP) without knowledge of the patient’s follow-up. Grade (1–3) was assigned according to the International Society of Urological Pathology5. The presence or absence of lymphatic, vascular, and perineural invasion were recorded. Lymphocytic infiltrate in H&E slides was evaluated as brisk/non-brisk + absent according to Clark´s scheme used in assessment of intratumoral lymphocytes in malignant melanoma12, as described in detail elsewhere9.
Immunohistochemistry
For immunohistochemistry, the tissue microarray (TMA) technique was used, using two cylindrical cores each measuring 2 mm in diameter from each case. Tissue sections of 4 μm thickness were stained using the Ventana BenchMark ULTRA autostainer (Ventana Medical Systems, USA). Monoclonal antibodies used included GATA3 (L50-823, CellMarque, ready to use/RTU), IMP3 (EP286, BioSB, 1:20), HIF-1-α (EP118, BioSB, 1:800), CK7 (OV-TL 12/30, BioSB, 1:500), CA-IX (EP161, BioSB, 1:100), HER2 (4B5, Roche, RTU), TTF-1 (SP141, Roche, RTU). The reactions were visualized using the Ultraview Detection System (Ventana Medical Systems) and counterstained with haematoxylin. The stained slides were then dehydrated and mounted in a xylene-based medium.
Immunohistochemistry evaluation
The immunohistochemistry slides were evaluated independently by two experienced surgical pathologists (JH and ZP) without knowledge of other variables and clinical outcome. Cytoplasmic (CA-IX, IMP3, HIF-1-α, CK7), nuclear (GATA3, TTF-1), and membranous (HER2) staining were noted. In each case, the percentage of positively stained cells was scored (0 = none, 1–9% = 1, 10–49% = 2, 50–79% = 3, 80–100% = 4), and staining intensity was semi-quantitatively classified into the following four categories: 0 = negative, 1 = weak, 2 = moderate, and 3 = strong positivity. In each core, an overall immunoreactivity score was calculated as proportion (0–4) × intensity (0–3), range 0–12 (Fig. 1). Each case rendered four scores: 2 cores from 2 pathologists. For the statistical analysis, the average value from individual cores was used. The immunoreactivity score was recorded for each core only if ≥ 100 invasive pSCC cells were found. If three or four cores were insufficient, the case was excluded from the study. In total, 11 cases were excluded from the study, and the final cohort size was n = 145.
Immunohistochemistry profiles of p53 and p16 antibodies were analyzed in whole sections and classified into mutated/wild type and block-positive/non-block-positive + negative, as described in detail elsewhere9. Programmed death ligand 1 (PD-L1) tumor proportion score was evaluated as described in detail elsewhere10, classified into negative (< 1%)/positive (1–100%).
Statistics
For the overall survival (OS) analysis, Kaplan–Meier analysis was conducted using the log-rank test, and confidence intervals were calculated using the log-log method. This was followed by a restricted mean survival time (RMST) analysis with a 95% confidence interval (CI). Univariate Cox regressions with 95% CIs were performed to calculate the hazard ratio (HR) for each parameter. The OS analyses were truncated at 5 years of follow-up, and in case of significant p value (< 0.05), separate analyses truncating at 3 years and 10 years of follow-up were performed. The significant results were subsequently analyzed by multivariate Cox regression adjusting the marker on the patient’s age and other clinical-pathological variables (see below). Multivariable analyses were conducted as pairwise models across key covariates due to limited events per variable. The markers with an impact on OS were further subjected to analysis of cancer-specific survival (CSS) using Fine Gray regression (a proportional hazards model for the subdistribution of a competing risk) distinguishing between penile cancer-related deaths and other causes of death, based on information from clinical documentation and Czech National Oncological Registry. The cohort was binarized according to immunoreactivity score for each marker using the surv_cut-point function (survminer) determining the optimal cut-point using the maximally selected rank statistics from the `maxstat` R package. According to the optimal cut-point, the cohort was classified into high and low for each marker. Interobserver agreement between the two pathologists was evaluated by calculating Cohen’s kappa. To assess association between immunohistochemistry findings and clinical-pathological variables, logistic regression/Pearson’s chi-squared test was performed. The clinical-pathological variables were grouped as follows: pT1 + pT2/pT3 + pT4; pNX + pN0/pN1 + pN2 + pN3; grade 1 + 2/grade 3; lymphatic invasion yes/no; vascular invasion yes/no; perineural invasion yes/no; lymphocytic infiltrate brisk/non-brisk + absent. The patients who underwent lymphadenectomy with negative lymph node histology were clustered together with patients without lymphadenectomy (pNX/cN0). The immunohistochemistry marker profiles have been compared with mutational profiles examined by next generation sequencing (NGS), whose results and methods have been described by our group elsewhere11. Genes which have been altered in ≥ 10 cases have been included in the analysis.
