Abstract
The prognostication of individual disease trajectory and selection of optimal therapy in patients with localized, low-grade prostate cancer often presents significant difficulty. The phosphatase and tensin homolog on chromosome 10 (PTEN) has emerged as a potential novel biomarker in this clinical context, based on its demonstrated prognostic significance in multiple retrospective studies. Incorporation into standard clinical practice necessitates exceptional diagnostic accuracy, and PTEN’s binary readout—retention or loss—suggests its suitability as a biomarker. This multi-institutional ring trial aimed to validate the diagnostic precision of PTEN immunohistochemistry in localized, low- to intermediate-risk prostate cancer, across ten university pathology institutes in Germany. The trial incorporated 90 cases of patients diagnosed with acinar adenocarcinoma of the prostate of grade groups 1 (n = 8, 8.9%) and 2 (n = 82, 91.1%) post-radical prostatectomy. Remarkably, the interpretation of PTEN immunohistochemistry displayed substantial variation (12.5–51.2% PTEN loss rates) within an identical cohort of prostate cancer. Fluorescence in situ hybridization analysis demonstrated PTEN hemizygous deletions in 5.5% (5/90) of cases. All cases with hemizygous deletions presented a distinct loss of PTEN expression by immunohistochemistry and were unanimously identified as PTEN loss by all participants (sensitivity 100%). However, negative (loss) immunohistochemistry was relatively non-specific for an underlying genomic deletion. Improved inter-observer agreement was observed in a subsequent ring trial. Finally, we identify S473-pAKT immunohistochemistry as a useful marker in equivocal cases. In summary, this multi-institutional ring trial illustrates surprisingly heterogeneous outcomes in defining PTEN status by immunohistochemistry.
Supplementary Information
The online version contains supplementary material available at 10.1007/s00428-024-03999-y.
Keywords: Prostate cancer, PTEN, Prognostic biomarker, Active surveillance, AKT
Introduction
The differentiation of lethal, curable, and insignificant tumors, ideally at the time of diagnosis, is a prerequisite for individualized therapy in men with prostate cancer. While the indication for definitive therapy is clear in the majority of patients, there is often disagreement about whether active surveillance therapy should be considered in a subset of patients with low- to early intermediate-risk tumors. Clinicopathological criteria for evaluating active surveillance therapy include Gleason score, tumor volume in core needle biopsies, percentage of biopsy core involvement and serum PSA level. Recently, histological features like cribriform Gleason pattern 4 and intraductal carcinoma (IDC-P) were found to prognosticate aggressive disease and thus should exclude patients from active surveillance therapy [1–5]. A comprehensive understanding of molecular alterations in prostate cancer led to the development of novel molecular biomarkers for disease prognostication that might aid clinical decision-making in low- to intermediate-risk tumors. In addition to Ki-67 labeling index and RNA-based assays, PTEN (phosphatase and tensin homolog on chromosome 10), a negative regulator of the oncogenic PI3K-AKT-mTOR pathway, turned out as a promising novel biomarker in this setting [6].
Patients with tumors with PTEN loss are more likely to have biochemical recurrence (BCR) after radical prostatectomy compared to patients with PTEN intact tumors [20–23]. Detection of PTEN loss in core needle biopsies of grade group 1 and grade group 2 tumors increases the likelihood of upgrading, non-organ-confined disease (NOCD), and BCR after radical prostatectomy [24, 25]. However, due to a lack of prospective validation studies on active surveillance cohorts, PTEN status has not yet been implemented in standard clinical practice for the evaluation of active surveillance therapy [6].
Once implemented in clinical testing algorithms, it is reasonable to evaluate PTEN status primarily via immunohistochemistry because of its fast and seemingly straightforward interpretation with a dichotomous staining pattern, its lower costs compared to DNA-based tests, the usability on small biopsy specimen, and the implementation of PTEN immunohistochemistry in aforementioned studies. In addition, immunohistochemical analysis of PTEN detects protein loss not only caused by genomic deletion but also caused by alternative mechanisms like gene mutation, epigenetic silencing, and posttranscriptional and posttranslational regulation, that would be missed by PTEN FISH analysis. Current testing algorithms propose PTEN FISH or sequencing for equivocal immunohistochemical testing results [28].
