Immunohistochemical Analysis of Androgen Receptor Expression Predicts the Prognosis of Metastatic Castration‐Sensitive Prostate Cancer Patients Receiving Abiraterone Acetate

ABSTRACT

Background

The efficacy of abiraterone acetate varies among patients with high‐risk metastatic castration‐sensitive prostate cancer (mCSPC). Both androgen receptor (AR) and cytokeratin 18 (CK18) are markers of the luminal lineage of prostate cancer, and their expression levels have been suggested to affect the response to androgen deprivation therapy (ADT). This study aimed to predict the efficacy of abiraterone acetate in high‐risk mCSPC via immunohistochemical staining of biopsy specimens obtained at the time of prostate cancer diagnosis.

Methods

We retrospectively analyzed 44 patients treated with abiraterone acetate in combination with ADT. AR and CK18 expression in prostate biopsy specimens were assessed using immunohistochemical staining.

Results

AR and CK18 staining was not significantly associated with overall survival (OS). However, low AR staining was significantly associated with a shorter time to castration‐resistant prostate cancer (TTCRPC) compared with high AR staining (log‐rank test, p = 0.018). Similarly, low CK18 staining was significantly associated with a shorter TTCRPC compared with high CK18 staining (log‐rank test, p = 0.037).

Conclusions

Immunohistochemical analysis of AR or CK18 expression in biopsy specimens may serve as a predictive biomarker of high‐risk mCSPC treated with abiraterone acetate.

Trial Registration: None

1. Introduction

Metastatic castration‐sensitive prostate cancer (mCSPC) has a poor prognosis. The incidence of metastatic prostate cancer is increasing in the United States [1]. Several randomized studies have demonstrated that the combination of androgen receptor signaling inhibitors (ARSIs) with androgen deprivation therapy (ADT) improves progression‐free survival (PFS) and overall survival (OS) in patients with mCSPC [2, 3, 4, 5]. In the LATITUDE trial, abiraterone acetate plus prednisone with ADT significantly improved OS and radiographic PFS in high‐risk patients with newly diagnosed mCSPC. However, even with abiraterone acetate plus prednisone, approximately 43% of patients with high‐risk mCSPC progress to castration‐resistant prostate cancer (CRPC) [2]. Therefore, identifying biomarkers that predict response to ARSI is essential.

The prostate consists of a bilayer epithelium comprising luminal secretory and outer basal cells. Luminal secretory cells express androgen receptor (AR) and cytokeratin 18 (CK18), whereas basal cells express p63, CK5, and CK14 [6]. Using PAM50 clustering, prostate cancer has been classified into luminal A, luminal B, and basal subtypes. Both luminal A and luminal B subtypes showed increased expression of AR and CK18. ADT improves the prognosis of patients with luminal B subtype but not those with basal subtype [7]. In this study, we investigated the expression of AR and CK18 as luminal subtype markers using immunohistochemistry in patients with mCSPC and analyzed their association with abiraterone acetate response.

2. Materials and Methods

2.1. Biopsy Specimens

We retrospectively analyzed 44 high‐risk mCSPC cases classified according to the LATITUDE trial criteria who began combination therapy with ADT and abiraterone acetate between January 2014 and December 2020 at Kindai University Hospital and Kindai University Nara Hospital. Clinicopathological characteristics were obtained from medical records and follow‐up data. Sixteen‐point trans‐perineal prostate needle biopsy was performed at the time of initial prostate cancer diagnosis. A total of 16 core needle biopsy samples were obtained from the prostate. Four cores were obtained from the peripheral zone, three from the transition zone, and one from the anterior fibromuscular stroma in each of the right and left lobes, totaling 16 cores. We evaluated cores with the highest Gleason scores. When tumors were present in multiple cores with equivalent Gleason scores, up to three cores were evaluated.

