Abstract
Background/Aim: For prostate cancer, positive surgical margins are considered an important predictor of biochemical recurrence. However, biochemical recurrence is observed in approximately 20% of cases, even with negative surgical margins, and some cases require salvage therapy. The elevated expression of low-molecular-weight protein tyrosine phosphatase (LMW-PTP, MW 18 kDa) is associated with a poor prognosis of certain cancers. In this study, we investigated whether the LMW-PTP expression levels could be used as a biomarker of recurrence in prostate cancer with negative surgical margins. Materials and Methods: The subjects of this retrospective study were 119 patients who underwent total prostatectomy with negative resection margins. LMW-PTP expression was categorized either as a high-expression group or as a low-expression group bye two pathologists. Subsequently, we examined the relationship between LMW-PTP expression levels and clinicopathological factors including biochemical recurrence. Results: Evaluation of the immunostained samples by two pathologists was highly reliable, with an Intraclass correlation (ICC) score for two distinct measurements of 0.77 and 0.98, respectively. Seventy-three patients (61.3%) were placed in the LMW-PTP high expression group; and 46 patients (38.7%) were placed in the low expression group. The log-rank test revealed early biochemical recurrence in the high LMW-PTP expression group (p=0.0001). In addition, pathological T stage (p=0.004), lymphatic invasion (p=0.0456), Ki-67 labeling index (p=0.0002), and biochemical recurrence (p<0.0001) were more frequently identified in the LMW-PTP high expression group. Furthermore, multivariate analyses revealed that a high LMW-PTP expression level was an independent prognostic factor for biochemical recurrence (HR=3.14, 95% CI=1.37-8.07, p=0.0057). In addition, Ki-67 labeling indices were significantly higher in the high-expression group compared to the low-expression group (p<0.0001). Conclusion: LMW-PTP can be assessed using a single immunostaining protocol in a highly reproducible fashion. Tt may, thus, be applied clinically to establish the required postoperative follow-up period and determine the necessity for salvage therapy in cases of prostate cancer with negative surgical margins. LMW-PTP has the potential to be a highly useful prognostic biomarker and a therapeutic target in conjunction with other factors, such as the Gleason Score, the pathological T stage and the PSA level.
- LMW-PTP
- prostate cancer
- protein phosphatase
- biomarker
- biochemical recurrence
Prostate cancer is the most common type of cancer in men in developed countries. In the United States, one in five new cases of diagnosed cancer is in the prostate, while in Japan, the increasingly widespread use of prostate-specific antigen (PSA) testing has led to prostate cancer becoming the most prevalent type of cancer in men in 2016 (1). Treatment options for localized prostate cancer include radical prostatectomy (2), radiotherapy (external & internal radiation) (3), and active surveillance (4). However, changes in surgical procedures (from open surgery to endoscopic/robot-assisted surgery) have led to an increase in the number of patients undergoing prostatectomy as a radical therapy (5).
When surgery is selected to treat localized prostate cancer, the prognosis is generally good. However, the postoperative biochemical recurrence is happening for a 16-31% in 5 years and for a 25-53% in 10 years (6, 7). A portion of these cases develop castrate-resistant prostate cancer following clinical recurrence, which often results in an unfortunate outcome (8). Thus, biochemical recurrence is one of the factors used to justify the use of salvage therapies such as endocrine therapy and radiotherapy. Several studies have identified a variety of predictive factors for a biochemical recurrence (9-12). Significant factors include a designation of positive surgical margins (RM1)(13), a preoperative PSA score (10, 11), a Gleason score at prostatectomy, and pathological staging. Of these, positive surgical margins (RM1) is the most important predictive factor for the biochemical recurrence (recurrence rate: 1.5-6.0x) (14). Even when negative surgical margins (RM0) are identified, which occurs in a large percentage of all patients, biochemical recurrence subsequently develops in approximately 20% of such cases, and these patients often require salvage therapy (15). Despite this, there are few reports presenting effective biomarkers that can be used to predict such cases. With the widespread use of PSA screening, prostate cancer is discovered at an earlier stage and thus therapeutic intervention is performed sooner (16). This has led to an increase in the percentage of cases with negative surgical margins (62-82%) (17). From all these, it is extremely important to identify a prognostic biomarker in cases of negative surgical margins, which comprise most of localized prostate cancer cases.