High diagnostic accuracy is crucial for the integration of a biomarker into standard clinical practice. Ten university hospitals that participate in a project aiming to collect molecular data on prostate cancer prospectively (“SUMUS” trial) planned to start collecting PTEN data in primary prostate cancer cases. Before commencing the actual data accrual, the participants wanted to compare their established PTEN immunohistochemistry assays in order to assure future comparability of the data.
Material and methods
Case selection and construction of TMA
Ninety patients diagnosed with acinar adenocarcinoma of the prostate including grade groups 1 (8/90, 8.9%) and 2 (82/90, 91.1%) after radical prostatectomy at a single institute (Institute of Pathology, University Hospital Bonn, Bonn, Germany) were included in this study. Table 1 states the clinicopathological data of the cohort. None of the patients received neoadjuvant therapy. Formalin-fixed, paraffin-embedded tissue was retrieved from the archive to construct a tissue microarray (TMA) for a first ring trial consisting of one tumor spot (diameter 1 mm) from each of the 90 patients. PTEN loss rates of all participating institutes and the correlation of immunohistochemical results to genomic PTEN FISH data were supplied to all participants at the end of the first ring trial. For the second ring trial, a smaller TMA comprising 19 cases selected from the initial cohort was constructed, consisting of four unequivocal loss cases, five unequivocal retained cases, and 10 cases with equivocal rating. Table 2 states the clinicopathological data of the second cohort. For both the first and the second cohorts, tissue microarrays were cut in the lead institute (3-µm thick) and mounted on superfrost slides (Menzel Gläser, Brunswick, Germany). Two unstained slides were sent to all participating institutes and stained for PTEN according to local routine procedure. S473-pAKT, CD24, and GP2 immunohistochemistry was performed on randomly selected prostate needle core biopsies in which the PTEN status was determined by immunohistochemistry.
Table 1.
Clinicopathological data of the cases included in the first ring trial
| Clinical feature | Finding (n = 90) |
|---|---|
| Age, median (range), years | 64 (49–76) |
| Preoperative PSA (ng/mL), median (range) | 6.8 (1.4–54) |
| Postoperative parameters | |
| Grade Group at RP, n (%) | |
| 1 (Gleason score 3 + 3 = 6), n (%) | 8 (8.9%) |
| 2 (Gleason score 3 + 4 = 7a), n (%) | 82 (91.1%) |
| Organ-confined disease, n (%) | 73 (81.1%) |
| Extraprostatic extension, n (%) | 17 (18.9%) |
| Seminal vesicle involvement, n (%) | 2 (2.2%) |
| Lymph node positive, n (%) | 1 (1.1%) |
| Positive margins, n (%) | 22 (24.4%) |
Table 2.
