- Email: [email protected]
Cancer stem cell markers in prostate cancer: an immunohistochemical study of ALDH1, SOX2 and EZH2
Pathology (December 2015) 47(7), pp. 622–628
ANATOMICAL PATHOLOGY
Cancer stem cell markers in prostate cancer: an immunohistochemical study of ALDH1, SOX2 and EZH2 ADMIRE MATSIKA1,2,3, BHUVANA SRINIVASAN1, CHRISTOPHER DAY1,2,3, SABRINA ANN MADER4, DEIRDRE MARGARET KIERNAN4, AMY BROOMFIELD1, JINLIN FU5, JOHN D. HOOPER5, JAMES G. KENCH6 AND HEMAMALI SAMARATUNGA3,7 1Department of Anatomical Pathology, Mater Health Services, South Brisbane, 2Pathology Queensland, Royal Brisbane and Women’s Hospital, Herston, 3Department of Pathology, University of Queensland, Brisbane, 4Department of Urology, Mater Adult Hospital, South Brisbane, 5Mater Research Institute – University of Queensland, Translational Research Institute, Woolloongabba, Qld, 6Department of Tissue Pathology and
Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown and Sydney Medical School, The University of Sydney, NSW, and 7Aquesta Pathology, Taringa, Qld, Australia
Summary The aims of this study were to investigate the immunohistochemical expression and potential prognostic significance of putative cancer stems cell markers ALDH1, EZH2 and SOX2 in prostate cancer. A total of 142 consecutive radical prostatectomies submitted to one laboratory with a diagnosis of prostatic adenocarcinoma between 2008 and 2012 were retrieved and retrospectively studied. Immunohistochemistry for the three markers was performed in each case and both univariate and multivariate analyses were undertaken to evaluate the correlation between the staining patterns and known histopathological prognostic features. ALDH1 showed a statistically significant association with tumour stage p < 0.001), extraprostatic extension ( p < 0.001) and lymphovascular invasion ( p ¼ 0.001). EZH2 correlated with Gleason score ( p ¼ 0.044) and lymph node metastases ( p ¼ 0.023). SOX2 showed a statistically significant correlation with lymphovascular invasion only ( p ¼ 0.018) in both univariate and multivariate analyses. Cancer stem cell markers are variably expressed in prostate adenocarcinoma and immunohistochemical staining for ALDH1 and EZH2 may have a role in predicting tumour aggressiveness before treatment of prostate cancer. Key words: Adenocarcinoma, ALDH1, androgen receptor, cancer stem cell theory, cancer stem cells, EZH2, immunohistochemistry, prognostic markers, prostate cancer, SOX2. Received 11 November 2014, revised 28 June, accepted 30 June 2015
INTRODUCTION The classical concept of cancer pathogenesis stipulates that tumours arise from stepwise accumulation of multiple mutations in mature somatic cells and that all cells in a cancer share similar molecular aberrations.1 The cancer stem cell (CSC) theory is an alternative, fairly new paradigm and hypothesises that distinct clones of cancer cells result from abnormalities that occur within specific cell types.2,3 In this theory, cells in a tumour do not have the same malignant potential but rather there are small clones of CSCs with selfrenewal capabilities, high proliferative potential and Print ISSN 0031-3025/Online ISSN 1465-3931 Copyright DOI: 10.1097/PAT.0000000000000325
#
pluripotency status that are responsible for tumour initiation, propagation and metastases.4 The self-propagating ability of these cells is demonstrated by their ability to establish xenografts in mice.5 Enzymatic assays and immunohistochemistry have identified several proteins and surface markers that are expressed by CSCs and may be used to identify them in patients.6 Aldehyde dehydrogenase 1A1 (ALDH1) is an intracellular enzyme that oxidises retinol to retinoic acid, an important regulator of cellular differentiation.7 This enzyme is also involved in detoxification and has been implicated in the emergence of drug resistance to chemotherapeutic agents.8 There is also evidence that ALDH1 is overexpressed in CSCs of various organs including haematopoietic,9,10 breast,11 pancreas, lung, colorectal and prostate.12 Fluorescence based biochemical studies have also shown that ALDH1 is a useful marker for prostate CSCs. Consistent with a role in prostate cancer, ALDH1 rich prostate cancer cells can initiate and propagate prostate cancers when transplanted into mice while those low in ALDH1 do not.13–15 Enhancer of zeste homolog 2 (EZH2), a catalytic subunit of the Polycomb Repressive Complex responsible for histone methylation, regulates gene transcription and chromatin structure.16 Studies to date suggest that EZH2, which is also overexpressed in CSCs, plays a critical role in stem cell renewal, maintenance, and differentiation into specific cell lineages.17 It is overexpressed in aggressive solid tumours, including metastatic prostate cancer and may be a useful prognostic marker in clinical practice18–20 with chemotherapeutic agents currently being developed that specifically target the enzyme.18 More than a dozen SOX genes have been described and are named after a shared motif called the SRY box, a region homologous to the DNA-binding domain of SRY, the mammalian sex determining gene.21 One of these genes encodes for a transcription factor SOX2 which has a well-known role in the maintenance and acquisition of stem cell features.22 In prostate cancer SOX2 is overexpressed in CSCs where it mediates tumourigenesis and has been linked to poor prognosis.23,24 Prostate cancer is the most commonly diagnosed non-skin cancer in men in the western world with mortality rates only second to lung cancer.25,26 It is generally accepted that not all prostate cancers require active aggressive treatment as some
2015 Royal College of Pathologists of Australasia. All rights reserved.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
STEM CELL MARKERS IN PROSTATE CANCER: IHC STUDY OF ALDH1, SOX2 AND EZH2
men die with their disease rather than from it. Some of the current radical treatment modalities for the disease are associated with considerable morbidity. Accordingly, there is a need for a diagnostic tool, for use in core biopsies, that can identify aggressive prostate cancers that will require radical treatment and distinguish them from indolent disease that can be managed conservatively with active surveillance.27,28 It is on this background that we have performed this study to evaluate the putative CSC markers: ALDH1, EZH2 and SOX2 as potential prognostic markers in prostate cancer. Androgen receptor (AR) staining of the prostate cancers was also performed to determine its association with these novel proteins.
MATERIALS AND METHODS Study design For this study 142 consecutive cases of prostate cancer that underwent radical prostatectomy between 2008 and 2012 were retrieved from the Mater Pathology archives. The study was approved by the Research Ethics Committee of our institution. Haematoxylin and eosin (H&E) stained sections of totally embedded prostatectomies were reviewed by two of the authors (AM, BS). A standard paraffin block with the highest Gleason score and largest tumour area from each case was selected for immunohistochemical staining. The following data were collected from histological and clinical records: patient age at time of prostatectomy, histological type, Gleason score, pathological stage (pT), nodal stage (pN); presence and extent of extraprostatic extension (EPE) and lymphovascular invasion (LVI). Immunohistochemistry and histopathological parameters Immunohistochemistry staining was performed on formalin fixed, paraffin embedded sections (4 mm). A positive control was present on each slide to determine the validity of the stain. All slides were stained using the Ventana Benchmark Ultra automated immunohistochemistry staining machine, using the Ventana Ultra View DAB detection system (Ventana, USA). The antibodies were: ALDH1, Clone 44/ALDH, 1/1000 dilution (BD Transduction Laboratories, USA); EZH2, Clone AP129, prediluted (Ventana); SOX2, Clone SP76, 1/20 dilution (Cell Marque, USA) and androgen receptor (AR), Clone AR441, 1/20 dilution (Cell Marque). All sections were subjected to heat-induced epitope retrieval in CC1 solution for 64 min at 958C and antibodies were incubated at 368C for 32 min. Stained slides were examined by two pathologists (BS, AM) blinded to the pathological prognostic parameters of the tumours. Only staining of malignant epithelial cells was evaluated in this study. The percentage of cells staining (0–100%) was recorded for each case and scored as follows: 0–33%, score 1; 34–66%, score 2 and >67%, score 3. The intensity of staining (0, none; 1, mild; 2, moderate; 3, strong) for each antibody was also recorded and an H-score like staining index (SI) was calculated for each antibody as follows: SI (0– 9) ¼ intensity score (0–3) percentage staining score (0–3). A modified Gleason score after Trock et al.29 that takes into account tertiary patterns (TGS5) and the prognostic differences of Gleason scores 4þ3 versus 3þ4 was used for statistical analysis.29 EPE was scored as follows as per Epstein’s proposal:30 0, absent; 1, focal; 2, extensive. LVI was also scored as follows: 0, absent; 1, focal; 2, extensive. Absence of nodal involvement (pN0) was scored 0 and pN1 as 1. pT category according to the American Joint Committee on Cancer, 7th edition,31 was recorded and scored as follows for statistical purpose: pT2 ¼ 2.0, pT3a ¼ 3.0, pT3b ¼ 3.5 and pT4 ¼ 4. Statistical analysis Mean and standard deviations were used to describe age at prostatectomy. Absolute counts and proportions were used for categorical variables. Spearman’s rank correlation was used to examine correlations between each of the markers and the known prognostic parameters. Correlation coefficients and significance levels were reported. Logistic and ordinal logistic regressions were also employed to detect the relationships between the variables. All analyses were performed using Stata/SE 12.1 for Windows. p values <0.05 were considered statistically significant.
