Abstract
Objective
In recent decades, studies have underscored nuclear proteins and signaling pathways in prostate cancer (PCa) development. Key biomarkers like Enhancer of zeste homolog 2 (EZH2) and Forkhead box M1 (FOXM1) are expressed in both healthy and malignant prostate cells. This study aimed to demonstrate the relationship between pathological characteristics, survival, recurrence, and tissue expression of EZH2 and FOXM1 in high-risk PCa patients.
Methods
PCa tissues were used in a retrospective analysis that spanned from September 2009 to August 2019. Inclusion criteria comprised pathological tumor stage (pT) 3 patients with positive surgical margins or tumor proximity to inked margins within 5 mm. After case selection, tissue slides were stained for EZH2 and FOXM1 antibodies, and Allred scores were calculated. Patients or relatives of deceased patients were contacted for signed agreements and disease follow-ups.
Results
The pT3b, ductal carcinoma component, and moderate EZH2 expression were associated with relapse (odds ratio [OR] 6.21, 95% confidence interval [CI] 1.41–27.27, p=0.016; OR 7.29, 95% CI 1.03–51.43, p=0.046; OR 5.96, 95% CI 1.09–32.48, p=0.039; respectively). The unilateral and bilateral seminal vesicle invasion increased the likelihood of recurrence by 9.98 times and 5.36 times, and the risk of death by around 9.78 times and 10.79 times, respectively. The pT3b was linked to higher death likelihood (OR 7.16, 95% CI 1.38–37.23, p=0.019), while moderate EZH2 expression did not show statistical significance (OR 4.54, 95% CI 0.87–23.60, p=0.072, marginally). Pathological regional lymph node stage (pN) 1 had significantly higher probability of mortality than pN unknown (3.9% vs. 27%, p<0.001). PCa in the neck and apex of the prostate gland increased death risk tenfold.
Conclusion
Sufficient immunoexpression of EZH2, ductal carcinoma component, and neoplastic proliferation in the seminal vesicles, apex and neck of the prostate gland correlates with elevated risks of recurrence and mortality. Clinicians should use these criteria for appropriate patient referrals, and a multicenter trial could provide accurate classifications.
Keywords: Death, Enhancer of zeste homolog 2, Forkhead box M1, Prostate cancer, Relapse
1. Introduction
In 2023, prostate cancer (PCa) is predicted to cause 34 700 fatalities and 288 300 new cases in the United States [1]. Clinical approaches to eliminate residual disease include radical prostatectomy (RP) combined with radiotherapy and/or androgen deprivation therapy [2]. A prostate-specific antigen level greater than 0.2 ng/mL during the follow-up is considered a biochemical relapse (BR), preceding symptoms by up to 5 years. BR can develop without histological confirmation or objective evidence of distant metastases or lymph node involvement. Positive surgical margin (PSM) is associated with a poorer prognosis; approximately 24% of RP patients reported PSM on their histology reports [3]. Men with PSM and/or pathological tumor stage (pT) 3 have improved survival and reduced BR or metastasis rates when radiotherapy is administered within 4 months of surgery [4]. Those with PSM have double the chance of experiencing BR compared to those with a negative margin [5]. Although the majority of PSM patients may not experience recurrence, the recurrence incidence is around 30%–35% [4,5]. Current biomarkers are not yet implemented in clinical settings, despite their prognostic and predictive value for PCa recurrence following or without radiotherapy [6]. Nuclear proteins and signaling pathways, including Enhancer of zeste homolog 2 (EZH2) and Forkhead box (FOX) M1 (FOXM1), play significant roles in the development of various cancers, including PCa [7,8]. Early identification of aggressive PCa and appropriate treatment are crucial to prevent disease progression.