Clinicopathological data of the cases included in the second ring trial
| Clinical feature | Finding (n = 19) |
|---|---|
| Age, median (range), years | 65.0 (50–76) |
| Preoperative PSA (ng/mL), median (range) | 8.0 (1.4–17.0) |
| Postoperative parameters | |
| Grade group at RP, n (%) | |
| 1. n (%) | 1 (5.3%) |
| 2. n (%) | 18 (94.7%) |
| Organ-confined disease, n (%) | 16 (84.2%) |
| Extraprostatic extension, n (%) | 3 (15.8%) |
| Seminal vesicle involvement, n (%) | 0 (0%) |
| Lymph node positive, n (%) | 1 (5.3%) |
| Positive margins, n (%) | 4 (21.1%) |
Participants, immunohistochemistry protocols, and readout
Altogether, 10 institutes of pathology involved in the German multicentric Network Study of Molecular Alterations in urological Tumors (“SUMUS”) participated in this ring trial. The idea of the trial was to determine the status quo of PTEN diagnostics; hence—analogous to most commercially available ring trials in Europe—no specific antibody or protocol was recommended. Three out of 10 institutes used the primary anti-PTEN antibody clone D4.3 XP, a rabbit monoclonal antibody (Cell Signaling Technology, Catalogue number 9188). Five out of 10 institutes used the primary anti-PTEN antibody clone SP218, a rabbit monoclonal antibody (Abcam, catalog number ab228466). Two out of 10 institutes used the primary anti-PTEN antibody clone 138G6 rabbit monoclonal antibody (Cell Signaling Technology, catalog number 9559). One institute used a primary anti-PTEN rabbit antibody (Cell Signaling Technology, catalog number 9552). Two institutes stained both slides provided with different antibodies (clone SP218 and clone 138G6). The scoring options were determined as either loss of PTEN expression or retained PTEN expression. Following local staining and interpretation, the results were returned to the lead institute in Bonn. Thereafter, feedback was supplied to all participating institutes including a comparison between the participating institutes and the correlation of immunohistochemical results to PTEN FISH data.
Phospho-AKT immunohistochemistry was performed in the lead institute using an anti-AKT1 rabbit monoclonal antibody against phosphorylated at Serine 473 (1: 50, Abcam, ab81283, Cambridge, UK). The CD24 immunohistochemistry was performed as described with clone SWA11) [29]. GP2 immunohistochemistry was performed using an anti-GP2 rabbit monoclonal antibody (1: 400, Biozol Diagnostica, Eching, Germany).
Definition of consensus
Cases rated as loss or retained by > 75% of all institutes were regarded as consensus voting [30].
PTEN FISH analysis
Dual-color FISH was performed on the tissue microarray using the Vysis LSI PTEN/CEP 10 FISH Probe Kit according to the manufacturer’s instructions (Abbott Laboratories, Order-Nr. 04N62-020). Hemizygous deletion of PTEN was defined as > 20% of tumor nuclei containing a single PTEN locus signal. A Leica DM5500 B fluorescence microscope (Leica, Wetzlar, Germany) was used for FISH analysis.
Statistical analysis and software
Statistical analysis was performed using the R software, version 4.1.0 (R Core Team (2021), R: A Language and Environment for Statistical Computing), R Foundation for Statistical Computing, Vienna, Austria. Inter-observer agreement was evaluated using Fleiss’ kappa with 95% confidence interval (CI). Fisher’s exact test was used for calculating statistical significance of S479-pAKT, CD24, and GP2 staining results. Graphs were generated with Prism, version 9.5.1, GraphPad Software, LLC.
Results
Analysis of PTEN expression in localized prostate cancer by immunohistochemistry shows low inter-observer agreement
Overall, 90 cases of patients diagnosed with acinar adenocarcinoma of the prostate of grade groups 1 (n = 8, 8.9%) and 2 (n = 82, 91.1%) after radical prostatectomy were included in this trial. A tissue microarray was constructed and provided to the 10 participating institutes. Two institutes evaluated staining results for two different anti-PTEN antibody clones resulting in 12 different data sets. Due to preprocessing artifacts (e.g., no tissue or no tumor on some TMA spots), of the 90 cases provided, the percentage of evaluable cases ranged from 66 to 88%. For 58 of the 90 cases (64.4%), all 12 datasets were entirely obtained.
Reported PTEN-loss rates among participating institutes ranged from 12.5 to 51.2% (mean 34.7% + / − 12.2 SD) (Fig. 1). Cases classified as loss or retained by > 75% of all institutes were regarded as consensus voting [30]. Using this definition of a consensus rating in total, 72% of cases (65/90 cases) showed a consensus rating for either PTEN loss or retained PTEN expression while an equivocal rating was found in 28% of the cases (25/90 cases). Consensus rating was preferentially found in cases showing either strong PTEN expression or total loss of PTEN expression (Fig. 2a, b). Equivocal cases showing low inter-observer agreement had particularly weak or doubtful PTEN expression (Fig. 2c). Fleiss’ kappa coefficient for the 58 datasets (64.4%), that were entirely obtained, was 0.54, 95% CI = [0.50; 0.57].
Fig. 1.
Results of the first ring trial. a Graphical and b tabular overview of PTEN immunohistochemistry results among the institutes in the first ring trial
Fig. 2.
Representative PTEN immunohistochemistry. a Homogenous loss of PTEN expression in tumor glands with intact staining in peritumoral stroma. These cases was rated ‘PTEN loss’ by all institutes. b Homogenous and strong PTEN expression in tumor glands and stroma. This case was rated ‘retained PTEN expression’ by all institutes. c Equivocal case with weak PTEN expression. This case was rated ‘PTEN loss’ by 5 institutes and ‘retained PTEN expression’ by 5 institutes
FISH analysis
To compare the immunohistochemical results with the genomic PTEN status, all 90 cases were analyzed for PTEN deletions by FISH analysis. PTEN hemizygous deletions were found in 5.6% of cases (5/90) whereas no homozygous deletions could be detected (Fig. 3). All five cases with hemizygous deletions showed a complete loss of PTEN expression by immunohistochemistry and were designated as PTEN loss by all participants. Therefore, a positive (retained pattern) immunohistochemistry result ruled out genomic PTEN loss with a sensitivity of 100%. However, a negative (loss pattern) immunohistochemistry result was rather unspecific for an underlying genomic deletion; the latter being found in 11.9 to 45.5% of all cases with PTEN loss. Thus, the specificity of immunohistochemistry to detect a genomic deletion thus ranged from 52.0 to 92.8% (Table 1).
Fig. 3.
PTEN FISH analysis. The LSI PTEN probe hybridizes to the 10q23 region on chromosome 10 (red signal). The CEP 10 SpectrumGreen probe hybridizes to chromosome 10 (green signal). a Tumor sample with retained PTEN expression and two intact PTEN alleles. b Tumor sample with PTEN loss and hemizygous PTEN deletion (red)
Repeated analysis of PTEN expression improves inter-observer agreement
As the rather poor inter-observer agreement seems unacceptable for clinical use, we aimed to test for a possibly improved inter-observer agreement in a subsequent second trial following feedback to the participants. Therefore, PTEN loss rates of all participating institutes and the correlation of immunohistochemical results to genomic PTEN FISH data were supplied to all participants. Next, a smaller tissue microarray comprising 19 cases selected from the cohort was constructed, consisting of four unequivocal loss cases, five unequivocal retained cases, and 10 cases with equivocal ratings. In all four loss cases (4/19, 21%), a hemizygous PTEN deletion was identified by FISH analysis. Again, two unstained slides were sent to all institutes for repeated evaluation and seven out of 10 institutes participated in the second ring trial. Similar to the first ring trial, all four cases with hemizygous deletions were designated as PTEN loss by all participants. Therefore, a positive (retained pattern) immunohistochemistry signal again ruled out genomic PTEN loss (sensitivity 100%). As expected, the mean of the PTEN loss rate was different due to the different composition of the second TMA (Fig. 4a). However, a marked harmonization of the inter-observer agreement was noted especially in equivocal cases (Fig. 4b, c). Fleiss’ kappa coefficient for the 16 datasets (82.4%) of the second ring trial that were entirely obtained increased to 0.64, 95% CI = [0.53; 0.75], compared to 0.54, 95% CI = [0.50; 0.57], in the first ring trial. When calculating the Fleiss’ kappa coefficient for the datasets of the first ring trial of only the participants of the second trial, the increase in Fleiss’ kappa coefficient was even more pronounced (0.40 vs. 0.64).
Fig. 4.