Table 1
623
Characteristics of patients
Characteristics Mean age, years (range) 61.1 (43–76) Gleason score 3þ3 ¼ 6 3þ3 ¼ 6 with TGS5 3þ4 ¼ 7 3þ4 ¼ 7 with TGS5 4þ3 ¼ 7 4þ3 ¼ 7 with TGS5 4þ4 ¼ 8 4þ4 ¼ 8 with TGS5 4þ5 ¼ 9 5þ4 ¼ 9 pT stage pT2 pT3a pT3b pN stage pN0 pN1 pNX LVI Absent Focal Extensive EPE Absent Focal Extensive
No. cases (%)
142 (100) 10 5 46 6 26 12 12 12 12 1
(7.0) (3.5) (32.4) (4.2) (18.3) (8.5) (8.5) (8.5) (8.5) (0.7)
67 (50.75) 57 (40.10) 13 (9.15) 111 (78.17) 13 (9.15) 18 (12.68) 120 (84.51) 16 (11.27) 6 (4.23) 72 (50.70) 46 (32.39) 24 (16.90)
EPE, extraprostatic extension; LVI, lymphovascular invasion; pN, pathological lymph nodal stage; pNX, unknown nodal status; pT, pathological tumour stage; TGS, tertiary Gleason score 5 pattern.
RESULTS Patient characteristics The study included 142 patients with a mean age of 61 years and range 43–76 years (Table 1). Gleason score 3þ4¼7 was the most common score in this cohort with 46 cases (32.4%). Nodal metastases were present in 9% (n ¼ 13) and in 18 cases it was unknown (pNX). LVI was seen in 22 cases, six of which had extensive involvement. Almost half of the prostatectomies (n ¼ 70) had some degree of EPE, and in 24 of these the extension had been reported as extensive by previously reported criteria.30,32 ALDH1 expression About half (n ¼ 69) of the 142 cases exhibited cytoplasmic staining for ALDH1. There were marked differences in the proportion of malignant cells expressing ALDH1, ranging from <5% up to more than 90%. As shown in Table 2, statistical analysis across the patient cohort indicated that ALDH1 staining index significantly correlated with pT ( p < 0.001), EPE ( p < 0.001) and LVI ( p ¼ 0.001). The association with Gleason score ( p ¼ 0.075) and nodal metastases ( p ¼ 0.280) did not reach statistical significance. ALDH1 positive epithelial tumour cells tended to be larger with more prominent nucleoli compared to other malignant cells of the same tumour (Fig. 1). ALDH1 also showed some positivity—albeit patchy and milder—in the stroma surrounding ALDH1 positive epithelial tumour cells (Fig. 2A). This stromal accentuation was not seen in acini of ALDH1 negative carcinomas. Incidentally, the antibody was invariably positive in seminal vesicles (100%) and always weakly positive in benign urothelium and ganglia
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
624 Table 2
Pathology (2015), 47(7), December
MATSIKA et al.
Summary of correlation between staining categories of ALDH1, EZH2 and SOX2 and pathological prognostic parameters ALDH1
GS EPE LVI pT pN
EZH2
SOX2
Spearman coefficient
p value
Spearman coefficient
p value
Spearman coefficient
p value
0.150 0.346 0.273 0.348 0.098
0.075 <0.001 <0.001 <0.001 0.280
0.240 0.071 0.036 0.112 0.204
0.004 0.405 0.674 0.184 0.023
0.102 0.129 0.199 0.144 0.137
0.228 0.125 0.018 0.088 0.130
p values <0.05 are in bold. EPE, extraprostatic extension; GS, Gleason score; LVI, lymphovascular invasion; pN, pathological lymph nodal stage; pT, pathological tumour stage.
incorporated in the examined sections (Fig. 2B). Benign prostatic acini were consistently negative for ALDH1 in this cohort.
2). There was no statistically significant association with any of the other histopathological parameters studied (Table 2). The antibody also strongly and uniformly stained basal cells of benign prostatic acini (99%), high grade PIN (Fig. 4C), benign urothelium and ganglia in almost all cases (Fig. 4D). As with the other two markers, no positive nuclear staining of benign prostatic epithelium was seen with SOX2 in all cases.
EZH2 expression Ninety-nine percent (n ¼ 139) of the prostate carcinomas showed at least 1% nuclear staining with EZH2 (Fig. 3A,B). EZH2 nuclear staining was present only in malignant cells and absent in benign acini (Fig. 3C). Of note, in each of the positive cases there was a sprinkling of more deeply staining malignant cells (Fig. 3D). Analysis of both staining intensity and percentage of cells staining showed that EZH2 nuclear staining in prostate carcinoma cells has a statistically significant association with Gleason score ( p ¼ 0.004) and nodal stage ( p ¼ 0.023). In contrast to ALDH1, there was no correlation between EZH2 staining and EPE, LVI or pT.
Correlation between ALDH1, EZH2 and SOX2 As shown in Table 3, there was a statistically significant correlation between ALDH1 and SOX2 ( p ¼ 0.001), as well as between EZH2 and SOX2 ( p ¼ 0.040). However, no significant association was observed between ALDH1 and EZH2. Androgen receptor expression Androgen receptor (AR) immunohistochemistry was performed on 138 cases and all but two (98.6%) showed at least moderate staining in the majority of tumour cells (not shown). There was no statistically significant correlation between AR staining and prognostic pathological parameters or staining with the three cancer stem cell markers.
SOX2 expression SOX2 immunohistochemistry showed nuclear positivity in epithelial tumour cells in 61 of the 142 (46.38%) (Fig. 4A,B). Staining of epithelial tumour cells showed a statistically significant association ( p ¼ 0.018) with LVI (Table
A
B
C
D
Fig. 1 ALDH1 immunohistochemistry staining in prostate carcinoma. (A) Low power, (B) high power. Benign acini are negative. (C) Note the tumour cells with larger nucleoli staining for ALDH1 while (D) smaller malignant cells in the same section are negative.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
STEM CELL MARKERS IN PROSTATE CANCER: IHC STUDY OF ALDH1, SOX2 AND EZH2
625
DISCUSSION We have investigated the immunohistochemical expression and potential prognostic significance of the three putative prostate cancer stem cell markers ALDH1, EZH2 and SOX2 in a cohort of 142 prostatectomies. While previous studies have utilised in vitro biochemical methods or small tissue samples on tissue microarrays (TMA), we have immunohistochemically stained standard sections from wholly embedded radical prostatectomies with commercially available antibodies for the three proteins as well as for the androgen receptor (AR). This cohort is larger than similar previous studies and the first to investigate CSC markers in standard sections from prostatectomies. The use of standard sections has allowed for better characterisation of the staining patterns including analysis of heterogeneity within an individual tumour. Tumour heterogeneity is expected with these markers as, according to the stem cell theory, only a small fraction of the tumour bulk has stem cell properties. Our study also addresses another major limitation of studies using TMA and core biopsies, which is the difficulty of examining other facets of tumour biology such as the interactions between tumour bulk and its stromal microenvironment. Stroma and benign tissue are usually not adequately represented in the miniature tissue fragments from TMA and core biopsies. Additionally, standard sections have also enabled us to study the immunohistochemical staining characteristics of malignant prostatic stroma as well as benign elements such as basal cells, stroma, seminal vesicle epithelium, nerves and ganglia. Consequently, we have also uncovered some potential utilities of ALDH1, EZH2 and SOX2 in diagnostic practice due to their unique staining of benign elements. Our study shows that the CSC marker ALDH1 is expressed in prostate carcinomas, and more likely so in cases with adverse pathological parameters, such as extraprostatic extension, lymphovascular invasion and higher pathological tumour stage. Previous studies have shown similar correlations between
A
B Fig. 2 ALDH1 immunohistochemistry in prostate carcinoma. (A) Stromal accentuation around malignant acini is characteristic. (B) Seminal vesicles (and neural tissue, not shown) also strongly stain for the marker in almost all cases. Adjacent prostatic acini are negative in this case.