EZH2, the catalytic subunit of Polycomb repressive complex 2 (PRC2), is overexpressed in human solid tumors including PCa, and its expression and activity are associated with disease progression [9]. Oncogenic features of EZH2 involve both PRC2-dependent and PRC2-independent actions [10,11]. The increased EZH2 expression is linked to poor prognosis in clinically localized or metastatic PCa [12]. EZH2 promotes PCa cell plasticity, aiding in the synthesis of biomarkers facilitating metastasis and antiandrogen resistance [13]. Consequently, EZH2 has become a target for cancer treatment, with small molecule inhibitors developed to block its enzymatic activity. The FOX superfamily, a conserved family of transcription factors, is critical for regulating various biological functions [14]. Among the FOX family, the FOXM1 transcription factor stands out, playing a significant role in embryonic development and associated with the initiation and progression of various cancer types [15]. FOXM1 presence leads to increased expression of DNA replication-related genes and becomes functionally active in tumor cells through transcriptional upregulation of FOXM1 mRNA and extracellular signal-regulated kinase-mediated phosphorylation of FOXM1 protein [16]. Overexpression of FOXM1 protein is associated with poor prognosis in human malignancies, promoting malignant characteristics and playing a role in cancer angiogenesis and invasion [17]. EZH2 and FOXM1 collaboratively enhance the expression of matrix metalloproteinase genes, promoting invasion, while their transcriptional consequences impact anti-apoptosis and cancer stemness [7,18].
This study optimized immunostaining for pathology and EZH2/FOXM1 expression in high-risk PCa patients with PSM and pT3. The aim was to explore the relevance of biomarkers to relapse and death in the Greek population. Primary findings suggested an EZH2-PCa relapse link, but not for overall survival. Relapse was defined as either distant or local metastases and/or BR. The pT3b, ductal carcinoma component, and moderate EZH2 expression were associated with relapse. Secondary findings showed a risk increase for pT3b versus pT3a in relapse or death. Ductal characteristics and apex and neck invasion can also raise the risk. Radiotherapy and biomarkers' role in relapse or death were assessed and will be detailed separately. Findings on these aspects will be presented separately due to the abundance of information and various interests. The study underscored EZH2 and FOXM1 as therapeutically relevant biomarkers for tailored therapy guidance.
2. Materials and methods
2.1. Patients
After obtaining the ethical approval (Ref. No 12626/30-08-09-19) from the Laiko Hospital of Athens, we conducted a retrospective cohort study on localized PCa patients diagnosed between September 2009 and August 2019 at the same hospital. Inclusion criteria encompassed localized PCa patients without metastasis and no prior cancer history. Specifically, PCa patients with pT3, pathological regional lymph node stage (pN) x/0/1, and PSM (recorded on medical reports as R+) or tumor margins within 5 mm of inked surfaces were selected. The study included 100 patients who underwent RP at the First Urology Clinic in the Laiko Hospital of Athens. Patient data, including age, postsurgical urinary issues, metastasis, family history, treatment, prostate-specific antigen-triggered hormone therapy initiation or change, and overall survival, were obtained from patients or their relatives. Pathological details such as tumor, node and metastasis (TNM) staging and Gleason score or Grade Group were sourced from electronic medical records at the First Department of Pathology, National and Kapodistrian University of Athens. Two pathologists independently reviewed hematoxylin and eosin-stained (H&E) slides to confirm tumor histology. For assessing extraprostatic extension, R+, or seminal vesicle invasion, at least three representative slides were chosen. The written informed consent was obtained from all enrolled patients or their relatives in the case of deceased patients via mail.