Results of the second ring trial in comparison to the first trial. Results of a all cases, b equivocal cases only and c and unequivocal cases only
Tentative ancillary surrogate markers of PTEN inactivation/loss
Despite the improvement in diagnostic accuracy for detecting PTEN loss through a repeated ring trial, individual cases still present diagnostic challenges. To address this, we investigated additional potential immunohistochemical markers that aid in the detection of PTEN loss in equivocal cases.
PTEN acts as a tumor suppressor by dephosphorylating PIP3 and antagonizing the PI3K-AKT pathway. PTEN loss results in the activation of PI3K, which subsequently leads to AKT activation via phosphorylation at two sites, T308 and S473, with maximal activation at S473 [31, 32]. We conducted immunohistochemical analysis of S473-pAKT in a total of 20 core biopsies of prostate cancer with both PTEN loss and retention. As anticipated, S473-pAKT was present in the majority of cancers with PTEN loss (7/9), whereas most cancers with retained PTEN expression showed no S473-pAKT expression (10/11) (Fig. 5a, b, d, e). However, few false negative and false positive results were noted (Fig. 5g, h). The sensitivity of S473-pAKT immunohistochemistry to detect PTEN loss was 77.8% with a high specificity of 90.9% (Table 3). Notably, the sensitivity was markedly decreased when tested on a tissue microarray of prostate cancer cases taken from prostatectomy specimens (16.3%). We attribute this to prolonged cold ischemia time in the center of the RPE specimen, as immediate fixation post-collection is essential for preservation of protein phosphorylation as shown in other tumors [33]. In concordance with this notion, preliminary results show a staining gradient for S473-pAKT in prostatectomy specimens, which we attribute to delayed fixation in the specimen center (Fig. S1). This gradient underscores the importance of immediate fixation post-collection, as previously noted for protein phosphorylation in other contexts.
Fig. 5.
Expression of PTEN, S473-pAKT, and CD24 in prostate needle core biopses. a–c Prostate cancer with retained PTEN expression, absent S473-pAKT, and no expression of CD24. d–f Prostate cancer with loss of PTEN expression, positivity for S473-pAKT, and expression of CD24. g–i Prostate cancer with retained PTEN expression, weak equivocal staining for S473-pAKT and diffuse overexpression of CD24
Table 3.
Staining results of PTEN, S473-pAKT, CD24, and GP2 on prostate needle core biopsies (p-values: S473-pAKT 0.0045, CD24 n.s., GP2 n.s.; Fisher’s exact test)
| Marker | PTEN loss | PTEN retained |
|---|---|---|
| S479-AKT + | 7 | 1 |
| S479-AKT − | 2 | 10 |
| CD24 + | 7 | 5 |
| CD24 − | 5 | 3 |
| GP2 + | 0 | 0 |
| GP2 − | 9 | 11 |
Additionally, we assessed CD24 expression as a potential immunohistochemical surrogate marker for PTEN loss, which we and others had found upregulated in cases with PTEN loss [7, 34]. We performed CD24 immunohistochemistry in the same core biopsies of prostate cancer with both PTEN loss and retention, which were already analyzed for S473-pAKT. CD24 expression was rather heterogenous compared to S473-pAKT expression and did not correlate with PTEN expression (Fig. 5c, f, i). The sensitivity of CD24 immunohistochemistry to detect PTEN loss was 58.3% with a rather low specificity of 37.5% (Table 3).
Thirdly, we picked GP2, an immune-associated gene, that also has been shown to be transcriptionally upregulated in prostate cancer with loss of PTEN expression in a prior screening study [35]. However, only focal expression of GP2 was found in one case of PTEN loss (Table 3). Thus, GP2 expression does not qualify as an immunohistochemical surrogate for PTEN loss.
Discussion
Determining the aggressiveness of a tumor or predicting the course of localized low-grade prostate cancer is a challenging task. This is because there is a range of treatment options available, from immediate definitive therapy to active surveillance. To facilitate treatment decisions, molecular biomarkers in addition to established serological, radiological, and histological parameters are desired. At the ISUP consensus conference on molecular biomarkers in genitourinary tumors in 2019, Ki-67 and PTEN were identified as the most promising markers [6]{Lotan, 2020 #76}.
However, the application of Ki-67 as a biomarker is complicated by interobserver variability due to the reflective nature of the proliferative activity, which requires a cut-off value to determine prognostic subgroups [36–38]. On the other hand, PTEN is one of the most frequently altered genes in prostate cancer, and its prognostic and potential therapeutic implications have been demonstrated in multiple retrospective studies [20–25, 39]. PTEN appears to be a more reliable biomarker than Ki-67 because its readout is dichotomous, meaning it is either retained or lost. Immunohistochemistry is the preferred modality for assessing PTEN status due to its easy interpretation, inexpensiveness, and wide implementation in pathology laboratories.
PTEN loss in prostate cancer is mostly due to PTEN biallelic genomic deletion whereas PTEN mutations occur less frequently and in association with hemizygous deletions [7–13]. Gene rearrangement comprising the PTEN locus is reported as an additional mechanism of PTEN inactivation [14]. PTEN loss or downregulation via promotor hypermethylation, miRNAs or other mechanisms is less well studied [15, 16]. Due to an association with Gleason score and tumor stage, the reported prevalence of PTEN loss in prostate cancer is variable across different cohorts and is reported in about 20% of primary and in about 40% of metastatic tumors [7, 8, 10, 11, 17–19].
Apart from its role as a prognostic biomarker, it is expectable that PTEN status might have a role as a predictive biomarker in PTEN-deficient advanced prostate cancer. Promising treatment options focus on the blockage of the PI3K-AKT-mTOR pathway in combination with established therapeutics [26]. An ongoing phase III randomized double-blind study (IPATential150) testing the AKT-inhibitor ipatasertib with abiraterone and prednisone compared with placebo plus abiraterone and prednisone in patients with metastatic castrate-resistant prostate cancer with PTEN loss showed an improved radiographic progression-free survival in ipatasertib-treated patients [27].
In preparation of a prospective multicentric collection of molecular prostate cancer data including PTEN immunohistochemistry, a ring trial was conducted to determine the status quo of the diagnostic accuracy of PTEN immunohistochemistry in localized low- to intermediate-risk prostate cancer among ten eminent university pathology institutes in Germany. Unexpectedly, the interpretation of PTEN immunohistochemistry in this study showed marked variation (12.5–51.2% PTEN loss rates) in an identical cohort of prostate cancer. This may not be surprising, as no harmonization of the PTEN assay was conducted prior to this ring trial. While there was a trend towards harmonization in the interobserver agreement in a subsequent smaller ring trial following feedback to the participants, even these results require further improvement; hence, we think this data argues against the uncritical use of PTEN immunohistochemistry to define PTEN status for clinical routine use. So far, the interobserver variability of PTEN immunohistochemistry has been studied in only a few other entities. Because of the frequent PTEN loss in endometrial cancer, PTEN immunohistochemistry is well implemented as a routine diagnostic biomarker in this entity. Comparing the interobserver variability of PTEN loss in endometrial cancer between two institutes, it was found to be highly reproducible with a kappa value of 0.8 [40].
FISH analysis of our cohort revealed PTEN hemizygous deletions in 5.5% (5/90) of all cases, which is in contrast to the reported prevalence of PTEN loss in about 20% of primary prostate cancers [7, 8, 10]. However, this finding is likely to be explained by the selection of only grade group 1 and 2 tumors [20, 23, 41].
In direct comparison of immunohistochemical results with FISH analysis, the positive predictive value of the genomic alteration ranged from 11.9 to 45.5%. All cases with a hemizygous PTEN deletion were totally negative on immunohistochemistry, and conversely, any positive PTEN immunoreactivity ruled out a genomic PTEN loss (sensitivity 100%). However, negative (loss) immunohistochemistry was rather unspecific for an underlying genomic deletion. Then, 6.7% (6/90) of the tumors with a loss immunophenotype showed no genomic loss in the FISH analysis, and therefore, alternative mechanisms of PTEN inactivation such as gene rearrangement, gene mutation, epigenetic silencing, and posttranscriptional or posttranslational regulation are likely present in these cases. It remains elusive whether FISH analysis should, as proposed, be defined as the gold standard in equivocal immunohistochemistry cases, because PTEN loss by other mechanisms than genomic loss could be missed.