This study was approved by the Institutional Review Board of Kindai University Faculty of Medicine (R02‐247) and was conducted in accordance with the principles of the Declaration of Helsinki. In this retrospective study, written informed consent was waived, and patients were informed of the opt‐out option via the hospital website.

2.2. Immunohistochemistry

Immunohistochemical staining was performed manually on 5‐μm sections of formalin‐fixed, paraffin‐embedded needle biopsy specimens mounted onto charged slides. Slides were deparaffinized, rehydrated, and subjected to antigen retrieval. The slides were steamed for 20 min in DAKO antigen retrieval solution (Agilent, catalog number S1699) and then cooled for 10 min. Endogenous peroxidase activity was blocked using 0.3% H2O2 in methanol (MeOH). The slides were blocked with the appropriate serum (goat serum for AR and horse serum for CK18) for 30 min and incubated with primary antibodies (AR [D6F11; Cell Signaling, USA] and CK18 [ab668; Abcam, UK]) overnight at 4°C. The antibody dilution for both AR and CK18 was set at 1:200. The slides were then incubated with secondary antibodies (rabbit for AR and mouse for CK18) for 30 min. Slides were washed and incubated with ABC reagent (VECTASTAIN Elite ABC Rabbit IgG Kit; Vector Laboratories, USA). Slides were developed using DAB (Invitrogen catalog #SK41) and counterstained with hematoxylin.

AR and CK18 expression in stained sections was determined by manual scoring performed by two researchers (M.N. and K.F.) who were blinded to sample identity. CK18 staining was assessed according to its cytoplasmic expression, and AR staining was assessed according to nuclear expression. Each marker was evaluated for staining intensity (negative [0], weak [1], moderate [2], or strong [3]) and distribution. Marker expression was quantified using the histochemical scoring (H‐score) method as follows: (3 × % of strongly stained cells) + (2 × % of moderately stained cells) + (1 × % of weakly stained cells) resulting in a score range of 0–300.

2.3. Statistical Analysis

Results are presented as medians (range). Comparisons between the two staining groups were performed using the Mann–Whitney U test and the χ ² test. Kaplan–Meier estimates and log‐rank tests were used to examine the relationship between parameters and patient survival. Multivariate analysis of patient age, immunohistochemical staining H‐score, and extension of disease (EOD) score as tumor burden indicators was performed using Cox proportional hazards analysis to predict OS and time to castration‐resistant prostate cancer (TTCRPC) and determine cutoff values for AR and CK18 [8]. The correlation between two variables was analyzed using the Spearman correlation test. All statistical analyses were conducted using JMP statistical software version 15 (SAS Institute Inc., Cary, NC, USA) and GraphPad Prism 9 (GraphPad Software, La Jolla, CA, USA) software. p‐values were two‐sided, with statistical significance set at p < 0.05. p‐values were two‐sided, with statistical significance set at p < 0.05.

3. Results

The patient characteristics are detailed in Table 1. The median age was 76 years (range, 53–88). The median prostate‐specific antigen (PSA) level upon prostate cancer diagnosis was 614.4 ng/mL (range, 4.6–11507.0). Gleason scores were 8 in 16 patients (36.4%), 9 in 20 patients (45.4%), and 10 in 8 patients (18.2%). Bone metastasis was found in 31 patients (70.5%), and bone and visceral metastasis in 13 patients (29.5%).

Table 1.