2.2. Immunohistochemical staining
Representative paraffin-embedded tissue blocks were selected for immunohistochemistry using commercially available antibodies for EZH2 and FOXM1 (anti-KMT6/EZH2 antibody [11/EZH2] and anti-FOXM1 antibody [EPR17379], both from Abcam, Cambridge, Cambridgeshire, the United Kingdom). Sections of archived paraffin blocks (5 μm thick) were cut and placed on coated slides. The slides were incubated overnight at 37 °C, followed by incubation at 60 °C for 30 min. Deparaffinization, hydration, and antigenicity exposure were performed using boiling at high pH (pH=9) in an automatic PT machine (PT Link, Dako North America, Inc., Via Real, CA, USA). After washing, the slides were treated with a 3% H2O2 solution to neutralize endogenous peroxidase. Incubation with rabbit monoclonal antibodies to FOXM1 (dilution 1:200) and mouse monoclonal antibodies to EZH2 (dilution 1:200) was carried out overnight at 4 °C. The slides were washed and incubated with a universal polymer reagent (horse anti-mouse/rabbit IgG) for 30 min at room temperature. The subsequent incubation with diaminobenzidine produced a noticeable brown color due to peroxidase polymerization. H&E staining was performed, followed by rinsing and incubation in a Tris-buffered saline solution. Dehydration was achieved using an increased alcohol series. The slides were cleared in xylene solution and underwent coating, observation, and evaluation by two pathologists under an optical microscope. Three slides for H&E staining, EZH2 and FOXM1 staining, were prepared for each patient. The ImmPRESS Polymer Kit from Vector Laboratories was used for identification.
2.3. Microscopic evaluation
Together with the other two independent pathologists, a third pathologist utilized the Allred score to visually assess the slides at magnifications ranging from 40× to 200×. This scoring system evaluated EZH2 and FOXM1 expression in carcinomas, considering the positive cell percentage and reaction product intensity. Malignancy is assigned an intensity score (0–3) denoting negative, mild, moderate, and strong staining. Tumors receive proportion ratings (0–5: no cells, ≤1%, 2%–10%, 11%–33%, 34%–66%, and 67%–100% of cells, respectively) based on the positive cell percentage. The Allred score, ranging 0–8, results from intensity and proportion factors. Scores categorize stained cells into negative (0–1), weak (2–3), moderate (4–6), and strong (7–8).
2.4. Statistical analysis
Quantitative variables were expressed as means with standard deviations, and medians and interquartile ranges, while categorical variables were shown as frequencies and percentages. The univariate analysis investigated the relationship between outcome variables (relapse: no vs. yes; death: no vs. yes) and demographic and clinical factors. For quantitative variables, the Student’s t-test or Mann-Whitney test was used, and for categorical variables, the Chi-square or Fisher exact test was used. Independent predictors of outcomes were identified through the multifactorial binary logistic regression using the enter technique, assigning odds ratios (ORs) and 95% confidence intervals (CIs) to model variables. Logistic regression assumptions were evaluated. A significance level of p<0.05 was used for all tests. The SPSS v21.00 (IBM Corporation, Armonk, NY, USA) was used to conduct analyses.
3. Results
Among our 100 patients, the median age was 66.0 (range 47.0–77.0) years, with a median follow-up of 7.62 years. Ten percent died, and 26% needed therapy for recurrence. The median survival time and recurrence time were 5.4 years and 2.6 years, respectively. Clinical and pathological data of the whole cohort are summarized in Table 1. Representative images of positive immunohistochemical staining for EZH2 and FOXM1 are shown in Figs. 1 and 2.
Table 1.
Clinical and pathological variables of patients (n=100).