To enhance the diagnostic accuracy of determining PTEN status by immunohistochemistry, we explored potential surrogate markers for PTEN loss in prostate cancer. We focused on the activation of the PI3K-AKT pathway using S473-pAKT immunohistochemistry, which is indicative of PTEN loss [31, 32]. In a cohort of 20 prostate cancer cases, we found that S473-pAKT demonstrated a sensitivity of 77.8% and specificity of 90.9% for detecting PTEN loss. Consequently, S473-pAKT may serve as a surrogate marker for PTEN loss in cases where PTEN immunohistochemistry results are equivocal. Importantly, the sensitivity was markedly decreased when tested on samples from radical prostatectomy specimen, because being a phosphoprotein, S473-pAKT is an unstable protein modification that is sensitive to prolonged cold ischemia time. Therefore, short times to fixation is of paramount importance in the correct evaluation of S473-pAKT. In addition, we tested CD24 and GP2 expressions as additional immunohistochemical surrogate markers for PTEN loss. However, CD24 expression was heterogenous and showed a rather low specificity compared to S473-pAKT immunohistochemistry. GP2 expression was only found focally in one case of PTEN loss. Thus, S473-pAKT but not CD24 or GP2 may serve as an additional marker in cases where PTEN immunohistochemistry results are unclear.
Strengths of this study are its participants that reflect a representative fraction of academic pathology institutes in Germany and the deliberate selection of low-grade prostate cancer cases—exactly the group in which prognostic biomarkers are most urgently needed. Weaknesses of this study are its small to medium cohort size (n = 90), the use of a tissue microarray which limits the amount of tumor that can be studied, and the lack of follow-up data.
In summary, this ring trial investigates the variability of PTEN immunohistochemistry in low- to intermediate-risk prostate cancer. The data recommends that every institute should establish a PTEN immunohistochemistry protocol in close correlation with PTEN genomics and genetics. It also indicates that only a complete loss of immunoreactivity is diagnostic of a PTEN loss, as even the faintest positivity, that may be perceived as background, is indicative of a retained PTEN status. In addition, in equivocal cases, S473-pAKT immunohistochemistry may serve as an ancillary surrogate marker for PTEN loss. Regular interlaboratory ring trials are necessary to further improve the reliability and reproducibility of the PTEN assay.
Supplementary Information
Below is the link to the electronic supplementary material.
Supplementary file1 Section from a basal margin of a radical prostectomy specimen showing cancer with PTEN loss (a). S473-pAKT staining shows a staining gradient with strong positivity at the specimen surface (lower image border) and reduced positivity in the center (b) (DOCX 741 KB)
Author contribution
GK and OH were responsible for conception of the study, writing and review of the manuscript, data interpretation, and review of cases. MB, TK, AS, MA, GB, JHB, FFD, RE, IE, AH, WH, BH, HK, ML, CLC, VS, HR, HUS, MS, US, PRS, VT, and FW were responsible for data collection, interpretation of data, and review of the manuscript. JB, CD, JE, JL, MS, KS, MS, AS, ALW, and BW were responsible for interpretation of data, and review of the manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Data availability
Data is available upon reasonable request.
Declarations
Ethics approval
This study was conducted under institutional ethics approval.
Conflict of interest
The authors declare no competing interests.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supplementary file1 Section from a basal margin of a radical prostectomy specimen showing cancer with PTEN loss (a). S473-pAKT staining shows a staining gradient with strong positivity at the specimen surface (lower image border) and reduced positivity in the center (b) (DOCX 741 KB)
Data Availability Statement
Data is available upon reasonable request.