Low-molecular-weight protein tyrosine phosphatase (LMW-PTP) is an 18-kDa enzyme that is encoded by ACP1 located on chromosome 2p25. It is normally expressed in a variety of human tissues and controls cell proliferation by dephosphorylating, and thus deactivating, a variety of tyrosine kinase receptors and docking proteins (18).
In recent years, LMW-PTP has also been found to be involved in cancer with related changes in expression. In colon cancer and neuroblastoma increased total LMW-PTP mRNA expression correlates with unfavorable outcome, while there was no correlation in lung cancer (19). Thus, it is considered an important factor involved with cancer progression. Protein phosphatases are generally involved in the regulation of intracellular signal transmissions, cell proliferation and cell differentiation, by dephosphorylating target proteins as a counterpart to protein kinases, and through this process they supposedly work towards suppressing tumor growth and development. However, in recent years it has been reported that some protein phosphatases promote tumor proliferation in a number of cancers (20). LMW-PTP is one of these protein phosphatases. Increased tumor expression of LMW-PTP in cancers such as colorectal cancer, breast cancer, and neuroblastoma has been reported to lead to poor prognosis (19, 21, 22). Additionally, it was recently reported that high expression of LMW-PTP is a factor related to poor prognosis in prostate cancer as well, suggesting that it may be an effective prognostic prediction factor (23, 24). Unfortunately, there have been no investigations on whether a high expression of LMW-PTP can be utilized as a biomarker to predict recurrence in cases of localized prostate cancer, and particularly, in cases of post-prostatectomy with negative surgical margins (RM0). In this study we evaluated LMW-PTP expression in cases of prostate cancer with negative surgical margins who had undergone surgical resection. We also investigated the feasibility of using serum PSA value in conjunction with Gleason score and various histopathological factors, as a predictive factor of biochemical recurrence.
Patients and Methods
This study was approved by the Research Ethics Committee of the Kurume University, which conforms to the guidelines of the Declaration of Helsinki (Number 17260).
Patients and tissue samples. The subjects of this retrospective study were 119 cases with negative surgical margins who were selected from 241 cases that underwent prostatectomy at the Kurume University Hospital (Kurume, Japan) between January 2007 and December 2011. As part of this study, the pathological diagnoses of the subjects were re-examined. Histopathological evaluations were performed by three pathologists (H.K., R.K. and H.Y.). Pathological diagnosis was performed according to the 2016 WHO Classification of Tumors of the Urinary System and Male Genital Organs (25). All patients were diagnosed with prostatic adenocarcinoma. The following were exclusion criteria: i) hormonal therapy and/or radiotherapy before surgery, ii) unclear resection margins (RM) when the pathological diagnosis was re-examined, iii) stage pT0 during surgery, and iv) inadequate specimen for tissue microarray (TMA).