| Variable | Value |
|---|---|
| Age, mean±SD (range), year | 66.0±6.6 (47.0–77.0) |
| Gleason score, mean±SD (range) | 8±1 (6–9) |
| Death, no/yes | 90 (90)/10 (10) |
| Relapse, no/yes | 74(74)/26 (26) |
| Radiotherapy, no/yes | 50(50)/50 (50) |
| Hormonotherapy, no/yes | 62 (62)/38 (38) |
| Secondary independent cancer, no/yes | 90 (90)/10 (10) |
| Heart disease, no/yes | 87 (87)/13 (13) |
| Diabetes mellitus, no/yes | 93 (93)/7 (7.0) |
| Family history, no/yes | 80 (80)/20 (20) |
| Grade Group (low/intermediate/high) | 6 (6.0)/60 (60)/34 (34) |
| High-grade prostatic intraepithelial neoplasia, no/yes | 37 (37)/63 (63) |
| Perineural invasion, no/yes | 23 (23)/77 (77) |
| Neoplastic vascular embolus, no/yes | 83 (83)/17 (17) |
| Neoplastic lymphatic embolus, no/yes | 89 (89)/11 (11) |
| Surgical margin, positive/negative | 87 (87)/13 (13) |
| pΤ3, a/b | 58 (58)/42 (42) |
| pΝ, unknown/negative/positive | 51 (51)/38 (38)/11 (11) |
| Extraprostatic extension, no/yes | |
| Bilateral | 46 (46)/54 (54) |
| Unilateral | 65 (65)/35 (35) |
| Seminal vesicle invasion, no/yes | |
| Bilateral | 77 (77)/23 (23) |
| Unilateral | 81 (81)/19 (19) |
| Apex or neck of the prostate gland, no/yes | 38 (38)/62 (62) |
| EZH2, negative/weak/moderate | 74 (74)/14 (14)/12 (12) |
| FOXM1, negative/weak/moderate | 62 (62)/8 (8)/30 (30) |
| Cancer type, acinar adenocarcinoma/ductal component | 91 (91)/9 (9.0) |
EZH2, Enhancer of zeste homolog 2; FOXM1, Forkhead box M1; SD, standard deviation; pΤ, pathological tumor stage; pN, pathological regional lymph node stage.
Note: all qualitative variables are presented as frequencies (percentages) except for ones specifically stated.
Figure 1.
The staining results performed on the same slide show a moderate Allred score of EZH2 staining. (A) Hematoxylin and eosin staining (40×); (B) Immunohistochemical staining of EZH2 showing moderately stained cells (200×, intensity of 2+ and 10% positivity, moderate Allred score); (C) Immunohistochemical staining of Forkhead box M1 showing weakly stained cells (40×, intensity of 1+ and 1% positivity, weak Allred score). EZH2, Enhancer of zeste homolog 2.
Figure 2.
The staining results performed on the same slide show a moderate Allred score of enhancer of FOXM1 staining. (A) Hematoxylin and eosin staining (40×); (B) Immunohistochemical staining for FOXM1 showing moderate positivity (100×, intensity 3+, 10% positivity, moderate Allred score); (C) Immunohistochemical staining for Enhancer of zeste homolog 2 showing weak positivity (40×, intensity 1+, 1% positivity, weak Allred score). FOXM1, Forkhead box M1.
The univariate analysis regarding relapse is summarized in Table 2 and shows significant differences between relapse and defined subgroups. Similarly, patients with pT3b and pNx staging had higher death risk in the univariate analysis, compared to pT3a (19% vs. 3.4%, p=0.016) and pN1 (3.9% vs. 27%, p<0.001), respectively (Supplementary Table 1).
Table 2.
Comparisons of patient and tumor characteristics between relapsed and non-relapsed patients.
| Variable | Relapse |
p-Value | |
|---|---|---|---|
| No (n=74) | Yes (n=26) | ||
| Age, mean±SD, year | 65.5±7.0 | 67.4±5.5 | 0.2a |
| Radiotherapy | 0.011 | ||
| No | 43 (86) | 7 (14) | |
| Yes | 31 (62) | 19 (38) | |
| Hormonotherapy | <0.001 | ||
| No | 59 (95) | 3 (4.8) | |
| Yes | 15 (39) | 23 (61) | |
| Secondary independent cancer | 0.7 | ||
| No | 67 (74) | 23 (26) | |
| Yes | 7 (70) | 3 (30) | |
| Heart disease | 1 | ||
| No | 64 (74) | 23 (26) | |
| Yes | 10 (77) | 3 (23) | |
| Diabetes mellitus | 0.4 | ||
| No | 70 (75) | 23 (25) | |
| Yes | 4 (57) | 3 (43) | |
| Family history associated with PCa | 1 | ||
| No | 59 (74) | 21 (26) | |
| Yes | 15 (75) | 5 (25) | |
| Grade Group | 0.017 | ||
| Low/intermediate | 54 (82) | 12 (18) | |
| High | 20 (59) | 14 (41) | |
| High-grade prostatic intraepithelial neoplasia | 0.2 | ||
| No | 30 (81) | 7 (19) | |
| Yes | 44 (70) | 19 (30) | |
| Perineural invasion | 0.17 | ||
| No | 20 (87) | 3 (13) | |
| Yes | 54 (70) | 23 (30) | |
| Neoplastic vascular embolus | 0.4 | ||
| No | 63 (76) | 20 (24) | |
| Yes | 11 (65) | 6 (35) | |
| Neoplastic lymphatic embolus | 0.032 | ||
| No | 69 (78) | 20 (22) | |
| Yes | 5 (45) | 6 (55) | |
| Surgical margin | 0.094 | ||
| Positive | 67 (77) | 20 (23) | |
| Negative | 7 (54) | 6 (46) | |
| Gleason score, mean±SD | 7.4±0.8 | 7.8±0.9 | 0.024 |
| pΤ3 | <0.001 | ||
| a | 52 (90) | 6 (10) | |
| b | 22 (52) | 20 (48) | |
| pΝ | 0.001 | ||
| Unknown | 46 (90) | 5 (9.8) | |
| 0 | 23 (61) | 15 (39) | |
| 1 | 5 (45) | 6 (55) | |
| Bilateral extraprostatic extension | 0.11 | ||
| No | 38 (83) | 8 (17) | |
| Yes | 36 (67) | 18 (33) | |
| Unilateral extraprostatic extension | 0.16 | ||
| No | 45 (69) | 20 (31) | |
| Yes | 29 (83) | 6 (17) | |
| Bilateral seminal vesicle invasion | 0.055 | ||
| No | 61 (79) | 16 (21) | |
| Yes | 13 (57) | 10 (44) | |
| Unilateral seminal vesicle invasion | 0.007 | ||
| No | 65 (80) | 16 (20) | |
| Yes | 9 (47) | 10 (53) | |
| Apex or neck of the prostate gland | 0.10 | ||
| No | 32 (84) | 6 (16) | |
| Yes | 42 (68) | 20 (32) | |
| EZH2 | 0.13 | ||
| Negative | 57 (77) | 17 (23) | |
| Weak | 11 (79) | 3 (21) | |
| Moderate | 6 (50) | 6 (50) | |
| FOXM1 | 0.8 | ||
| Negative | 47 (76) | 15 (24) | |
| Weak | 6 (75) | 2 (25) | |
| Moderate | 21 (70) | 9 (30) | |
| Cancer type | 0.2 | ||
| Acinar | 69 (76) | 22 (24) | |
| Ductal component | 5 (56) | 4 (44) | |
pT, pathological tumor stage; pN, pathological regional lymph node stage; PCa, prostate cancer; EZH2, Enhancer of zeste homolog 2; FOXM1, Forkhead box M1; SD, standard deviation.
Note: all qualitative variables are presented as frequencies (percentages) except for ones specifically stated.
The Student’s t-test.
We found that the only significant variable associated with relapse and death, in the multivariate analysis of significant pathological and clinical characteristics, was hormonotherapy. We were able to observe the following effects of the other variables in our analysis by excluding hormonotherapy due to medical and scientific reasons. For analyzing the remaining most impactful clinical and pathological variables associated with death, a multiple logistic regression model with the Forward logistic regression method was used, including variables with p-values under 0.2 from the univariate analysis. Patients with pT3b stage had a 7.16-fold increased risk of death compared to patients staged as pT3a (OR 7.16, 95% CI 1.38–37.23, p=0.019) and unfortunately, moderate EZH2 expression (OR 4.54, 95% CI 0.87–23.60, p=0.072, marginally) did not show statistically significant (Supplementary Table 2). Respectively, in the multivariate analysis (Table 3), pT3b stage was linked to 6.21 times higher relapse risk compared to pT3a stage. Additionally, prostate ductal carcinoma component was associated with 7.29-fold higher relapse likelihood than acinar adenocarcinoma, and moderate EZH2 expression correlated with 5.96 times higher relapse likelihood compared to negative and weak expression.