All analyses were performed using R version 4.2.2 (2022-10-31), with survival analyses carried out using the survival package version 3.4–0.17; p values < 0.05 were considered statistically significant.
Ethics
The study was approved by the institutional review board and by the University Hospital Královské Vinohrady ethics committee, approval number EK-VP1261012020. Informed consent was waived due to the retrospective nature of the study. The entire research was performed in accordance with the Declaration of Helsinki.
Results
Optimal cut-points
The cohort description is summarized in Table 1. All research data are listed in Supplementary Table 1 (Table S1). Samples from all 145 cases were HER2- and TTF-1-negative. The remaining six markers showed varying intensity and percentage of staining. In the HIF-1-α, only cytoplasmic staining without any nuclear staining was observed. The optimal cut-points using the survminer function were set at immunoreactivity score 0.5 in GATA3, 0.75 in IMP3, 3.0 in HIF-1-α, 3.25 in CK7, and 8.75 in CA-IX.
The cohort (n = 145) was classified according to the optimal cut-points as low/n (%) as follows: GATA3 = 60/145 (42%), IMP3 = 70/145 (48%), HIF-1-α = 122/144 (85%), CK7 = 129/145 (87%), CA-IX = 126/143 (88%). Detailed description of the mutual associations is in Supplementary Table 2 (Table S2). Cohen’s kappa reached for binarized markers: GATA3 K = 0.78, IMP3 Κ = 0.84, HIF-1-α Κ = 0.65, CK7 Κ = 0.92, CA-IX Κ = 0.72. This calculation showed almost perfect interobserver agreement in CK7 and IMP3, and substantial agreement in GATA3, HIF-1-α and CA-IX.
OS analysis
The results of Kaplan-Meier analysis, RMST, and univariate Cox regression are summarized in Table 2, the results of Kaplan-Meier analysis in Fig. 2. The results of multivariate Cox regression are summarized in Table 3.
Patients with GATA3-low tumors displayed significantly worse OS in the univariate (Hazard ratio/HR = 1.69, 95%CI = 1.02–2.78, p = 0.042), but not in the multivariate age-adjusted (HR = 1.41, 95%CI = 0.85–2.33, p = 0.2) Cox regression. The Cox regression truncated at 3 years of follow-up displayed significant negative prognostic impact of GATA3-low status (univariate HR = 2.08, 95%CI = 1.22–3.57, p = 0.007; age-adjusted HR = 1.75, 95%CI = 1.03–3.03, p = 0.04). GATA3-low status was a significant negative prognostic marker also in 10 years of follow-up in the univariate (HR = 1.82, 95%CI = 1.12–2.94, p = 0.016) but not in the age adjusted (HR = 1.49, 95%CI = 0.92–2.44, p = 0.1) analysis. GATA3-low status remained significantly associated with shorter 3 years OS if adjusted on pT stage, pN stage, grade, lymphatic, venous, and perineural invasion, lymphocytic infiltrate, p16 status, p53 profile, and PD-L1 expression (Table 2). In the multivariate Cox regression, the following variables were negative prognostic factors independently from GATA3 status: pN stage, lymphatic, venous, and perineural invasion, lymphocytic infiltrate, and p53 profile (Table 2).
IMP3-high tumors displayed an insignificant trend towards worse OS in the univariate 5 years Cox regression (HR = 1.48, 95%CI = 0.89–2.44, p = 0.13). HIF-1-α-high tumors tended insignificantly to show better OS (HR = 0.75, 95%CI = 0.39–1.44, p = 0.4). CK7-high expression was associated with an insignificant trend towards shorter OS (HR = 1.46, 95%CI = 0.69–3.07, p = 0.3). CA-IX-high status showed an insignificant negative prognostic impact (HR = 1.53, 95%CI = 0.73–3.22, p = 0.3).