Immunohistochemical analysis. Specimens for TMA were stained from 119 prostate cancer patients. Immunostaining of 2 TMA samples per patient was performed to resolve the high degree of heterogeneity of prostate cancer. Paraffin-embedded tissue samples were cut to a thickness of 4 μm, examined on a coated glass-slide, and were labeled with anti-LMW-PTP (sc-100343, 1:100; Santa Cruz Biotechnologies, Dallas Tx, USA) and anti-Ki-67 antibodies (NCL-Ki67-MM1; dilution 1:200; Leica Biosystems, Nussloch, Germany) using a BenchMark ULTRA (Ventana Automated Systems, Inc., Tucson, AZ, USA). Briefly, the slides were heat-treated using Ventana's ULTRA cell conditioning 1 retrieval solution (CC1; Ventana Automated Systems, Inc.) for 36 min at 95°C and were incubated with anti-LMW-PTP and anti-Ki-67 antibodies for 32 min at 37°C. An automated system with a Ventana UltraVIEW 3,3’-diaminobenzidine (DAB) detection kit was used. We considered only the cytoplasmic expression of LMW-PTP as positive, and only the nuclear expression of Ki-67 as positive. Representative IHC microphotographs of LMW-PTP staining are shown in Figure 1. Expression was classified as: i) 0, when very low or with no staining, ii) 1, when intensity was just above background level, iii) 2, when clearly visible at a low magnification, and iv) 3, when striking at a low magnification. All immunohistochemical analyses were evaluated by two experienced pathologists who were unaware of the patients' clinical condition. The scores of the two pathologists were added, and each patient was given a total expression score consisting of 13 levels from 0 to 12.
Statistical analysis. We examined the association between LMW-PTP expression and clinicopathological characteristics, such as: i) age at diagnosis, ii) serum PSA level at diagnosis, iii) D'Amico risk stratification, iv) Gleason Score at RP, v) pathological T stage, vi) lymphatic invasion, vii) peripheral nerve invasion, viii) Ki-67 LI, and ix) biochemical recurrence, using the χ2 test or Fisher's exact test. Cancer survival analysis was performed using the Kaplan-Meier method, log-rank test, and Cox's proportional hazards model. The threshold for statistical significance was set at p<0.05. Biochemical recurrence was defined as an increase in PSA level over 0.2 ng/ml after two different measurements at least 3 months apart. Finally, because cell proliferation affects the relationship between LMW-PTP expression and prognosis, we used Spearman rank correlation testing to investigate the correlation between Ki-67 LI, which is an index of cell proliferation, and LMW-PTP. The statistical software used was JMP® Pro 13 (SAS Institute Inc., Cary, NC, USA).
Results
Patients' characteristics. The clinicopathological characteristics of 119 patients are summarized in Table I. The median postoperative follow-up period was 72 months (range, 39-84). The median age was 67 years (range, 53-77) and median PSA level at initial diagnosis was 6.44 ng/ml (range, 2.13-62.34). D'Amico risk stratification was low in 31 patients (26.0%), intermediate in 56 patients (47.1%), and high in 32 patients (26.9%). Gleason Scores at prostatectomy were ≤6 in 20 patients (16.8%), 3+4=7 in 49 patients (41.2%), 4+3=7 in 38 patients (31.9%), and ≥8 in 12 patients (10.1%). Pathological stages were T2a, b in 22 patients (18.6%), T2c in 77 patients (65.3%), and T3 in 19 patients (16.1%). The numbers of patients with lymphatic and peripheral nerve invasion were 4 (3.4%) and 41 (34.5%), respectively. Overall, 44 patients (37.0%) experienced biochemical recurrence.
Immunohistochemical analysis of LMW-PTP expression. The distribution of LMW-PTP expression score is shown in Figure 2. The evaluation of immunostained samples by the two pathologists was highly reliable and reproducible, with an intraclass correlation coefficient (ICC) for two distinct measurements of 0.77 and 0.98 (excellent), respectively. By constructing a receiver-operating characteristic curve, LMW-PTP expression was categorized into either a high expression group (score: ≥5) or a low expression group (score: ≤4). Of the 119 patients, 73 (61.3%) were placed in the LMW-PTP high expression group and 46 (38.7%) were placed in the low LMW-PTP expression group.
Association between LMW-PTP expression and clinicopathological characteristics. The association between LMW-PTP expression and clinicopathological characteristics are summarized in Table II. Pathological T stage (p=0.004), lymphatic invasion (p=0.0456), Ki-67 LI (p=0.0002), and biochemical recurrence (p<0.0001) were more frequently identified in the LMW-PTP high expression group. However, there was no association between LMW-PTP expression and the Gleason Score (p=0.3334).