Patient characteristics.

n (%)	44
Age, median (years)	76 (53–88)
ECOG‐PS
0	27 (61.4)
1	17 (38.6)
PSA, median (ng/mL)	614.4 (4.6–11507.0)
Gleason score
8	16 (36.4)
9	20 (45.4)
10	8 (18.2)
Gleason pattern 5 involved	29 (65.9)
T2	7 (15.9)
T3	23 (52.3)
T4	14 (31.8)
Lymph node metastasis, n (%)
N0	12 (27.3)
N1	32 (72.7)
Distant metastasis, n (%)
M1a	0 (0)
M1b	31 (70.5)
M1c	13 (29.5)
Extent of disease, n (%)
0	4 (9.1)
1	13 (29.5)
2	14 (31.8)
3	5 (11.4)
4	8 (18.2)

Immunohistochemical analysis showed that AR was expressed in the nuclei of prostate cancer cells, and CK18 was primarily expressed in the cytoplasm of prostate cancer cells (Figure 1). The 5‐year CRPC‐free survival rate was 48.3% in the overall cohort. The median TTCRPC time was 55 months (Figure 2A). The 5‐year OS rate was 72.4% in the overall cohort. The median OS time was not reached (Figure 2B). High‐risk patients with mCSPC treated with abiraterone acetate were divided into two groups based on high and low AR staining, and based on high and low CK18 staining. The cutoff for AR H‐score was 150 and the cutoff for CK18 H‐score was 110, as identified using the Cox proportional hazards model for TTCRPC. Low AR staining was significantly associated with a shorter TTCRPC compared with high AR staining (log‐rank test, p = 0.018) (Figure 2C). Similarly, low CK18 staining was significantly associated with a shorter TTCRPC compared with high CK18 staining (log‐rank test, p = 0.037) (Figure 2E). However, AR and CK18 staining was not significantly associated with OS (Figure 2D,F). Supporting Information S1: Figure 1 shows the TTCPRC stratified by AR and CK18 expression. There were 12 cases with low AR expression, among which 4 cases had high CK18 expression, and 8 cases had low CK18 expression. No significant differences were observed between the AR‐Low/CK18‐High and AR‐Low/CK18‐Low groups. Eight patients with low AR and CK18 levels had a median PSA level of 747.6 ng/mL (range: 9.7–1529), with Gleason scores of 8 in 3, 9 in 3, and 10 in 2 cases. The median LDH level was 244 IU/L (range: 137–1865), and the median ALP level was 242 IU/L (range: 84–1532). Additionally, histological analysis revealed no evidence of neuroendocrine differentiation. Supporting Information S1: Table 1 details the differences in patient background between the high and low AR H‐score groups. There were no significant differences between the two groups. These 8 cases with low AR and CK18 levels were not considered to have neuroendocrine features.

Figure 1.

Immunohistochemical analysis of AR and CK18 expression of prostate cancer. Representative photomicrographs of formalin‐fixed paraffin‐embedded needle core biopsy specimens immunostained for AR and CK18. AR and CK18 are expressed in the nuclei and cytoplasm, respectively. Panels show differences in the staining patterns of AR and CK18 in prostate cancer cells ranging from negative to high. The scale bar represents 100 μm. AR, androgen receptor; CK18, cytokeratin 18. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2.

Kaplan–Meier curve analysis of patients with metastatic castration‐sensitive prostate cancer receiving abiraterone acetate. Kaplan–Meier plots of time to CRPC (A) and overall survival (B) for all patients. Kaplan–Meier plots showing time to CRPC (C) and overall survival (D) according to AR expression. Kaplan–Meier plots showing time to CRPC (E) and overall survival (F) according to CK18 expression. CRPC, castration‐resistant prostate cancer. AR, androgen receptor; CK18, cytokeratin18.

Table 2 shows the univariate and multivariate Cox proportional hazards analyses for TTCRPC. In the univariate analysis, the following factors showed significant differences: age at diagnosis (HR, 1.088; 95% CI: 1.017–1.163; p = 0.013), EOD score > 2 (HR, 3.291; 95% CI: 1.175–9.215; p = 0.023), AR H‐score (HR, 2.777; 95% CI: 1.143–6.745; p = 0.024), and CK18 H‐score (HR, 2.522; 95% CI: 1.020–6.233; p = 0.045). Eastern Cooperative Oncology Group‐Performance Status (ECOG‐PS) (HR, 2.047; 95% CI: 0.8456–4.956; p = 0.112), PSA level at diagnosis (HR, 1.000; 95% CI: 0.999–1.000; p = 0.739), and presence of Gleason Pattern 5 (HR, 1.852; 95% CI: 0.715–4.800; p = 0.204) did not show significant differences. As shown in Supporting Information S1: Figure 2, the AR and CK18 H‐scores were strongly correlated. Additionally, a higher AR or CK18 staining intensity was associated with better prognosis (Supporting Information S1: Figure 3). In the multivariate analysis, EOD score > 2 (HR, 3.214; 95% CI: 1.180–9.240; p = 0.030) and AR H‐score (HR, 3.658; 95% CI: 1.422–9.411; p = 0.007) showed significant differences. The relationship between AR and CK18 H‐scores and PSA levels at diagnosis is shown in Supporting Information S1: Figure 4. No correlations were found between AR and PSA or between CK18 and PSA (Spearman correlation test, p = 0.825 and p = 0.244, respectively). In the analysis of OS using the Cox proportional hazards model, none of the factors showed significant differences. Similarly, none of the factors showed significant differences in multivariate analysis (Supporting Information S1: Table 2).

Table 2.

The univariate and multivariate Cox proportional hazards analyses for TTCRPC prediction.

Variables	Univariate analysis			Multivariate analysis
	HR	95% CI	p value	HR	95% CI	p value
Age (years)	1.088	1.017, 1.163	0.013	1.076	0.998, 1.160	0.054
ECOG‐PS ≥ 1 vs. 0	2.047	0.845, 4.956	0.112
PSA, median (ng/ml)	1.000	0.999, 1.000	0.739
Gleason pattern 5 present	1.852	0.715, 4.800	0.204
EOD ≥ 2 vs. ≤ 1	3.291	1.175, 9.215	0.023	3.214	1.118, 9.240	0.030
AR H‐score (> 150 vs. ≤ 150)	2.777	1.143, 6.745	0.024	3.658	1.422, 9.411	0.007
CK18 H‐score (> 110 vs. ≤ 110)	2.522	1.020, 6.234	0.045

Abbreviation: TTCRPC, Time to castration‐resistant prostate cancer.

4. Discussion

ARSIs such as abiraterone acetate, enzalutamide, and apalutamide improve OS and progression‐free survival (PFS) in patients with mCSPC [2, 3, 4, 5]. Abiraterone is an inhibitor of CYP17A1 that plays an important role in de novo androgen biosynthesis in the adrenal gland and tumors. Abiraterone blocks prostate cancer progression more effectively than conventional hormone therapy. In the LATITUDE trial, the abiraterone acetate treatment group for mCSPC extended PFS from 14.7 months in the placebo group to 33.1 months [2]. A retrospective propensity score‐matched analysis also showed a 2‐year PFS of 76.1% with abiraterone acetate plus prednisone (AAP) with ADT, compared to 38.6% with combined androgen blockade therapy [9]. However, despite ARSI treatment, one‐third of patients progress to CRPC due to PSA recurrence or radiographic progression within 2 years. For patients who progress to CRPC early, more effective and potent treatment strategies, such as triplet therapy, including docetaxel, are desired. One factor influencing the efficacy of abiraterone acetate on OS in high‐risk patients with mCSPC was the presence of Gleason Pattern 5 in the biopsy specimens [10]. In this study, high‐risk mCSPC patients with high AR expression in biopsy specimens had a longer time to CRPC than those with low AR expression.

The prostate epithelium comprises a bilayer of luminal secretory cells and an outer basal layer. Wang et al. demonstrated that prostate cancer originates from the luminal secretory cell layer using markers such as AR and CK18 (= KRT18) (markers for the secretory cell layer) and p63, CK5, and CK14 (markers for the basal cell layer) [6]. Lu et al. suggested that both basal and luminal cell populations could serve as the origins of prostate cancer, with different potentials for aggressiveness [11]. Basal cells exhibit higher expression of transcription factors that induce epithelial‐mesenchymal transition and possess stem/progenitor cell characteristics than luminal cells [12].