A
B
C
D
Fig. 3 EZH2 immunohistochemistry staining in prostate carcinoma. (A) Low power, (B) high power. (C) Adjacent benign acini are negative. (D) Note the sprinkling of more deeply staining malignant cells which were found to be characteristic of this antibody.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
626
Pathology (2015), 47(7), December
MATSIKA et al.
A
B
C
D
Fig. 4 SOX2 immunohistochemistry staining in prostate carcinoma. (A) Low power, (B) medium power. The basal cells in benign acini and malignant epithelia are positive while benign epithelial cells do not stain. (C) High grade PIN positive for SOX2. (D) Ganglia and neural tissue nuclei are strongly positive.
ALDH1 and poor prognosticators as well as disease free survival13,33,34 on prostate core biopsies and TMAs. The CSC theory is controversial and not unanimously accepted.6 The immunohistochemical staining pattern observed in this cohort whereby some but not all of the tumour cells overexpress ALDH1, EZH2 and/or SOX2 adds weight to this theory. There appears to be small subsets of tumour cells in aggressive prostate cancers that produce CSC proteins. The associations of these CSC proteins with histopathological staging parameters in this study suggest that these cells may be drivers of tumour progression and metastases. If confirmed to be correct, therapeutic agents could be developed that target CSCs rendering the tumours more indolent. A peculiar observation in our study was that ALDH1 positive epithelial tumour cells tended to be larger with more prominent nucleoli compared to other malignant cells in the same tumours (Fig. 1C,D). We interpret this as morphology-genotype correlation evidence of tumour cell diversity and further support for the CSC theory in prostate cancer. The behaviour of tumour cells is also influenced by the microenvironment, such as the stromal component, blood vessels and inflammatory cells.35,36 Immunohistochemistry Table 3
ALDH1 EZH2 SOX2
Correlations among CSC markers in prostate carcinoma ALDH1
EZH2
SOX2
– (1.000) 0.177 (0.114) 0.001 (0.284)
– (1.000) 0.040 (0.173)
– (1.000)
The p values are italicised and the correlation coefficients are in parentheses. The bold values indicate a statistically significant result.
with ALDH1 showed some positivity—albeit patchy and milder—in the stroma surrounding ALDH1 positive epithelial tumour cells (Fig. 2). This ‘stromal blushing’ was absent in ALDH1 negative sections or tumours and suggests that the stroma around CSCs could be abnormal at a molecular level, even though it may appear unremarkable on light microscopy. Other authors have come to the same conclusion in the past using different techniques35,37 and have concluded that the stroma (microenvironment) probably plays a more significant role in tumourigenesis and propagation than is currently appreciated. Our study shows a statistically significant association between EZH2, Gleason score and pathological nodal stage. Wolters et al. have previously also showed a significant association between EZH2 and high Gleason score, tumour volume and EPE in a study of 86 prostate core biopsies.38 Other authors have reported an association between EZH2 expression and disease free survival39,40 while some have found that it is not an independent prognosticator.41 These studies have used TMAs and prostate core biopsies which, by their nature, limit tumour sampling and do not take into account the tumour heterogeneity and patchy staining expected of stem cell markers. Subtle differences between our data and these reports may also be explained by the use of different EZH2 antibody clones with different specificities to target antigens. The antibody we used proved to be a very sensitive marker for prostatic malignant cells with nuclear staining of variable extent and intensity in 98% of the tumours while sparing benign prostatic epithelium in the same sections. Thus, it may have a role in diagnostic pathology to distinguish malignant prostatic from benign prostatic acini and urothelium. Karanikolas et al. have suggested that EZH2 is required for tumour progression in androgen independent but not androgen dependent prostate cancers.42,43 Therefore, EZH2 would be expected to have prognostic value in androgen receptor positive
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
STEM CELL MARKERS IN PROSTATE CANCER: IHC STUDY OF ALDH1, SOX2 AND EZH2
tumours. In our patient cohort, androgen receptor immunohistochemistry was performed in 138 cases and all but one (99.3%) showed at least moderate staining in the majority of tumour cells. We did not find any association between EHZ2 staining and androgen receptor expression. The complex role of EZH2 in prostate cancer requires further study as potential therapeutic agents targeting its pathway are being developed.44 Interestingly the prognostic parameters associated with EZH2 (Gleason score and pN stage) in this study differ from those associated with ALDH1 expression (EPE, LVI and pT). In CSC theory this may be explained by the observations from other studies that tumours have different CSC clones with different roles as well as biochemical and molecular properties.5 One study for example has shown that ALDH7A1, another aldehyde dehydrogenase isoform, is a requisite for bone metastases in prostate cancer.15,45 EZH2 has been shown to be overexpressed in metastatic prostate cancers and is currently being studied as a target for new chemotherapeutic agents in prostate cancer.18 Our observation that EZH2 predicts prostate cancers with higher Gleason score and nodal metastases in this study appears to confirm observations by others linking it to aggressive prostate cancers with high metastatic potential.40,46 ALDH1 expressing and EZH2 expressing cells probably contribute to prostate cancer progression via different pathways that are yet to be discovered. In our cohort the two markers rarely stained the same tumour cells in a case. SOX2 has been shown to correlate with tumour progression and high Gleason score in prostate adenocarcinoma in previous studies.24,47 In this study, only the association between neoplastic epithelial cell staining for SOX2 and lymphovascular invasion was statistically significant ( p ¼ 0.018). The protein is believed to have a role in tumour progression and metastases.48 It has previously been shown that the SOX2 gene is androgen dependent and up-regulated in late stages of castration treated prostate cancer, thereby promoting treatment resistance. In contrast, in our study no association between AR and SOX2 immunohistochemical staining was found. However, we have not investigated the relationship between treatment modalities, expression of AR and the three putative CSC markers. Incidentally, SOX2 stained basal cells in almost all cases (99%), suggesting it may be a useful alternative to basal cell markers in current use (p63, PIN4) for differentiating benign from malignant acini in prostate biopsies. The antibody also showed stronger nuclear positivity for benign urothelium and ganglion cells and further studies may be required to determine if it can be useful in distinguishing urothelial carcinoma from prostatic adenocarcinoma. Limitations of this study include its retrospective nature and a short follow-up duration which precluded evaluation of each marker’s association with mortality, biochemical recurrence and survival outcomes. In addition, while immunohistochemical prognostic markers are useful if applied to small biopsies before radical surgery, our study was carried out on whole sections of the largest tumour nodule in each case after prostatectomy. Future studies will need to look at the practical utility of ALDH1 and EZH2 in prostate core biopsies to predict tumour aggressiveness before radical treatment is rendered. In conclusion, similar to other solid and haematopoietic malignancies, prostate adenocarcinomas variably express the CSC markers ALDH1, EZH2 and SOX2. Immunopositivity for these proteins correlate with adverse pathological prognostic parameters and may be predictive of tumour aggressiveness in
627
prostate cancer. Further studies are required to determine if these markers have a role in triaging of prostate cancer in core biopsies and whether they can be future targets for treatment of aggressive prostate cancers. Acknowledgements: The authors thank Associate Professor Jane Armes (Mater Pathology, South Brisbane, Qld) and Dr Kris Kerr (Sullivan Nicolaides Pathology, Taringa, Qld) for peer review of the manuscript. Conflict of interest and sources of funding: The study was funded by the Mater Medical Research Institute, South Brisbane. The authors state that there are no conflicts of interest to disclose. Address for correspondence: Dr A. Matsika, Mater Pathology, Mater Health Services, Level 3, Duncombe Building, Raymond Terrace, South Brisbane, Qld 4101, Australia. E-mail: [email protected]
References 1. Hahn WC, Weinberg RA. Rules for making human tumor cells. N Engl J Med 2002; 347: 1593–603. 2. Tu SM, Lin SH. Prostate cancer stem cells. Clin Genitourin Cancer 2012; 10: 69–76. 3. De Marzo AM, Nelson WG, Meeker AK, Coffey DS. Stem cell features of benign and malignant prostate epithelial cells. J Urol 1998; 160: 2381–92. 4. Alison MR, Lim SM, Nicholson LJ. Cancer stem cells: problems for therapy? J Pathol 2011; 223: 147–61. 5. La Porta CA. Thoughts about cancer stem cells in solid tumors. World J Stem Cells 2012; 4: 17–20. 6. Sharpe B, Beresford M, Bowen R, Mitchard J, Chalmers AD. Searching for prostate cancer stem cells: markers and methods. Stem Cell Rev 2013; 9: 721–30. 7. Yoshida A, Hsu LC, Dave V. Retinal oxidation activity and biological role of human cytosolic aldehyde dehydrogenase. Enzyme 1992; 46: 239– 44. 8. Moreb JS, Mohuczy D, Ostmark B, Zucali JR. RNAi-mediated knockdown of aldehyde dehydrogenase class-1A1 and class-3A1 is specific and reveals that each contributes equally to the resistance against 4-hydroperoxycyclophosphamide. Cancer Chemother Pharmacol 2007; 59: 127– 36. 9. Armstrong L, Stojkovic M, Dimmick I, et al. Phenotypic characterization of murine primitive hematopoietic progenitor cells isolated on basis of aldehyde dehydrogenase activity. Stem Cells 2004; 22: 1142–51. 10. Cheung AM, Wan TS, Leung JC, et al. Aldehyde dehydrogenase activity in leukemic blasts defines a subgroup of acute myeloid leukemia with adverse prognosis and superior NOD/SCID engrafting potential. Leukemia 2007; 21: 1423–30. 11. De Brot M, Rocha RM, Soares FA, Gobbi H. Prognostic impact of the cancer stem cell related markers ALDH1 and EZH2 in triple negative and basal-like breast cancers. Pathology 2012; 44: 303–12. 12. Yoshida A, Hsu LC, Yanagawa Y. Biological role of human cytosolic aldehyde dehydrogenase 1: hormonal response, retinal oxidation and implication in testicular feminization. Adv Exp Med Biol 1993; 328: 37–44. 13. Li T, Su Y, Mei Y, et al. ALDH1A1 is a marker for malignant prostate stem cells and predictor of prostate cancer patients’ outcome. Lab Invest 2010; 90: 234–44. 14. Nishida S, Hirohashi Y, Torigoe T, et al. Gene expression profiles of prostate cancer stem cells isolated by aldehyde dehydrogenase activity assay. J Urol 2012; 188: 294–9. 15. van den Hoogen C, van der Horst G, Cheung H, et al. High aldehyde dehydrogenase activity identifies tumor-initiating and metastasis-initiating cells in human prostate cancer. Cancer Res 2010; 70: 5163–73. 16. Chen H, Rossier C, Antonarakis SE. Cloning of a human homolog of the Drosophila enhancer of zeste gene (EZH2) that maps to chromosome 21q22. 2. Genomics 1996; 38: 30–7. 17. Chou RH, Yu YL, Hung MC. The roles of EZH2 in cell lineage commitment. Am J Transl Res 2011; 3: 243–50. 18. Yang YA, Yu J. EZH2, an epigenetic driver of prostate cancer. Protein Cell 2013; 4: 331–41. 19. Bryant RJ, Cross NA, Eaton CL, Hamdy FC, Cunliffe VT. EZH2 promotes proliferation and invasiveness of prostate cancer cells. Prostate 2007; 67: 547–56.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
628
MATSIKA et al.
20. Bachmann IM, Halvorsen OJ, Collett K, et al. EZH2 expression is associated with high proliferation rate and aggressive tumor subgroups in cutaneous melanoma and cancers of the endometrium, prostate, and breast. J Clin Oncol 2006; 24: 268–73. 21. Stevanovic M, Zuffardi O, Collignon J, Lovell-Badge R, Goodfellow P. The cDNA sequence and chromosomal location of the human SOX2 gene. Mamm Genome 1994; 5: 640–2. 22. Boyer LA, Lee TI, Cole MF, et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 2005; 122: 947–56. 23. Rybak AP, Tang D. SOX2 plays a critical role in EGFR-mediated selfrenewal of human prostate cancer stem-like cells. Cell Signal 2013; 25: 2734–42. 24. Jia X, Li X, Xu Y, et al. SOX2 promotes tumorigenesis and increases the anti-apoptotic property of human prostate cancer cell. J Mol Cell Biol 2011; 3: 230–8. 25. Australian Institute of Health and Welfare (AIHW). Cancer in Australia: an overview 2012. Cat no. CAN 70. Canberra: AIHW, 2012. 26. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin 2013; 63: 11–30. 27. Ahmed HU. Prostate cancer: Time for active surveillance of intermediaterisk disease? Nat Rev Urol 2013; 10: 6–8. 28. Dall’Era MA, Albertsen PC, Bangma C, et al. Active surveillance for prostate cancer: a systematic review of the literature. Eur Urol 2012; 62: 976–83. 29. Trock BJ, Guo CC, Gonzalgo ML, Magheli A, Loeb S, Epstein JI. Tertiary Gleason patterns and biochemical recurrence after prostatectomy: proposal for a modified Gleason scoring system. J Urol 2009; 182: 1364–70. 30. Epstein JI, Carmichael MJ, Pizov G, Walsh PC. Influence of capsular penetration on progression following radical prostatectomy: a study of 196 cases with long-term followup. J Urol 1993; 150: 135–41. 31. Edge SB, Byrd DR, Compton CC. AJCC Cancer Staging Manual. 7th ed. New York: Springer, 2010; 649. 32. Wheeler TM, Dillioglugil O, Kattan MW, et al. Clinical and pathological significance of the level and extent of capsular invasion in clinical stage T1-2 prostate cancer. Hum Pathol 1998; 29: 856–62. 33. Bremer B, Tacha D. ALDH1A1 as a marker of stem cells in prostate cancer: correlation with Gleason scores and tumor stage and relevance for patient outcome. Arch Pathol Lab Med 2011; 135: 1102–230; (Abstr 761). 34. Le Magnen C, Bubendorf L, Rentsch CA, et al. Characterization and clinical relevance of ALDHbright populations in prostate cancer. Clin Cancer Res 2013; 19: 5361–71. 35. Tuxhorn JA, Ayala GE, Rowley DR. Reactive stroma in prostate cancer progression. J Urol 2001; 166: 2472–83.
Pathology (2015), 47(7), December
36. Chung LW, Baseman A, Assikis V, Zhau HE. Molecular insights into prostate cancer progression: the missing link of tumor microenvironment. J Urol 2005; 173: 10–20. 37. Dakhova O, Rowley D, Ittmann M. Genes upregulated in prostate cancer reactive stroma promote prostate cancer progression in vivo. Clin Cancer Res 2013; 22: 22. 38. Wolters T, Vissers KJ, Bangma CH, Schroder FH, van Leenders GJ. The value of EZH2, p27(kip1), BMI-1 and MIB-1 on biopsy specimens with low-risk prostate cancer in selecting men with significant prostate cancer at prostatectomy. BJU Int 2010; 106: 280–6. 39. Laitinen S, Martikainen PM, Tolonen T, Isola J, Tammela TL, Visakorpi T. EZH2, Ki-67 and MCM7 are prognostic markers in prostatectomy treated patients. Int J Cancer 2008; 122: 595–602. 40. Varambally S, Dhanasekaran SM, Zhou M, et al. The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature 2002; 419: 624–9. 41. Rhodes DR, Sanda MG, Otte AP, Chinnaiyan AM, Rubin MA. Multiplex biomarker approach for determining risk of prostate-specific antigen-defined recurrence of prostate cancer. J Natl Cancer Inst 2003; 95: 661–8. 42. Karanikolas BD, Figueiredo ML, Wu L. Comprehensive evaluation of the role of EZH2 in the growth, invasion, and aggression of a panel of prostate cancer cell lines. Prostate 2010; 70: 675–88. 43. Saramaki OR, Tammela TL, Martikainen PM, Vessella RL, Visakorpi T. The gene for polycomb group protein enhancer of zeste homolog 2 (EZH2) is amplified in late-stage prostate cancer. Genes Chromosomes Cancer 2006; 45: 639–45. 44. Tamgue O, Chai CS, Hao L, et al. Triptolide inhibits histone methyltransferase EZH2 and modulates the expression of its target genes in prostate cancer cells. Asian Pac J Cancer Prev 2013; 14: 5663–9. 45. van den Hoogen C, van der Horst G, Cheung H, Buijs JT, Pelger RC, van der Pluijm G. The aldehyde dehydrogenase enzyme 7A1 is functionally involved in prostate cancer bone metastasis. Clin Exp Metastasis 2011; 28: 615–25. 46. Hoffmann MJ, Engers R, Florl AR, Otte AP, Muller M, Schulz WA. Expression changes in EZH2, but not in BMI-1, SIRT1, DNMT1 or DNMT3B are associated with DNA methylation changes in prostate cancer. Cancer Biol Ther 2007; 6: 1403–12. 47. Kregel S, Kiriluk KJ, Rosen AM, et al. Sox2 is an androgen receptorrepressed gene that promotes castration-resistant prostate cancer. PLoS One 2013; 8: 11. 48. Li X, Xu Y, Chen Y, et al. SOX2 promotes tumor metastasis by stimulating epithelial-to-mesenchymal transition via regulation of WNT/beta-catenin signal network. Cancer Lett 2013; 336: 379–89.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
ANATOMICAL PATHOLOGY
Cancer stem cell markers in prostate cancer: an immunohistochemical study of ALDH1, SOX2 and EZH2 ADMIRE MATSIKA1,2,3, BHUVANA SRINIVASAN1, CHRISTOPHER DAY1,2,3, SABRINA ANN MADER4, DEIRDRE MARGARET KIERNAN4, AMY BROOMFIELD1, JINLIN FU5, JOHN D. HOOPER5, JAMES G. KENCH6 AND HEMAMALI SAMARATUNGA3,7 1Department of Anatomical Pathology, Mater Health Services, South Brisbane, 2Pathology Queensland, Royal Brisbane and Women’s Hospital, Herston, 3Department of Pathology, University of Queensland, Brisbane, 4Department of Urology, Mater Adult Hospital, South Brisbane, 5Mater Research Institute – University of Queensland, Translational Research Institute, Woolloongabba, Qld, 6Department of Tissue Pathology and
Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown and Sydney Medical School, The University of Sydney, NSW, and 7Aquesta Pathology, Taringa, Qld, Australia
Summary The aims of this study were to investigate the immunohistochemical expression and potential prognostic significance of putative cancer stems cell markers ALDH1, EZH2 and SOX2 in prostate cancer. A total of 142 consecutive radical prostatectomies submitted to one laboratory with a diagnosis of prostatic adenocarcinoma between 2008 and 2012 were retrieved and retrospectively studied. Immunohistochemistry for the three markers was performed in each case and both univariate and multivariate analyses were undertaken to evaluate the correlation between the staining patterns and known histopathological prognostic features. ALDH1 showed a statistically significant association with tumour stage p < 0.001), extraprostatic extension ( p < 0.001) and lymphovascular invasion ( p ¼ 0.001). EZH2 correlated with Gleason score ( p ¼ 0.044) and lymph node metastases ( p ¼ 0.023). SOX2 showed a statistically significant correlation with lymphovascular invasion only ( p ¼ 0.018) in both univariate and multivariate analyses. Cancer stem cell markers are variably expressed in prostate adenocarcinoma and immunohistochemical staining for ALDH1 and EZH2 may have a role in predicting tumour aggressiveness before treatment of prostate cancer. Key words: Adenocarcinoma, ALDH1, androgen receptor, cancer stem cell theory, cancer stem cells, EZH2, immunohistochemistry, prognostic markers, prostate cancer, SOX2. Received 11 November 2014, revised 28 June, accepted 30 June 2015
INTRODUCTION The classical concept of cancer pathogenesis stipulates that tumours arise from stepwise accumulation of multiple mutations in mature somatic cells and that all cells in a cancer share similar molecular aberrations.1 The cancer stem cell (CSC) theory is an alternative, fairly new paradigm and hypothesises that distinct clones of cancer cells result from abnormalities that occur within specific cell types.2,3 In this theory, cells in a tumour do not have the same malignant potential but rather there are small clones of CSCs with selfrenewal capabilities, high proliferative potential and Print ISSN 0031-3025/Online ISSN 1465-3931 Copyright DOI: 10.1097/PAT.0000000000000325
#
pluripotency status that are responsible for tumour initiation, propagation and metastases.4 The self-propagating ability of these cells is demonstrated by their ability to establish xenografts in mice.5 Enzymatic assays and immunohistochemistry have identified several proteins and surface markers that are expressed by CSCs and may be used to identify them in patients.6 Aldehyde dehydrogenase 1A1 (ALDH1) is an intracellular enzyme that oxidises retinol to retinoic acid, an important regulator of cellular differentiation.7 This enzyme is also involved in detoxification and has been implicated in the emergence of drug resistance to chemotherapeutic agents.8 There is also evidence that ALDH1 is overexpressed in CSCs of various organs including haematopoietic,9,10 breast,11 pancreas, lung, colorectal and prostate.12 Fluorescence based biochemical studies have also shown that ALDH1 is a useful marker for prostate CSCs. Consistent with a role in prostate cancer, ALDH1 rich prostate cancer cells can initiate and propagate prostate cancers when transplanted into mice while those low in ALDH1 do not.13–15 Enhancer of zeste homolog 2 (EZH2), a catalytic subunit of the Polycomb Repressive Complex responsible for histone methylation, regulates gene transcription and chromatin structure.16 Studies to date suggest that EZH2, which is also overexpressed in CSCs, plays a critical role in stem cell renewal, maintenance, and differentiation into specific cell lineages.17 It is overexpressed in aggressive solid tumours, including metastatic prostate cancer and may be a useful prognostic marker in clinical practice18–20 with chemotherapeutic agents currently being developed that specifically target the enzyme.18 More than a dozen SOX genes have been described and are named after a shared motif called the SRY box, a region homologous to the DNA-binding domain of SRY, the mammalian sex determining gene.21 One of these genes encodes for a transcription factor SOX2 which has a well-known role in the maintenance and acquisition of stem cell features.22 In prostate cancer SOX2 is overexpressed in CSCs where it mediates tumourigenesis and has been linked to poor prognosis.23,24 Prostate cancer is the most commonly diagnosed non-skin cancer in men in the western world with mortality rates only second to lung cancer.25,26 It is generally accepted that not all prostate cancers require active aggressive treatment as some
2015 Royal College of Pathologists of Australasia. All rights reserved.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
STEM CELL MARKERS IN PROSTATE CANCER: IHC STUDY OF ALDH1, SOX2 AND EZH2
men die with their disease rather than from it. Some of the current radical treatment modalities for the disease are associated with considerable morbidity. Accordingly, there is a need for a diagnostic tool, for use in core biopsies, that can identify aggressive prostate cancers that will require radical treatment and distinguish them from indolent disease that can be managed conservatively with active surveillance.27,28 It is on this background that we have performed this study to evaluate the putative CSC markers: ALDH1, EZH2 and SOX2 as potential prognostic markers in prostate cancer. Androgen receptor (AR) staining of the prostate cancers was also performed to determine its association with these novel proteins.
MATERIALS AND METHODS Study design For this study 142 consecutive cases of prostate cancer that underwent radical prostatectomy between 2008 and 2012 were retrieved from the Mater Pathology archives. The study was approved by the Research Ethics Committee of our institution. Haematoxylin and eosin (H&E) stained sections of totally embedded prostatectomies were reviewed by two of the authors (AM, BS). A standard paraffin block with the highest Gleason score and largest tumour area from each case was selected for immunohistochemical staining. The following data were collected from histological and clinical records: patient age at time of prostatectomy, histological type, Gleason score, pathological stage (pT), nodal stage (pN); presence and extent of extraprostatic extension (EPE) and lymphovascular invasion (LVI). Immunohistochemistry and histopathological parameters Immunohistochemistry staining was performed on formalin fixed, paraffin embedded sections (4 mm). A positive control was present on each slide to determine the validity of the stain. All slides were stained using the Ventana Benchmark Ultra automated immunohistochemistry staining machine, using the Ventana Ultra View DAB detection system (Ventana, USA). The antibodies were: ALDH1, Clone 44/ALDH, 1/1000 dilution (BD Transduction Laboratories, USA); EZH2, Clone AP129, prediluted (Ventana); SOX2, Clone SP76, 1/20 dilution (Cell Marque, USA) and androgen receptor (AR), Clone AR441, 1/20 dilution (Cell Marque). All sections were subjected to heat-induced epitope retrieval in CC1 solution for 64 min at 958C and antibodies were incubated at 368C for 32 min. Stained slides were examined by two pathologists (BS, AM) blinded to the pathological prognostic parameters of the tumours. Only staining of malignant epithelial cells was evaluated in this study. The percentage of cells staining (0–100%) was recorded for each case and scored as follows: 0–33%, score 1; 34–66%, score 2 and >67%, score 3. The intensity of staining (0, none; 1, mild; 2, moderate; 3, strong) for each antibody was also recorded and an H-score like staining index (SI) was calculated for each antibody as follows: SI (0– 9) ¼ intensity score (0–3) percentage staining score (0–3). A modified Gleason score after Trock et al.29 that takes into account tertiary patterns (TGS5) and the prognostic differences of Gleason scores 4þ3 versus 3þ4 was used for statistical analysis.29 EPE was scored as follows as per Epstein’s proposal:30 0, absent; 1, focal; 2, extensive. LVI was also scored as follows: 0, absent; 1, focal; 2, extensive. Absence of nodal involvement (pN0) was scored 0 and pN1 as 1. pT category according to the American Joint Committee on Cancer, 7th edition,31 was recorded and scored as follows for statistical purpose: pT2 ¼ 2.0, pT3a ¼ 3.0, pT3b ¼ 3.5 and pT4 ¼ 4. Statistical analysis Mean and standard deviations were used to describe age at prostatectomy. Absolute counts and proportions were used for categorical variables. Spearman’s rank correlation was used to examine correlations between each of the markers and the known prognostic parameters. Correlation coefficients and significance levels were reported. Logistic and ordinal logistic regressions were also employed to detect the relationships between the variables. All analyses were performed using Stata/SE 12.1 for Windows. p values <0.05 were considered statistically significant.
Table 1
623
Characteristics of patients
Characteristics Mean age, years (range) 61.1 (43–76) Gleason score 3þ3 ¼ 6 3þ3 ¼ 6 with TGS5 3þ4 ¼ 7 3þ4 ¼ 7 with TGS5 4þ3 ¼ 7 4þ3 ¼ 7 with TGS5 4þ4 ¼ 8 4þ4 ¼ 8 with TGS5 4þ5 ¼ 9 5þ4 ¼ 9 pT stage pT2 pT3a pT3b pN stage pN0 pN1 pNX LVI Absent Focal Extensive EPE Absent Focal Extensive
No. cases (%)
142 (100) 10 5 46 6 26 12 12 12 12 1
(7.0) (3.5) (32.4) (4.2) (18.3) (8.5) (8.5) (8.5) (8.5) (0.7)
67 (50.75) 57 (40.10) 13 (9.15) 111 (78.17) 13 (9.15) 18 (12.68) 120 (84.51) 16 (11.27) 6 (4.23) 72 (50.70) 46 (32.39) 24 (16.90)
EPE, extraprostatic extension; LVI, lymphovascular invasion; pN, pathological lymph nodal stage; pNX, unknown nodal status; pT, pathological tumour stage; TGS, tertiary Gleason score 5 pattern.
RESULTS Patient characteristics The study included 142 patients with a mean age of 61 years and range 43–76 years (Table 1). Gleason score 3þ4¼7 was the most common score in this cohort with 46 cases (32.4%). Nodal metastases were present in 9% (n ¼ 13) and in 18 cases it was unknown (pNX). LVI was seen in 22 cases, six of which had extensive involvement. Almost half of the prostatectomies (n ¼ 70) had some degree of EPE, and in 24 of these the extension had been reported as extensive by previously reported criteria.30,32 ALDH1 expression About half (n ¼ 69) of the 142 cases exhibited cytoplasmic staining for ALDH1. There were marked differences in the proportion of malignant cells expressing ALDH1, ranging from <5% up to more than 90%. As shown in Table 2, statistical analysis across the patient cohort indicated that ALDH1 staining index significantly correlated with pT ( p < 0.001), EPE ( p < 0.001) and LVI ( p ¼ 0.001). The association with Gleason score ( p ¼ 0.075) and nodal metastases ( p ¼ 0.280) did not reach statistical significance. ALDH1 positive epithelial tumour cells tended to be larger with more prominent nucleoli compared to other malignant cells of the same tumour (Fig. 1). ALDH1 also showed some positivity—albeit patchy and milder—in the stroma surrounding ALDH1 positive epithelial tumour cells (Fig. 2A). This stromal accentuation was not seen in acini of ALDH1 negative carcinomas. Incidentally, the antibody was invariably positive in seminal vesicles (100%) and always weakly positive in benign urothelium and ganglia
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
624 Table 2
Pathology (2015), 47(7), December
MATSIKA et al.
Summary of correlation between staining categories of ALDH1, EZH2 and SOX2 and pathological prognostic parameters ALDH1
GS EPE LVI pT pN
EZH2
SOX2
Spearman coefficient
p value
Spearman coefficient
p value
Spearman coefficient
p value
0.150 0.346 0.273 0.348 0.098
0.075 <0.001 <0.001 <0.001 0.280
0.240 0.071 0.036 0.112 0.204
0.004 0.405 0.674 0.184 0.023
0.102 0.129 0.199 0.144 0.137
0.228 0.125 0.018 0.088 0.130
p values <0.05 are in bold. EPE, extraprostatic extension; GS, Gleason score; LVI, lymphovascular invasion; pN, pathological lymph nodal stage; pT, pathological tumour stage.
incorporated in the examined sections (Fig. 2B). Benign prostatic acini were consistently negative for ALDH1 in this cohort.
2). There was no statistically significant association with any of the other histopathological parameters studied (Table 2). The antibody also strongly and uniformly stained basal cells of benign prostatic acini (99%), high grade PIN (Fig. 4C), benign urothelium and ganglia in almost all cases (Fig. 4D). As with the other two markers, no positive nuclear staining of benign prostatic epithelium was seen with SOX2 in all cases.
EZH2 expression Ninety-nine percent (n ¼ 139) of the prostate carcinomas showed at least 1% nuclear staining with EZH2 (Fig. 3A,B). EZH2 nuclear staining was present only in malignant cells and absent in benign acini (Fig. 3C). Of note, in each of the positive cases there was a sprinkling of more deeply staining malignant cells (Fig. 3D). Analysis of both staining intensity and percentage of cells staining showed that EZH2 nuclear staining in prostate carcinoma cells has a statistically significant association with Gleason score ( p ¼ 0.004) and nodal stage ( p ¼ 0.023). In contrast to ALDH1, there was no correlation between EZH2 staining and EPE, LVI or pT.
Correlation between ALDH1, EZH2 and SOX2 As shown in Table 3, there was a statistically significant correlation between ALDH1 and SOX2 ( p ¼ 0.001), as well as between EZH2 and SOX2 ( p ¼ 0.040). However, no significant association was observed between ALDH1 and EZH2. Androgen receptor expression Androgen receptor (AR) immunohistochemistry was performed on 138 cases and all but two (98.6%) showed at least moderate staining in the majority of tumour cells (not shown). There was no statistically significant correlation between AR staining and prognostic pathological parameters or staining with the three cancer stem cell markers.
SOX2 expression SOX2 immunohistochemistry showed nuclear positivity in epithelial tumour cells in 61 of the 142 (46.38%) (Fig. 4A,B). Staining of epithelial tumour cells showed a statistically significant association ( p ¼ 0.018) with LVI (Table
A
B
C
D
Fig. 1 ALDH1 immunohistochemistry staining in prostate carcinoma. (A) Low power, (B) high power. Benign acini are negative. (C) Note the tumour cells with larger nucleoli staining for ALDH1 while (D) smaller malignant cells in the same section are negative.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
STEM CELL MARKERS IN PROSTATE CANCER: IHC STUDY OF ALDH1, SOX2 AND EZH2
625
DISCUSSION We have investigated the immunohistochemical expression and potential prognostic significance of the three putative prostate cancer stem cell markers ALDH1, EZH2 and SOX2 in a cohort of 142 prostatectomies. While previous studies have utilised in vitro biochemical methods or small tissue samples on tissue microarrays (TMA), we have immunohistochemically stained standard sections from wholly embedded radical prostatectomies with commercially available antibodies for the three proteins as well as for the androgen receptor (AR). This cohort is larger than similar previous studies and the first to investigate CSC markers in standard sections from prostatectomies. The use of standard sections has allowed for better characterisation of the staining patterns including analysis of heterogeneity within an individual tumour. Tumour heterogeneity is expected with these markers as, according to the stem cell theory, only a small fraction of the tumour bulk has stem cell properties. Our study also addresses another major limitation of studies using TMA and core biopsies, which is the difficulty of examining other facets of tumour biology such as the interactions between tumour bulk and its stromal microenvironment. Stroma and benign tissue are usually not adequately represented in the miniature tissue fragments from TMA and core biopsies. Additionally, standard sections have also enabled us to study the immunohistochemical staining characteristics of malignant prostatic stroma as well as benign elements such as basal cells, stroma, seminal vesicle epithelium, nerves and ganglia. Consequently, we have also uncovered some potential utilities of ALDH1, EZH2 and SOX2 in diagnostic practice due to their unique staining of benign elements. Our study shows that the CSC marker ALDH1 is expressed in prostate carcinomas, and more likely so in cases with adverse pathological parameters, such as extraprostatic extension, lymphovascular invasion and higher pathological tumour stage. Previous studies have shown similar correlations between
A
B Fig. 2 ALDH1 immunohistochemistry in prostate carcinoma. (A) Stromal accentuation around malignant acini is characteristic. (B) Seminal vesicles (and neural tissue, not shown) also strongly stain for the marker in almost all cases. Adjacent prostatic acini are negative in this case.
A
B
C
D
Fig. 3 EZH2 immunohistochemistry staining in prostate carcinoma. (A) Low power, (B) high power. (C) Adjacent benign acini are negative. (D) Note the sprinkling of more deeply staining malignant cells which were found to be characteristic of this antibody.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
626
Pathology (2015), 47(7), December
MATSIKA et al.
A
B
C
D
Fig. 4 SOX2 immunohistochemistry staining in prostate carcinoma. (A) Low power, (B) medium power. The basal cells in benign acini and malignant epithelia are positive while benign epithelial cells do not stain. (C) High grade PIN positive for SOX2. (D) Ganglia and neural tissue nuclei are strongly positive.
ALDH1 and poor prognosticators as well as disease free survival13,33,34 on prostate core biopsies and TMAs. The CSC theory is controversial and not unanimously accepted.6 The immunohistochemical staining pattern observed in this cohort whereby some but not all of the tumour cells overexpress ALDH1, EZH2 and/or SOX2 adds weight to this theory. There appears to be small subsets of tumour cells in aggressive prostate cancers that produce CSC proteins. The associations of these CSC proteins with histopathological staging parameters in this study suggest that these cells may be drivers of tumour progression and metastases. If confirmed to be correct, therapeutic agents could be developed that target CSCs rendering the tumours more indolent. A peculiar observation in our study was that ALDH1 positive epithelial tumour cells tended to be larger with more prominent nucleoli compared to other malignant cells in the same tumours (Fig. 1C,D). We interpret this as morphology-genotype correlation evidence of tumour cell diversity and further support for the CSC theory in prostate cancer. The behaviour of tumour cells is also influenced by the microenvironment, such as the stromal component, blood vessels and inflammatory cells.35,36 Immunohistochemistry Table 3
ALDH1 EZH2 SOX2
Correlations among CSC markers in prostate carcinoma ALDH1
EZH2
SOX2
– (1.000) 0.177 (0.114) 0.001 (0.284)
– (1.000) 0.040 (0.173)
– (1.000)
The p values are italicised and the correlation coefficients are in parentheses. The bold values indicate a statistically significant result.
with ALDH1 showed some positivity—albeit patchy and milder—in the stroma surrounding ALDH1 positive epithelial tumour cells (Fig. 2). This ‘stromal blushing’ was absent in ALDH1 negative sections or tumours and suggests that the stroma around CSCs could be abnormal at a molecular level, even though it may appear unremarkable on light microscopy. Other authors have come to the same conclusion in the past using different techniques35,37 and have concluded that the stroma (microenvironment) probably plays a more significant role in tumourigenesis and propagation than is currently appreciated. Our study shows a statistically significant association between EZH2, Gleason score and pathological nodal stage. Wolters et al. have previously also showed a significant association between EZH2 and high Gleason score, tumour volume and EPE in a study of 86 prostate core biopsies.38 Other authors have reported an association between EZH2 expression and disease free survival39,40 while some have found that it is not an independent prognosticator.41 These studies have used TMAs and prostate core biopsies which, by their nature, limit tumour sampling and do not take into account the tumour heterogeneity and patchy staining expected of stem cell markers. Subtle differences between our data and these reports may also be explained by the use of different EZH2 antibody clones with different specificities to target antigens. The antibody we used proved to be a very sensitive marker for prostatic malignant cells with nuclear staining of variable extent and intensity in 98% of the tumours while sparing benign prostatic epithelium in the same sections. Thus, it may have a role in diagnostic pathology to distinguish malignant prostatic from benign prostatic acini and urothelium. Karanikolas et al. have suggested that EZH2 is required for tumour progression in androgen independent but not androgen dependent prostate cancers.42,43 Therefore, EZH2 would be expected to have prognostic value in androgen receptor positive
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
STEM CELL MARKERS IN PROSTATE CANCER: IHC STUDY OF ALDH1, SOX2 AND EZH2
tumours. In our patient cohort, androgen receptor immunohistochemistry was performed in 138 cases and all but one (99.3%) showed at least moderate staining in the majority of tumour cells. We did not find any association between EHZ2 staining and androgen receptor expression. The complex role of EZH2 in prostate cancer requires further study as potential therapeutic agents targeting its pathway are being developed.44 Interestingly the prognostic parameters associated with EZH2 (Gleason score and pN stage) in this study differ from those associated with ALDH1 expression (EPE, LVI and pT). In CSC theory this may be explained by the observations from other studies that tumours have different CSC clones with different roles as well as biochemical and molecular properties.5 One study for example has shown that ALDH7A1, another aldehyde dehydrogenase isoform, is a requisite for bone metastases in prostate cancer.15,45 EZH2 has been shown to be overexpressed in metastatic prostate cancers and is currently being studied as a target for new chemotherapeutic agents in prostate cancer.18 Our observation that EZH2 predicts prostate cancers with higher Gleason score and nodal metastases in this study appears to confirm observations by others linking it to aggressive prostate cancers with high metastatic potential.40,46 ALDH1 expressing and EZH2 expressing cells probably contribute to prostate cancer progression via different pathways that are yet to be discovered. In our cohort the two markers rarely stained the same tumour cells in a case. SOX2 has been shown to correlate with tumour progression and high Gleason score in prostate adenocarcinoma in previous studies.24,47 In this study, only the association between neoplastic epithelial cell staining for SOX2 and lymphovascular invasion was statistically significant ( p ¼ 0.018). The protein is believed to have a role in tumour progression and metastases.48 It has previously been shown that the SOX2 gene is androgen dependent and up-regulated in late stages of castration treated prostate cancer, thereby promoting treatment resistance. In contrast, in our study no association between AR and SOX2 immunohistochemical staining was found. However, we have not investigated the relationship between treatment modalities, expression of AR and the three putative CSC markers. Incidentally, SOX2 stained basal cells in almost all cases (99%), suggesting it may be a useful alternative to basal cell markers in current use (p63, PIN4) for differentiating benign from malignant acini in prostate biopsies. The antibody also showed stronger nuclear positivity for benign urothelium and ganglion cells and further studies may be required to determine if it can be useful in distinguishing urothelial carcinoma from prostatic adenocarcinoma. Limitations of this study include its retrospective nature and a short follow-up duration which precluded evaluation of each marker’s association with mortality, biochemical recurrence and survival outcomes. In addition, while immunohistochemical prognostic markers are useful if applied to small biopsies before radical surgery, our study was carried out on whole sections of the largest tumour nodule in each case after prostatectomy. Future studies will need to look at the practical utility of ALDH1 and EZH2 in prostate core biopsies to predict tumour aggressiveness before radical treatment is rendered. In conclusion, similar to other solid and haematopoietic malignancies, prostate adenocarcinomas variably express the CSC markers ALDH1, EZH2 and SOX2. Immunopositivity for these proteins correlate with adverse pathological prognostic parameters and may be predictive of tumour aggressiveness in
627
prostate cancer. Further studies are required to determine if these markers have a role in triaging of prostate cancer in core biopsies and whether they can be future targets for treatment of aggressive prostate cancers. Acknowledgements: The authors thank Associate Professor Jane Armes (Mater Pathology, South Brisbane, Qld) and Dr Kris Kerr (Sullivan Nicolaides Pathology, Taringa, Qld) for peer review of the manuscript. Conflict of interest and sources of funding: The study was funded by the Mater Medical Research Institute, South Brisbane. The authors state that there are no conflicts of interest to disclose. Address for correspondence: Dr A. Matsika, Mater Pathology, Mater Health Services, Level 3, Duncombe Building, Raymond Terrace, South Brisbane, Qld 4101, Australia. E-mail: [email protected]