Identification of LMW-PTP as a biomarker of prostatic biochemical recurrence. The time to biochemical recurrence was significantly shorter in the high LMW-PTP expression group compared to the low LMW-PTP expression group (p=0.0001) (Figure 3). Univariate analysis for the time to biochemical recurrence revealed that: i) LMW-PTP expression (high vs. low; HR=4.20, 95% CI=1.98-10.3, p<0.0001), ii) pathological T stage (T2a,b vs. T2c; HR=1.23, 95% CI=0.54-3.14: T2a,b vs. T3; HR=5.40, 95% CI=2.15-14.9, p=0.0002) and iii) Ki-67 LI (<1% vs. 1-5%; HR=1.06, 95% CI=0.43-2.28: <1% vs. > 5%; HR=3.77, 95% CI=1.41-8.56, p=0.0388) were all significant predictors for biochemical recurrence (Table III). Multivariate analysis demonstrated that pathological T stage (T2a,b vs. T2c; HR=1.02, 95% CI=0.43-2.75: T2a,b vs. T3; HR=2.87, 95% CI=1.05-8.54, p=0.021) as well as the LMW-PTP expression levels (high vs. low; HR=3.14, 95% CI=1.37-8.07, p=0.0057) were independent poor prognostic factors for biochemical recurrence (Table III).
Correlation between LMW-PTP expression and Ki-67 index. Spearman's ranked correlation analysis revealed a coefficient of 0.443 (p<0.0001) as the standard of significance, indicating a strong positive correlation between LMW-PTP and Ki-67 expression.
Discussion
The resection margin status is an important prognosis predictor following prostatectomy (14). However, although factors for negative surgical margins, such as the Gleason Score, the pathological T stage, the preoperative PSA level, and the PSA mass ratio have been reported to be prognostic, there is as yet no consensus on their usefulness (26, 27). In this study, we evaluated the LMW-PTP expression levels in biopsies from prostate cancer with negative surgical margins and assessed the feasibility of using the results as a predictive factor with respect to biochemical recurrence. The results of this study are the first to suggest that patients in the LMW-PTP high-expression group exhibit a poor prognosis compared to those in the low-expression group, and the results of univariate and multivariate analyses also suggest that high LMW-PTP expression may be a useful prognostic factor for biochemical recurrence in case of negative surgical margins. The Ki-67 LI in the LMW-PTP high-expression group was significantly higher compared to the low-expression group, and our results indicate that LMW-PTP is related to cells' proliferative ability. In addition, based on the ICC, the immunostaining assessments of LMW-PTP conducted by two pathologists had a high degree of reliability and reproducibility. Based on these findings, histological evaluation of LMW-PTP appears to be useful regarding the postoperative treatment strategy.
Prostate cancer, which is a typical example of a cancer with a high degree of heterogeneity, both suppresses and stimulates the functions of multiple signal transduction pathways to varying levels, with the final output of these processes giving prostate cancer its characteristics. One such product of signal transduction, protein tyrosine kinase (PTK), is known to have functional and expression-related abnormalities in many malignancies. For example, some findings suggest that the increased phosphorylation of HER-2 in cases of prostate cancer is closely related to androgen-independent tumor cell proliferation (28-30). One substance that plays a role in the negative regulation of tyrosine phosphorylation is protein tyrosine phosphatase (PTP). Although Igawa et al. have reported that prostatic acid phosphatase is involved in the negative control of prostate cancer cell proliferation through regulation of PTKs, there are far fewer reports on PTPs in prostate cancer than there are on PTKs (31, 32), hence, the details of the mechanism of action remain largely unknown.