Zhao et al. reported increased expression of the luminal markers AR, CK18, and NKX3.1 in both luminal A and luminal B subtypes in whole‐mount prostatectomy specimens [7]. Using PAM50 clustering, they classified prostate cancer into three subtypes: luminal A (hormone receptor‐positive with low Ki‐67 expression), luminal B (hormone receptor‐positive with high Ki‐67 expression), and basal (hormone receptor‐negative, high Ki‐67 expression with high expression of basal cell markers CK5/6, EGFR, p63), thereby elucidating clinical and genetic differences. They reported improved prognosis for luminal B subtype after postoperative ADT. Although luminal B and basal subtypes have similar proliferation scores, the proliferation score alone does not correlate with ADT treatment response.

Nanda et al. demonstrated that cytoplasmic AR is an independent predictor of biochemical recurrence, regardless of Gleason score, in immunohistochemical staining of tissue microarrays of normal prostate cells, prostate cancer, and CRPC [13]. In our study, patients with high AR expression, regardless of the presence of Gleason Pattern 5, showed extended TTCRPC after ADT plus abiraterone acetate compared with those with low AR expression. Conversely, CK18 expression was not correlated with TTCRPC. Although both are markers of luminal cells, AR, which directly participates in the hormonal treatment of prostate cancer, appears to be more strongly associated with progression. Hamid et al. reported that luminal B type prostate cancer improved OS with ADT plus docetaxel compared with ADT monotherapy, whereas the basal type cancer did not show OS improvement with ADT plus docetaxel compared with ADT monotherapy [14].

Our findings suggest that ARSI might benefit prostate cancer patients with high AR expression. However, for patients with low AR expression, ADT plus abiraterone acetate may be insufficient, warranting consideration of ADT plus docetaxel or ADT plus ARSI plus docetaxel. ADT plus docetaxel improved both OS and TTCRPC regardless of AR expression levels compared to ADT monotherapy. Patients with low AR expression may rapidly progress to CRPC with ARSI alone, indicating that initial treatment may be inadequate.

The limitations of this study include the small sample size of 44 cases. The small sample size of this study may have influenced the results, potentially limiting the ability to accurately predict the prognosis of patients treated with abiraterone acetate. Furthermore, this study did not clearly classify luminal A, luminal B, and basal subtypes using PAM50 clustering. In this study, both AR and CK18 were associated with prognosis, and AR and CK18 exhibited a strong correlation. Prostate cancer cells with low AR and low CK18 staining may represent a basal subtype. In contrast, some patients had a discrepancy in AR and CK18 expression (e.g., low AR and high CK18, or high AR and low CK18). Santamaria et al. demonstrated the heterogeneity of CK18 expression in normal prostate and prostate cancer cells [15]. The cutoff values for AR and CK18 expression might affect the stratification into luminal or basal subtypes. Our classification based on high or low AR expression via immunohistochemistry was simple, inexpensive, and allowed for quick determination.

5. Conclusion

Immunohistochemical staining of AR or CK18 in biopsy specimens identified patients with mCSPC who would benefit from abiraterone acetate and ADT. Because mCSPC patients with low AR expression progress to mCRPC earlier than those with high AR expression, mCSPC patients with low AR expression might have benefit from docetaxel or docetaxel plus ARSI.

Disclosure

The authors have nothing to report.

Ethics Statement

This study was approved by the Institutional Review Board of Kindai University Faculty of Medicine (R02‐247) and was conducted in accordance with the principles of the Declaration of Helsinki.

Consent

In this retrospective study, written informed consent was waived, and patients were informed of the opt‐out option via the hospital website.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting information.

Data Availability Statement

The authors have nothing to report.