CSS analysis of GATA3
In Fine Gray regression, GATA3-low status showed insignificant trends towards worse survival in 3 years (HR = 1.85, 95%CI = 0.88–4.88, p = 0.1) and 5 years (HR = 1.82, 95%CI = 0.9–3.7, p = 0.094) follow-up, but significantly worse CSS in 10 years follow-up (HR = 2.04, 95%CI = 1.03–4.17, p = 0.039).
Logistic regression/Pearson’s Chi squared test – immunohistochemistry and clinical pathological variables
Complete list of associations between analyzed variables is presented in Supplementary 2 (Table S2).
GATA3-low tumors (negative prognostic feature) were significantly associated with locally advanced (pT3 + 4) tumor stage (Odds ratio/OR = 4.17, 95%CI = 1.54–12.5, p = 0.002), lymphatic invasion (OR = 2.17, 95%CI = 2.15–4.76, p = 0.031), non-brisk lymphocytic infiltrate (OR = 2.5, 95%CI = 2.5–5.56, p = 0.01), HIF-1-α-low status (OR = 3.42, 95%CI = 1.23–11.1, p = 0.01), and borderline insignificantly also with p53 mutated profile (OR = 2.17, 95%CI = 0.87–5.54, p = 0.087).
IMP3-high status was significantly more frequent in patients with regional lymph node metastases (OR = 4.16, 95%CI = 1.47–13.7, p = 0.004), borderline insignificantly more in G3 tumors (OR = 2.05, 95%CI = 0.96–4.50, p = 0.051), PD-L1 + tumors (OR = 2.11, 95%CI = 0.88–5.32, p = 0.075), and tumor with mutated p53-profiles (OR = 2.17, 95%CI = 0.88–5.88, p = 0.092).
HIF-1-α-high profile was significantly associated with beneficial prognostic parameters - low stage pT1 + 2 (OR = 0.14, 95%CI = 0–0.95.95, p = 0.028), low grade G1 + 2 (OR = 0.13, 95%CI = 0.01–0.56, p = 0.001), and borderline insignificantly with absent lymphatic (OR = 0.40, 95%CI = 0.11–1.19, p = 0.11) and absent venous (OR = 0.30 95%CI = 0.03–1.33, p = 0.11) invasion.
CK7-high status was significantly associated with G3 (OR = 6.06, 95%CI = 1.78–23.9, p = 0.001), p16 positivity (OR = 12.5, 95%CI = 1.88-∞, p = 0.002), CA-IX-high status (OR = 6.09, 95%CI = 1.52–23.2, p = 0.005), and p53 wild type status (HR = ∞, 95%CI = 0-∞, p = 0.042).
CA-IX-high status was significantly associated with G3 (OR = 4.9, 95%CI = 1.52–17.5, p = 0.004), lymphatic invasion (OR = 4.07, 95%CI = 1.24–14.7, p = 0.01), with regional lymph node metastases (OR = 4.14, 95%CI = 1.18–14.0.18.0, p = 0.013) and borderline insignificantly with perineural invasion (OR = 3.10, 95%CI = 0.63–12.6, p = 0.087), non-brisk lymphocytic infiltrate (OR = 2.58, 95%CI = 0.74–11.5, p = 0.12), and p16 positivity (OR = 2.7, 95%CI = 0.77–12.5, p = 0.12).
No further associations with p < 0.15 were found.
Logistic regression/Pearson’s Chi squared test – immunohistochemistry and NGS variables
Complete list of associations between analyzed variables is presented in Supplementary Tablet 2 (Table S2).
GATA3-low status was associated with mutations in NOTCH1 (OR = 0.45, 95%CI = 0.19–1.06, p = 0.048) and ATM (OR = 0.12, 95%CI = 0.01–0.60, p = 0.003). IMP3-low status has been linked to HRAS mutation (OR = 0.09, 95%CI = 0.00–0.66.00.66, p = 0.008). CK7-high status was associated with PIK3CA mutation (OR = 4.92, 95%CI = 1.40–18.6, p = 0.005) but with absent CDKN2A mutation (OR = 0, 95%CI = 0–0.87.87, p = 0.021). There were no significant associations between CA-IX, HIF-1-α, and genomic variables.