Table 3.
The multivariate analysis with the Forward logistic regression method of significant pathological and clinical characteristics in relation to relapse.
| Variable | Reference category | OR | 95% CI | p-Value |
|---|---|---|---|---|
| Radiotherapy (yes) | No | 1.75 | 0.49–6.21 | 0.4 |
| Grade Group (high) | Moderate | 0.63 | 0.15–2.71 | 0.5 |
| Surgical margin (positive) | Negative | 0.87 | 0.14–5.31 | 0.9 |
| pΤ3 (b) | a | 6.21 | 1.41–27.27 | 0.016 |
| pΝ | 0.2 | |||
| 0 | Unknown (x) | 2.95 | 0.70–12.33 | 0.14 |
| 1 | 5.04 | 0.66–38.54 | 0.12 | |
| EZH2 (moderate) | Negative and weak | 5.96 | 1.09–32.48 | 0.039 |
| FOXM1 (moderate) | Negative and weak | 0.79 | 0.22–2.83 | 0.7 |
| Cancer type (ductal component) | Acinar | 7.29 | 1.03–51.43 | 0.046 |
pT, pathological primary tumor stage; pN, pathological regional lymph node stage; EZH2, Enhancer of zeste homolog 2; FOXM1, Forkhead box M1; OR, odds ratio; CI, confidence interval.
Using the enter method, a multiple logistic regression model was employed to assess the influence of pT3 variables on death and relapse. According to Table 4, neoplastic proliferation in neck and apex of the prostate gland (OR 9.87, 95% CI 0.99–98.7, p=0.051, marginally), bilateral seminal vesicle invasion (OR 10.79, 95% CI 1.28–90.62, p=0.028), and unilateral seminal vesicle invasion (OR 9.78, 95% CI 1.40–68.25, p=0.021) correlated with increased death risk. Similarly, bilateral seminal vesicle invasion (OR 5.36, 95% CI 1.52–18.92, p=0.001) and unilateral seminal vesicle invasion (OR 9.98, 95% CI 2.76–36.08, p<0.001) were tied to higher relapse risk. Notably, no statistically significant difference was found in the association between relapse or death and the expression of EZH2 and FOXM1 among patients who tested positive for only one biomarker or were negative for both.
Table 4.
Pathology variables defining the pT3 and associated with relapse and death.
| Variable | Reference category | Relapse |
Death |
||||
|---|---|---|---|---|---|---|---|
| OR | 95% CI | p-Value | OR | 95% CI | p-Value | ||
| BEE | No | 1.81 | 0.31–10.55 | 0.5 | 0.56 | 0.05–6.64 | 0.6 |
| UEE | No | 1.09 | 0.15–7.87 | 0.9 | 2.51 | 0.19–32.71 | 0.5 |
| BSVI | No | 5.36 | 1.52–18.92 | 0.001 | 10.79 | 1.28–90.62 | 0.028 |
| USVI | No | 9.98 | 2.76–36.08 | <0.001 | 9.78 | 1.40–68.25 | 0.021 |
| Apex or neck of the prostate gland | No | 2.33 | 0.74–7.35 | 0.15 | 9.87 | 0.99–98.7 | 0.051 |
BEE, bilateral extraprostatic extension; UEE, unilateral extraprostatic extension; BSVI, bilateral seminal vesicle invasion; USVI, unilateral seminal vesicle invasion; OR, odds ratio; CI, confidence interval.
4. Discussion
This study aimed to explore the link between relapse and death in high-risk PCa patients following RP, specifically focusing on EZH2 and FOXM1 protein expression. Previous studies show their correlation, motivating us to assess them independently and together. With accessible protein antibodies, we could detect these markers affordably, pinpointing patients needing closer monitoring and personalized care. Emerging molecularly targeted therapies can benefit those who might benefit from innovative treatments. EZH2 overexpression suggests potential for EZH2 inhibitor therapy in relapse or mortality cases. Conversely, reduced EZH2 expression may favor immunotherapy initiation, according to Du et al.’s [19] study. Our study delves into the association between EZH2 and FOXM1 immunoreactivity and recurrence and death.
Both increased rates of relapse and mortality were associated with sufficient immunoexpression of EZH2, although the latter finding had only marginal statistical significance. In our analysis, 12% of patients exhibited moderate immunoexpression of EZH2, which was associated with a 5.96-fold higher risk of disease relapse and a 4.54-fold higher risk of mortality. The expression of EZH2 is associated with the methylation of DNA histones, which facilitates replication. Adequate expression of EZH2 is believed to promote the survival of cancer cells and accelerate the proliferation of cells in cancer clones. Studies have consistently demonstrated an association between EZH2 protein expression, including gene activation, and increased rates of relapse and mortality [12,[20], [21], [22]]. Notably, Huber et al. [23] found that the expression of EZH2 was not statistically significant in patients with a Gleason score of less than 7. Immunoexpression of EZH2 has the potential to be incorporated into routine clinical testing procedures, enabling the identification of patients, especially those with a Gleason score of higher than 7, who are at a greater risk of mortality and relapse. Additionally, 14% of patients exhibited weak immunoexpression of EZH2, suggesting that they may benefit from immunotherapy. This information can provide valuable insights for patient management.
According to the findings of our study, patients with acinar adenocarcinoma who also exhibited a prostate ductal carcinoma component in their pathological examination had a higher likelihood of relapse. These patients, who comprised 9% of our research sample and all had sporadic detection of ductal carcinoma component, have a relapse risk that is 7.29 times higher than that of patients whose microscopic investigations revealed acinar adenocarcinoma without evidence of a ductal component. Pure or component ductal carcinoma in PCa has been associated with a worse prognosis and a lower survival rate [[24], [25], [26]], which aligns with our findings. The presence of ductal PCa development may indicate enhanced caution in assessment of pT3 patients for a higher risk of relapse.
It is well-established that patients classified as pT3b have a poorer prognosis compared to those classified as pT3a [27,28], which is consistent with our study findings. Patients diagnosed with pT3b have a 6.21-fold increased risk of relapse and a 7.16-fold increased risk of death compared to patients staged as pT3a. Additionally, our study revealed that patients with involvement of the seminal vesicles, whether bilateral or unilateral, had a 10.79-fold and 9.78-fold higher risk of death, relatively. The presence of cancer in bilateral seminal vesicles increased the likelihood of recurrence by 5.36 times, while unilateral involvement increased it by about tenfold compared to patients without bilateral or unilateral extension. The differences in our findings regarding the likelihood of recurrence in pT3 patients based on the presence of cancer in the seminal vesicles, whether unilateral or bilateral, cannot be directly compared, as they pertain to separate groups of patients without extension in the studied anatomical structure. The identification of distinct tissue-like organization in various types of cancer has prompted a reevaluation of the clonal concept of tumor progression [29,30]. It is now believed that most, if not all, solid tumors contain a subset of cancer stem cells that are organized in a hierarchical manner [31]. Potentially, instead of viewing cancer cells as individual entities, it is important to emphasize their collective behavior as a cohesive unit during growth and spread. According to this perspective, cancers develop hierarchies of tumor-initiating cells that possess the ability to self-renew, divide indefinitely, and generate differentiated cells within the tumor mass. This process results in the generation of distinct cell states and contributes to the intratumoral heterogeneity observed in cancer [32]. Tumor-initiating cells are known to exhibit high metastatic potential and a slow-proliferating nature, both of which are associated with treatment resistance and relapse [33]. This viewpoint aligns with our findings, as we observed that patients with PCa detected in the apex and neck, despite non-statistical significance, and bilateral or unilateral seminal vesicle invasion had up to about tenfold higher risk of mortality compared to patients without such involvement.