References 1. Hahn WC, Weinberg RA. Rules for making human tumor cells. N Engl J Med 2002; 347: 1593–603. 2. Tu SM, Lin SH. Prostate cancer stem cells. Clin Genitourin Cancer 2012; 10: 69–76. 3. De Marzo AM, Nelson WG, Meeker AK, Coffey DS. Stem cell features of benign and malignant prostate epithelial cells. J Urol 1998; 160: 2381–92. 4. Alison MR, Lim SM, Nicholson LJ. Cancer stem cells: problems for therapy? J Pathol 2011; 223: 147–61. 5. La Porta CA. Thoughts about cancer stem cells in solid tumors. World J Stem Cells 2012; 4: 17–20. 6. Sharpe B, Beresford M, Bowen R, Mitchard J, Chalmers AD. Searching for prostate cancer stem cells: markers and methods. Stem Cell Rev 2013; 9: 721–30. 7. Yoshida A, Hsu LC, Dave V. Retinal oxidation activity and biological role of human cytosolic aldehyde dehydrogenase. Enzyme 1992; 46: 239– 44. 8. Moreb JS, Mohuczy D, Ostmark B, Zucali JR. RNAi-mediated knockdown of aldehyde dehydrogenase class-1A1 and class-3A1 is specific and reveals that each contributes equally to the resistance against 4-hydroperoxycyclophosphamide. Cancer Chemother Pharmacol 2007; 59: 127– 36. 9. Armstrong L, Stojkovic M, Dimmick I, et al. Phenotypic characterization of murine primitive hematopoietic progenitor cells isolated on basis of aldehyde dehydrogenase activity. Stem Cells 2004; 22: 1142–51. 10. Cheung AM, Wan TS, Leung JC, et al. Aldehyde dehydrogenase activity in leukemic blasts defines a subgroup of acute myeloid leukemia with adverse prognosis and superior NOD/SCID engrafting potential. Leukemia 2007; 21: 1423–30. 11. De Brot M, Rocha RM, Soares FA, Gobbi H. Prognostic impact of the cancer stem cell related markers ALDH1 and EZH2 in triple negative and basal-like breast cancers. Pathology 2012; 44: 303–12. 12. Yoshida A, Hsu LC, Yanagawa Y. Biological role of human cytosolic aldehyde dehydrogenase 1: hormonal response, retinal oxidation and implication in testicular feminization. Adv Exp Med Biol 1993; 328: 37–44. 13. Li T, Su Y, Mei Y, et al. ALDH1A1 is a marker for malignant prostate stem cells and predictor of prostate cancer patients’ outcome. Lab Invest 2010; 90: 234–44. 14. Nishida S, Hirohashi Y, Torigoe T, et al. Gene expression profiles of prostate cancer stem cells isolated by aldehyde dehydrogenase activity assay. J Urol 2012; 188: 294–9. 15. van den Hoogen C, van der Horst G, Cheung H, et al. High aldehyde dehydrogenase activity identifies tumor-initiating and metastasis-initiating cells in human prostate cancer. Cancer Res 2010; 70: 5163–73. 16. Chen H, Rossier C, Antonarakis SE. Cloning of a human homolog of the Drosophila enhancer of zeste gene (EZH2) that maps to chromosome 21q22. 2. Genomics 1996; 38: 30–7. 17. Chou RH, Yu YL, Hung MC. The roles of EZH2 in cell lineage commitment. Am J Transl Res 2011; 3: 243–50. 18. Yang YA, Yu J. EZH2, an epigenetic driver of prostate cancer. Protein Cell 2013; 4: 331–41. 19. Bryant RJ, Cross NA, Eaton CL, Hamdy FC, Cunliffe VT. EZH2 promotes proliferation and invasiveness of prostate cancer cells. Prostate 2007; 67: 547–56.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
628
MATSIKA et al.
20. Bachmann IM, Halvorsen OJ, Collett K, et al. EZH2 expression is associated with high proliferation rate and aggressive tumor subgroups in cutaneous melanoma and cancers of the endometrium, prostate, and breast. J Clin Oncol 2006; 24: 268–73. 21. Stevanovic M, Zuffardi O, Collignon J, Lovell-Badge R, Goodfellow P. The cDNA sequence and chromosomal location of the human SOX2 gene. Mamm Genome 1994; 5: 640–2. 22. Boyer LA, Lee TI, Cole MF, et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 2005; 122: 947–56. 23. Rybak AP, Tang D. SOX2 plays a critical role in EGFR-mediated selfrenewal of human prostate cancer stem-like cells. Cell Signal 2013; 25: 2734–42. 24. Jia X, Li X, Xu Y, et al. SOX2 promotes tumorigenesis and increases the anti-apoptotic property of human prostate cancer cell. J Mol Cell Biol 2011; 3: 230–8. 25. Australian Institute of Health and Welfare (AIHW). Cancer in Australia: an overview 2012. Cat no. CAN 70. Canberra: AIHW, 2012. 26. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin 2013; 63: 11–30. 27. Ahmed HU. Prostate cancer: Time for active surveillance of intermediaterisk disease? Nat Rev Urol 2013; 10: 6–8. 28. Dall’Era MA, Albertsen PC, Bangma C, et al. Active surveillance for prostate cancer: a systematic review of the literature. Eur Urol 2012; 62: 976–83. 29. Trock BJ, Guo CC, Gonzalgo ML, Magheli A, Loeb S, Epstein JI. Tertiary Gleason patterns and biochemical recurrence after prostatectomy: proposal for a modified Gleason scoring system. J Urol 2009; 182: 1364–70. 30. Epstein JI, Carmichael MJ, Pizov G, Walsh PC. Influence of capsular penetration on progression following radical prostatectomy: a study of 196 cases with long-term followup. J Urol 1993; 150: 135–41. 31. Edge SB, Byrd DR, Compton CC. AJCC Cancer Staging Manual. 7th ed. New York: Springer, 2010; 649. 32. Wheeler TM, Dillioglugil O, Kattan MW, et al. Clinical and pathological significance of the level and extent of capsular invasion in clinical stage T1-2 prostate cancer. Hum Pathol 1998; 29: 856–62. 33. Bremer B, Tacha D. ALDH1A1 as a marker of stem cells in prostate cancer: correlation with Gleason scores and tumor stage and relevance for patient outcome. Arch Pathol Lab Med 2011; 135: 1102–230; (Abstr 761). 34. Le Magnen C, Bubendorf L, Rentsch CA, et al. Characterization and clinical relevance of ALDHbright populations in prostate cancer. Clin Cancer Res 2013; 19: 5361–71. 35. Tuxhorn JA, Ayala GE, Rowley DR. Reactive stroma in prostate cancer progression. J Urol 2001; 166: 2472–83.
Pathology (2015), 47(7), December
36. Chung LW, Baseman A, Assikis V, Zhau HE. Molecular insights into prostate cancer progression: the missing link of tumor microenvironment. J Urol 2005; 173: 10–20. 37. Dakhova O, Rowley D, Ittmann M. Genes upregulated in prostate cancer reactive stroma promote prostate cancer progression in vivo. Clin Cancer Res 2013; 22: 22. 38. Wolters T, Vissers KJ, Bangma CH, Schroder FH, van Leenders GJ. The value of EZH2, p27(kip1), BMI-1 and MIB-1 on biopsy specimens with low-risk prostate cancer in selecting men with significant prostate cancer at prostatectomy. BJU Int 2010; 106: 280–6. 39. Laitinen S, Martikainen PM, Tolonen T, Isola J, Tammela TL, Visakorpi T. EZH2, Ki-67 and MCM7 are prognostic markers in prostatectomy treated patients. Int J Cancer 2008; 122: 595–602. 40. Varambally S, Dhanasekaran SM, Zhou M, et al. The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature 2002; 419: 624–9. 41. Rhodes DR, Sanda MG, Otte AP, Chinnaiyan AM, Rubin MA. Multiplex biomarker approach for determining risk of prostate-specific antigen-defined recurrence of prostate cancer. J Natl Cancer Inst 2003; 95: 661–8. 42. Karanikolas BD, Figueiredo ML, Wu L. Comprehensive evaluation of the role of EZH2 in the growth, invasion, and aggression of a panel of prostate cancer cell lines. Prostate 2010; 70: 675–88. 43. Saramaki OR, Tammela TL, Martikainen PM, Vessella RL, Visakorpi T. The gene for polycomb group protein enhancer of zeste homolog 2 (EZH2) is amplified in late-stage prostate cancer. Genes Chromosomes Cancer 2006; 45: 639–45. 44. Tamgue O, Chai CS, Hao L, et al. Triptolide inhibits histone methyltransferase EZH2 and modulates the expression of its target genes in prostate cancer cells. Asian Pac J Cancer Prev 2013; 14: 5663–9. 45. van den Hoogen C, van der Horst G, Cheung H, Buijs JT, Pelger RC, van der Pluijm G. The aldehyde dehydrogenase enzyme 7A1 is functionally involved in prostate cancer bone metastasis. Clin Exp Metastasis 2011; 28: 615–25. 46. Hoffmann MJ, Engers R, Florl AR, Otte AP, Muller M, Schulz WA. Expression changes in EZH2, but not in BMI-1, SIRT1, DNMT1 or DNMT3B are associated with DNA methylation changes in prostate cancer. Cancer Biol Ther 2007; 6: 1403–12. 47. Kregel S, Kiriluk KJ, Rosen AM, et al. Sox2 is an androgen receptorrepressed gene that promotes castration-resistant prostate cancer. PLoS One 2013; 8: 11. 48. Li X, Xu Y, Chen Y, et al. SOX2 promotes tumor metastasis by stimulating epithelial-to-mesenchymal transition via regulation of WNT/beta-catenin signal network. Cancer Lett 2013; 336: 379–89.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.