Discussion
This study is a retrospective analysis of several immunohistochemical markers on a relatively large cohort of patients with pSCC (n = 145), whose prognostic significance has not yet been described in this context. Although some markers showed borderline trends towards a worse prognosis, only the GATA3-low status reached a p-value below 5% in our survival analysis.
GATA3
GATA binding protein 3 (GATA3) is a zinc-finger transcription factor playing a key role in cell differentiation during development. In the epidermis, GATA3 suppresses cell proliferation and induces expression of differentiation markers13.
Over the past ten years, GATA3 immunohistochemistry has been widely used by pathologists to identify breast or urothelial origin of cancer14. Moreover, GATA3 is expressed in cutaneous adnexal tumors (including basal cell carcinoma), trophoblastic and endodermal sinus tumors13,15. Miettinen et al. performed an extensive study on 2500 various tumors in terms of nuclear GATA3 expression, regarding at least 5% of staining tumor cell nuclei positive. In SCC, there was a relatively frequent GATA3 expression: 81% of cutaneous SCC, with a median of 50% tumor cells positive (range, 5–100%), 33% cervical carcinomas (33% of cases, nearly half of them showing positivity in > 50% of tumor cells), 16% laryngeal and 12% pulmonary SCCs. Half of the GATA3-positive examples of the pulmonary tumors and 2 of 8 of the laryngeal ones showed positivity in > 50% of tumor cells15. Reportedly, the expression of GATA3 in SCCs varies by site, being frequent in skin tumors16 and less common in respiratory SCCs13,17. Loss of GATA3 has been described as a part of HPV-independent carcinogenesis towards SCC, as a helpful hallmark of differentiated (HPV-negative) vulvar intraepithelial lesion (dVIN) distinguishing this from HPV-associated dysplasia18, and as a correlate of sun induced skin dysplasia and cancer19. Prognostic significance of GATA3 expression varies among various tumors. GATA3 expression has been linked to poorer survival in lung adenocarcinoma20, pancreatic adenocarcinoma21, high grade serous ovarian carcinoma22, and soft tissue sarcomas23.
In our study on penile cancer, we used immunoreactivity score cut-off value 0.5 (mean from two cores and two pathologists), which counts almost any staining as positive, and there were 85/145 (58%) positive/high cases. The prognostic implications of GATA3 expression in SCC are equivocal. There was a negative prognostic impact of GATA3 expression in esophageal cancer24. On the other hand, knockdown of GATA3 stimulated proliferation and invasion in oral SCC cell cultures, whereas anticancer function of MEG3 was GATA3-dependent25. Vaziri Fard et al. described GATA3 loss in 40% of 86 vulvar SCC26, which is etiologically, morphologically, and prognostically similar to pSCC. In our data, there were 42% of GATA-low (negative) cases. In vulvar SCC, associations between GATA3 loss, HPV-independency, p53 abnormal profile and shorter OS were found. In line with this, our study showed negative prognostic implications of GATA3 loss in terms of shorter survival and association with advanced stage and lymphatic invasion. In vulvar pathology, GATA3 loss seems to be associated with dVIN-associated HPV-independent cancerogenesis, based on the findings of Vaziri Fard et al.26. As vulvar SCC is etiologically, morphologically, and prognostically similar to pSCC, analogous association of GATA3 expression with HPV/p16 positivity could be expected. Nevertheless, no association of GATA3 status with p16/HPV profile was found in our study.
IMP3
IMP3 is a member of the RNA-binding protein family, involved in RNA trafficking and stabilization, as well as cell growth and migration during the early stages of embryonic development27. Numerous studies have demonstrated that IMP3 is specifically expressed in malignant tumors and functions as an important cancer-specific gene involved in various aggressive and advanced cancers28. There is growing evidence that upregulated IMP3 expression in tumor tissues is correlated with poor clinical outcome and can serve as a prognostic factor. Chen et al. performed a robust meta-analysis comprising 53 studies on 8937 patients with a broad spectrum of malignant tumors. High IMP3 expression was significantly associated with shorter OS (HR = 2.08, 95%CI = 1.80–2.42, p < 0.001); the prognostic impact was found in the majority of tumor types except for ovarian cancer29.
Among SCC, there is an association of IMP3 positivity with epithelial dysplasia and carcinoma, together with negative impact on prognosis SCC of the uterine cervix, where the vast majority of dysplasias and carcinomas are HPV-associated30,31. Similarly, IMP3 expression signifies poor prognosis in esophageal carcinoma, which is typically HPV-independent32. Head and neck SCC (HNSCC) may arise in both HPV-associated and HPV-independent pathways, and IMP3 has been described as negative prognostic marker in several studies, together with IMP3-upregulation in squamous epithelium dysplasia33,34,35,36,37,38.
In the first study focusing on IMP3 in pSCC, IMP3-high status showed a borderline insignificant trend towards worse survival in a relatively large cohort. Although IMP3 did not reach statistical significance in the survival analysis, it showed associations with several adverse prognostic features such as lymph node metastases, high grade, and p53-mutated profile, whose negative prognostic role in pSCC has been well documented9,39,40,41,42,43,44, together with the role of p53 in epithelial-mesenchymal transition45 and its association with tumor budding46, both p53 and tumor budding being strong negative prognostic markers9. These findings suggest that IMP3 expression may reflect broader tumor aggressiveness but does not independently predict survival. Its potential role as a surrogate marker of aggressive tumor biology warrants further study.
HIF-1-α
Hypoxia-inducible factors (HIFs) are key regulators of oxygen homeostasis vital for each cell in the human body. Cancer cells utilize HIFs to drive malignant progression. In relation to HIF-1-α prognostic impact, abundant literature is available. Negative prognostic implications of HIF-1-α results were described in meta-analyses on SCC of the esophagus47,48, head and neck49,50, and uterine cervix51.
Notably, findings in our study on HIF-1-α are strikingly discordant with the cited literature. We observed an insignificant tendency to better OS in HIF-1-α-high tumors, and associations with low risk clinical-pathological variables (stage, grade, lymphovascular invasion). The plausible explanation could be that many cited studies lack information regarding HIF-1-α compartmentalization, making it unclear whether the antibody reaction is occurring in the cytoplasm or the nucleus. In uterine cervix SCC, which is etiologically comparable to pSCC, only nuclear HIF-1-α positivity has been associated with a worse prognosis51. This has not been observed in our samples. The role of cytoplasmic staining in SCC requires further investigation, but its prognostic value appears to be quite limited in pSCC.
CK7
CK7 is found in various ductal and glandular epithelia; most adenocarcinomas express CK7, except for those originating from the colon, prostate, kidney, thymus, carcinoid tumors, and Merkel cell tumors of the skin52. The anomalous expression of CK7 is linked to tumor progression and metastasis, whereas the mechanism is not yet clearly understood53. In several cancer types, CK7 (over)expression plays a prognostic role. Approximately 70% of urothelial carcinomas express CK754, the overexpression has been linked to advanced stage, poor OS, and shorter disease-free survival55. In adenocarcinomas, the negative prognostic implications of abnormal expression have been described in tubo-ovarian high-grade serous carcinoma56, pancreatic adenocarcinoma57, and colorectal carcinoma58,59.
Compared to adenocarcinomas, CK7 expression is less frequent in SCCs. In HNSCC, CK7 positivity has been found in 29%60, the authors described that CK7 expression is linked to HPV positive status, like we did in this study, but no prognostic impact of CK7 has been found in HNSCC61,62. In uterine cervix tumors, which are mostly HPV-dependent, CK7 expression has been described in 57–66% of invasive carcinomas and in 42–100% of dysplasias63,64, and the CK7 expression has been linked to low grade non-progressive dysplasia65 and favorable clinical course64. Hashiguchi et al. report potential negative prognostic impact of decreased CK7 expression in cervical SCC66. On the other hand, Lee et al. suggest CK7 functions as a protector of HPV-induced E7 transcript, thereby, CK7 might be essential for production of proteins for viral replication, and oncogenic transformation. The authors propose CK7 staining as a marker for predicting the physical status of HR HPV and E7 level in SCC and related dysplasias63.
We present the first description of CK7 prognostic implications in penile cancer, identifying only insignificant trends. In line with studies on cervical SCC, we identified tight associations of CK7 expression with HPV/p16 positivity, CA-IX overexpression, and high grade, whereas most HPV + SCCs with basaloid morphology are evaluated as grade 3. In line with this, there was a negative association between CK7-high status and p53 mutated profile, as the overwhelming majority of HPV/p16 + cases are p53 wild type9,11. Products of TP53 and CDKN2A are usually silenced by viral oncoproteins E6 and E7 in HPV-associated pSCCs, and these genes are usually wild type at DNA level67. Our result of logistic regression of CK7 and p53 with HR = ∞ shows the absence of CK7 + p53 mutated cases. Although in line with the known negative association of HPV and p53 and novel association of CK7 + with HPV/p16 positivity, the true effect size cannot be reliably determined and further studies in larger cohorts are warranted.
CA-IX
Carbonic anhydrases are metallo-enzymes involved in various pathophysiological processes that require tissue pH regulation. CA-IX, a tumor-associated isoform, is induced by hypoxia and aids tumor cells in adapting to acidosis. Multiple tumor-driving pathways can induce CA-IX expression, which is linked to cancer cell invasion, metastatic properties, stem-like characteristics, and drug resistance. High CA-IX expression has been described as a marker of poor prognosis in breast, lung, ovarian, and bladder carcinomas, but a low expression is a negative prognostic marker in clear cell renal cell carcinoma68. A meta-analysis including 147 studies with 24,523 patients suffering from various cancers reported that high CA-IX expression was associated with a worse OS (HR = 1.76, 95%CI = 1.58–1.98, p < 0.0001)69. Focusing on SCC, upregulated CA-IX has been linked to postoperative recurrence and poorer outcome in oral cavity70,71,72, tongue73, and sinonasal SCC74. To the best of our knowledge, only one study aiming to explore the prognostic role of CA-IX specifically in pSCC has been published prior to this article. Zhu et al. analyzed approximately half the number of cases (n = 73) compared to our study using an arbitrary cutoff of 10% of positively stained tumor cells. The authors found high expression in 42% cases. The expression was not associated with lymph node metastases, T stage, grade, lymphovascular invasion, Ki-67 expression, and cancer-specific survival, but was associated with p53 expression75. Unlike Zhu et al., our study found a lower number of CA-IX-high tumors (11.8%) due to the use of optimal cut-point value. CA-IX-high status was significantly associated with high grade and lymphatic invasion, and borderline significantly with p16/HPV positivity, whereas p16/HPV positivity, CK7-high and CA-IX high profiles were mutually associated. Nevertheless, like Zhu et al., our analysis failed to prove a significant impact on survival rendering just an insignificant trend towards worse OS.
Associations of immunohistochemistry profiles with NGS data
Comparing the immunohistochemistry marker results with previously published NGS data11, several associations have been found: GATA3-low status was associated with mutations in NOTCH1 and ATM. IMP3-low status was inversely associated with HRAS mutation. CK7-high status was positively associated with PIK3CA mutation but inversely associated with CDKN2A mutation (OR = 0, 95% CI = 0–0.87.87, p = 0.021), indicating absence of CDKN2A mutation in CK7-high cases.
Some of these associations are in line with other results of this study. As already mentioned, CK7 is almost exclusively expressed in HPV/p16 + SCCs, which are characterized by low prevalence of TP53 and CDKN2A mutations (canonical mutations in HPV-independent pSCC cancer pathway)11,67. CK7 is a hallmark marker of many adenocarcinomas including breast cancer, with 98% of breast carcinomas showing CK7 positivity. PIK3CA mutations are common in estrogen receptor-positive breast cancer (ca. 35%)76, particularly in luminal subtypes. Also, urothelial carcinoma, which is constitutively positive for CK777, displays relatively frequently PIK3CA mutation (8–25%)78, and CDKN2A mutation (10–12%)79. However, CK7-high status is not specifically correlated with PIK3CA and CDKN2A mutations in breast cancer and urothelial cancer studies, as CK7 is nearly universally expressed. Also, association of underexpressed IMP3 (correlate of aggressive phenotype in many cancers) and mutated HRAS (without prognostic significance in pSCC)11 has not been reported in the literature. ATM and NOTCH1 mutation occur in ca. 1–7% and 2% of breast cancers, respectively80,81,82, both alterations are enriched in triple negative breast cancer83,84, which is characterized by decreased or absent GATA3 expression85. Nevertheless, it is questionable to apply these findings on pSCC, and further studies are warranted.
Limitations
Although it is a cost-effective method, the use of TMAs may represent a potential limiting factor of our study, as the expression of immunohistochemistry markers may vary within the tumor and the area of expression may be potentially missed. However, the same issue concerns small diagnostic biopsy samples in routine practice. For statistical analysis, pNX (clinically cN0) cases were grouped with pN0, acknowledging this as a limitation. In view of the limited number of events and to minimize overfitting, we employed pairwise multivariable models including GATA3 and a single covariate; this approach supports robustness across key factors but does not replace a fully specified joint model adjusting all variables mutually. Larger cohorts are required to confirm GATA3 as a strictly independent prognostic factor.
Conclusion
This study evaluated the prognostic and clinicopathologic relevance of eight immunohistochemical markers in the largest penile cancer cohort to date. Among these, only GATA3 loss demonstrated a statistically significant and independent association with worse overall and cancer-specific survival, as well as correlations with adverse pathological features such as advanced tumor stage and lymphatic invasion. The other markers, including IMP3, CK7, and CA-IX, did not show significant prognostic impact but were associated with relevant tumor characteristics such as high grade, HPV/p16 status, and lymph node metastases, suggesting potential roles in tumor biology. While these findings contribute to the growing understanding of pSCC heterogeneity, they warrant further validation in independent cohorts and mechanistic studies before clinical application.
immunohistochemistry profiles of penile squamous cell carcinoma (pSCC), 200x.a: pSCC evaluated as GATA3-low (negative) by both pathologists, IMP3-high (intensity 3 in 85% and 100%), HIF-1-α negative, CK7-high (intensity 3 in 100% and 95%), CA-IX-high (2 in 60%, 3 in 60%).b: pSCC evaluated as GATA3-high by both pathologists (intensity 3 in 90% and 2 in 80%), IMP3 negative, HIF-1-α negative, CK7-high (3 in 100% and 95%), CA-IX-high (3 in 80% and 90%).c: pSCC evaluated as GATA3-high (intensity 2 in 60% by both pathologists), IMP3-low (zero and 1 in 10%), HIF-1-α-high (intensity 3 in 30% and 2 in 15%), CK7-low (2 in 5% and 1 in 30%), CA-IX-low (2 in 10% by both pathologists).d: pSCC evaluated as negative/low in all markers by both pathologists. The microphotographs from four selected cases illustrate the highly variable immunoreactivity with studied markers in pSCC.
Kaplan-Meier curves demonstrating the prognostic effects of six immunohistochemistry markers (GATA3, IMP3, HIF-1-α, CK7, CA-IX) on 5 year overall survival (OS) in penile squamous cell carcinoma (pSCC). Note the significant negative impact of GATA3-low status (a), insignificant trends towards worse prognosis in IMP3-high (b), HIF-1-α-low (c), CK7-high (d), and CA-IX-high (e) tumors.
Data availability
Research data are available in the Supplementary tables, full NGS data are available per email request from the corresponding author.
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Funding
This work was supported by the Ministry of Health of the Czech Republic (projects Conceptual Development of Research Organization, General University Hospital, Prague (VFN, 00064165) and Thomayer University Hospital (FTN, 00064190) and the Czech Health Research Council (Grant number NU21J-03-00019) and the Charles University program Cooperatio Medical Diagnostics.
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JHr contributed to the study design, funding management, cohort acquisition, immunohistochemistry and histopathological analysis, result interpretation, and manuscript writing. ZP was responsible for immunohistochemistry and histopathological analysis. RM was involved in study design, funding management, cohort acquisition, histopathological analysis, result interpretation, and manuscript writing. JHo contributed to the study design, funding management, NGS studies, and interpretation of results. MKB contributed to cohort acquisition and follow-up data acquisition. DČ and NZ were responsible for follow-up data acquisition. PW conducted statistical analysis and contributed to result interpretation. All authors read and approved the final version of the manuscript.
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Hrudka, J., Prouzová, Z., Matěj, R. et al. Low GATA3 predicts worse survival in penile cancer. Sci Rep 15, 40321 (2025). https://doi.org/10.1038/s41598-025-24166-6
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DOI: https://doi.org/10.1038/s41598-025-24166-6