Given the known impact of higher Gleason score and/or Grade Group on disease prognosis [34], it is reasonable to expect that patients who received radiotherapy and hormone therapy immediately after surgery, and who had a high Gleason score and were classified in the high or very high Grade Group (4 or 5), were more likely to relapse. This finding is consistent with previous research on the subject, which has shown that patients who underwent direct radiotherapy and hormone therapy were at a significant risk of relapse and death [35,36]. Patients with lymphatic tumor dissemination and those with involvement of local lymph nodes had a higher incidence of recurrence. Neoplastic lymphatic embolus and lymph node involvement (pN) have both been associated with disease progression and recurrence [34,37].
In our study, we did not find any association between FOXM1 expression and recurrence or death in patients diagnosed with pT3. Furthermore, none of the associations between FOXM1 and the other variables we evaluated yielded significant results. According to the study by Kim et al. [38], FOXM1 primarily influenced the prognosis of PCa by controlling benign prostatic hyperplasia, PCa stages (pT1–pT3), and castration-resistant PCa rather than specifically affecting a particular disease stage. In the study, they reported high expression of FOXM1 in 17.3% of pT3a cases and 21.2% of pT3b cases. In our study, the overall percentage of FOXM1 protein expression in pT3 was 30%. However, we were unable to establish a clear association between the percentage of FOXM1 protein expression and the stage of the disease. It is important to note that FOXM1 protein is involved in numerous signaling pathways, which could introduce a bias in our analysis [[15], [16], [17]]. Additionally, a study has demonstrated the ability of herbs such as Baicalin to deactivate FOXM1 [39]. It is possible that the Mediterranean diet followed by our cohort, in contrast to other studies, could be a contributing factor to the lack of correlation with FOXM1. Based on our findings, it is not feasible to recommend or implement routine clinical monitoring of patients based on FOXM1 protein expression. It is important to note that our study utilized patient archive data, and the fixation procedures employed may result in the loss of protein molecules including FOXM1 [40].
Limitations of our retrospective study include potential bias due to confounding variables, which was partially mitigated by a multiple variable analysis. A larger sample size would enhance reliability, but broader participation was restricted. Nevertheless, our study offers valuable insights despite these limitations. Utilizing archives can pose challenges to the accuracy of protein expression detection. Additionally, as only one ethnic group was studied nationally, generalizing findings to other ethnicities might be complex. Further research is necessary for validation.
5. Conclusion
Our study identified high-risk features in localized PCa patients linked to increased relapse and mortality risk. Physicians should consider factors like EZH2 expression, prostate component ductal carcinoma, and cancer spread to specific structures, alongside established criteria. A multicenter prospective study involving PCa patients might yield more accurate risk classifications and aid in personalized treatment decisions.
Author contributions
Study concept and design: Sotirios Koubardas, Harikleia Gakiopoulou, Christos Alamanis, Andreas C. Lazaris.
Data analysis: Evangelia Krikou, Ourania Mpatsi, Sotirios Koubardas.
Drafting of manuscript: Sotirios Koubardas, Dimitrios Goutas, Iliana Mani.
Critical revision of the manuscript: Harikleia Gakiopoulou, Christos Alamanis, Andreas C. Lazaris.
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
We would like to express our gratitude to all our colleagues at the First Department of Pathology and First Urology Clinic at National and Kapodistrian University of Athens, whose contributions were instrumental in completing this project. We also acknowledge the support provided by the National and Kapodistrian University of Athens.
Footnotes
Peer review under responsibility of Tongji University.
Supplementary data to this article can be found online at https://doi.org/10.1016/j.ajur.2024.09.007.
Appendix A. Supplementary data
The following is the Supplementary data to this article:
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