LMW-PTPs are a group of 18-kDa enzymes that belong to the class II cysteine-based PTPs and are expressed abundantly in tissues (33, 34). LMW-PTP has been largely considered a negative regulator of growth factor-induced cell proliferation, due to its interaction with molecules such as the platelet-derived growth factor receptor (35), β-catenin (36), Janus kinase 2 (37), signal transducer and activator of transcription 5 (STAT5) (38), the fibroblast growth factor receptor (39), and the insulin receptor (40). Only in few instances it acts as a positive regulator, such as the Ephrin Receptor A2 (EphA2), which seems to be involved in the regulation of carcinogenesis through LMW-PTP signaling (20, 41, 42). Hyperexpression of EphA2, which is a receptor tyrosine kinase, has a higher enzymatic activity when in the dephosphorylated state compared to its phosphorylated state. It has been reported, that when EphA2 is dephosphorylated, it regulates tumor cell proliferation, migration, infiltration, and other cell behaviors, while in a large number of cancers it functions as a positive regulator of the tumor (43, 44). LMW-PTP is an important negative regulator of EphA2 phosphorylation, and therefore, it is involved in the proliferation, invasion, and migration of tumors through the dephosphorylation of EphA2 (18). The Ki-67 LI is recognized as a marker of cell proliferation, and Ki-67 LI scores were significantly higher in the LMW-PTP high-expression group, compared to the low-expression group in this study. This suggests a positive correlation between LMW-PTP with cell proliferative capacity. These findings indicate that cell proliferation may also be induced in prostate cancer as a result of dephosphorylation of EphA2. There are no previous studies in prostate cancer that show a correlation between LMW-PTP and EphA2 expression, but this would be an interesting field of exploration for the future.
In the present study, LMW-PTP was found to be a prognostic factor, whereas serum PSA values and Gleason scores were not reliable, despite the Gleason score being an important prognostic factor used in routine clinical practice. Surprisingly, in this study, no significant correlation was observed between LMW-PTP expression and the Gleason score. The Gleason score is a metric used in the histological evaluation of tumor cells (45, 46). However, it is difficult to characterize the biological properties of tumor cells based on tissue morphology alone. In this study, the early biochemical recurrence observed in the LMW-PTP high-expression group and high LMW-PTP expression level strongly correlated with Ki-67 expression. In the patients we examined, LMW-PTP expression was found to be a promising biomarker for evaluating malignancies that cannot be identified based on tissue morphology alone. Overall, our findings indicate the feasibility of using LMW-PTP expression as a biochemical recurrence factor in combination with the Gleason score.
In general, biochemical recurrence serves as an indicator of the early stages of relapse, since local recurrence and distant metastasis may occur after biochemical recurrence (47, 48). The fact that a large number of tumor cells strongly expressing LMW-PTP correlate with the tumor proliferation ability may suggest that these cells exhibit tumor characteristics that are likely to result in recurrence. Since LMW-PTP expression in resected specimens is a strong predictor of biochemical recurrence, the extent of LMW-PTP expression could provide useful information for the determination of post-operative treatment strategy. The value of LMW-PTP expression as a predictive biomarker may though be hindered, depending on the size of the biopsy tissue that could be difficult to assess as well as due to cancer heterogeneity. For this reason, our future aim is to measure LMW-PTP expression in different size prostate biopsy tissues and add this assessment to the initial diagnostic tests, in order to better explore the clinical applications of this biomarker. The results of immunostaining of LMW-PTP conducted in this study suggest that LMW-PTP is a useful predictive factor for biochemical recurrence in cases of prostate cancer with negative surgical margins. LMW-PTP expression can be assessed using a single immunostaining protocol, and the assessments are highly reproducible. Thus, it might be applied in clinical practice to establish the required postoperative follow-up period and determine the necessity of salvage therapy in cases of prostate cancer with negative surgical margins. LWM-PTP has the potential to be a highly useful prognostic biomarker in conjunction with the Gleason score, the pathological T stage and the PSA level. We hope to conduct further studies to correlate LMW-PTP with EphA2 in cases of prostate cancer.
Footnotes
Conflicts of Interest
All Authors have declared no conflicts of interest.
- Received December 24, 2018.
- Revision received January 7, 2019.
- Accepted January 17, 2019.
- Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved