GSTP1 positive prostatic adenocarcinomas are more common in Black than White men in the United States.

GSTP1 is a member of the Glutathione-S-transferase (GST) family silenced by CpG island DNA hypermethylation in 90-95% of prostate cancers. However, prostate cancers expressing GSTP1 have not been well characterized. We used immunohistochemistry against GSTP1 to examine 1673 primary prostatic adenocarcinomas on tissue microarrays (TMAs) with redundant sampling from the index tumor from prostatectomies. GSTP1 protein was positive in at least one TMA core in 7.7% of cases and in all TMA cores in 4.4% of cases. The percentage of adenocarcinomas from Black patients who had any GSTP1 positive TMA cores was 14.9%, which was 2.5 times higher than the percentage from White patients (5.9%; P < 0.001). Further, the percentages of tumors from Black patients who had all TMA spots positive for GSTP1 (9.5%) was 3-fold higher than the percentage from White patients (3.2%; P<0.001). In terms of association with other molecular alterations, GSTP1 positivity was enriched in ERG positive cancers among Black men. By in situ hybridization, GSTP1 mRNA expression was concordant with protein staining, supporting the lack of silencing of at least some GSTP1 alleles in GSTP1-positive tumor cells. This is the first report revealing that GSTP1-positive prostate cancers are substantially over-represented among prostate cancers from Black compared to White men. This observation should prompt additional studies to determine whether GSTP1 positive cases represent a distinct molecular subtype of prostate cancer and whether GSTP1 expression could provide a biological underpinning for the observed disparate outcomes for Black men.

Introduction

The pi class glutathione S transferase (encoded by the GSTP1 gene) is a member of the cytosolic superfamily of glutathione-S-transferases. These phase II detoxification enzymes catalyze the conjugation of glutathione to diverse endogenous and exogenous electrophiles [1]. In the prostate, GSTP1 is constitutively expressed at high levels in basal epithelial cells, variably expressed in luminal epithelial cells, and epigenetically silenced in approximately 90 to 95% of adenocarcinomas by somatic hypermethylation of its regulatory CpG island [2-5]. Hypermethylation of GSTP1 appears to occur early in prostatic carcinogenesis since it is already present in approximately 70% of high-grade PIN lesions [6,7], the presumed precursor of most invasive carcinomas, where it is silenced specifically in luminal epithelial cells [7,8]. Studies in human prostate cancer cells suggest a role for GSTP1 as a caretaker gene whose loss increases cell survival in response to protracted oxidative injury [9]. In mouse models of carcinogen exposure induced cancers of the lung and skin, Gstp1/2 behaves as a tumor suppressor [10,11]. In a mouse model of early prostate cancer development induced by human MYC, Gstp1/2 also functions as a tumor suppressor [12].

GSTP1 acts as a homodimer to catalyze reactions between reactive oxygen species, anti-cancer drugs or carcinogens, and glutathione, sometimes ‘detoxifying’ and at other times ‘toxifying’ the substrates [13]. By contrast to silencing GSTP1, an induction of expression of GSTP1 protein is used to detect early cancer formation in response to carcinogenic exposure in the rat liver [14]. Moreover, expression and overexpression of GSTP1 are common in several human tumor types, where it has been implicated in promoting resistance to a number of chemotherapeutic agents [15]. Although GSTP1 expression has been estimated to be maintained in 5–10% of prostate cancer cases, little is known about the molecular and clinical features of these cases and only a few studies have directly asked the question of the frequency of positive GSTP1 Immunohistochemical (IHC) staining in prostate cancer [16,17].

Black men are more likely to develop prostate cancer than White men, and are more likely to have poor outcomes if diagnosed with the disease [18]. The reasons for the disparity are multifaceted, including inequities in access to high-quality prostate cancer screening, detection, diagnosis, and treatment. In addition, there appear to be at least some biological differences in disease pathogenesis and malignant progression, potentially based on associations of perceived race with genetic variation, or, as a result of different exposures. For example, when compared to prostate cancers from predominantly White men, prostate cancers from Black men contain fewer TMPRSS2-ERG fusions [19-22], fewer PTEN deletions [20-23], and more SPOP mutations [21,22].

In this study we comprehensively surveyed the frequency of GSTP1 protein expression in human clinical prostate cancers by IHC using tissue microarrays (TMAs). Our goal was to begin to determine whether GSTP1-positive prostate cancer represents a distinct molecular subtype, and, whether the prevalence of GSTP1-positivity differs between Black and White men.

Materials and methods

Study population

The study population consisted of 1673 patients who underwent radical prostatectomy (RRP) at a single center between 1993 and 2019, with an age range from 40 to 75 years old and whose prostatectomy tissue was arrayed across 45 TMA blocks. The cancers encompassed all Gleason grade groups and a spectrum of pathological stages. For IHC staining for GSTP1, we used the following TMA sets. TMA set 1 consists of prostatectomy tissue enriched for different Gleason grade groups and pathological stages from 476 cases operated on between 1997 and 2005 (N = 11 TMA blocks). TMA set 2 consists of prostatectomy tissue from 726 patients operated on between 1993 to 2000 (N = 16 TMA blocks) [24]. TMA set 3 consists of prostatectomy tissue from 343 Black and White men, operated on between 1993 and 2019 and matched on Gleason score, pathological stage and date of surgery within 2 years (N = 9 TMA blocks) [25]. TMA set 4 is newly designed and consists of prostatectomy tissue from 353 men operated on between 2007 and 2015 from a case-cohort design (N = 9 TMA blocks; detailed design to be described in a separate publication). These TMAs were all constructed as described [24,26,27] from the index tumor (highest grade) with a 3–4 fold sampling redundancy. To compare IHC staining and the in situ hybridization assay for GSTP1 mRNA, we used a novel TMA (TMA set 5), which consists of prostatectomy tissue from 31 patients operated on less than 1 year before TMA construction in which TMA slides are stored at -20°C, which improves RNA quality for hybridizations [28]. All clinical and pathology data used for the study were obtained from the pathology archives of the Johns Hopkins. The Johns Hopkins University School of Medicine Institutional Review Board approved this study.

IHC staining

We analytically validated an automated IHC assay against human GSTP1 protein using well-known cell lines and controls and human prostate cancer tissues using a mouse monoclonal antibody (Cell Signaling Technologies, #3369) at 1:400 dilution on a Ventana DISCOVERY ULTRA Autostainer using the DISCOVERY anti-HQ HRP kit as described [8]. Cell line FFPE blocks were prepared and utilized as described [29,30]. LNCaP cells were negative and DU145 and PC3 prostate cancer cell lines were positive for GSTP1 protein as expected [2]. In a subset of the above TMAs, we also performed IHC staining for ERG (rabbit recombinant monoclonal antibody; ABCAM EPR3864, Cambridge, UK) and PTEN (rabbit monoclonal; Cell Signaling Technologies, Danvers, MA), which were performed in an automated assay on the Ventana DISCOVERY ULTRA using the DISCOVERY HQ+ Amp kit. Genetically validated cell line controls were used for antibody specificity for ERG (VCAP cells are positive and PC3, LNCaP and DU145 are negative) and PTEN (PC3 and LNCaP cells are negative and DU145 cells are positive) [31,32]. Immunostaining for p63 was carried out using the mouse monoclonal antibody (clone 4A4, Biocare Medical, Concord,CA, USA) as part of a singleplex stain [33] or as part of a cocktail [34] (PIN4 staining, Biocare Medical) on the Ventana DISCOVERY ULTRA platform.

Slide scanning, image management and scoring

Whole TMA slides were scanned on a Hamamatsu Nanozoomer, and imported into Concentriq (from Proscia). Composite images were exported from Concentriq and imported into and visualized in TMAJ [28]. For quality control of IHC staining in the TMA spots, we used the fact that a high fraction of non-neoplastic stromal cells routinely stained positively for GSTP1. We used these stromal cells as internal positive controls such that TMA cores that lacked any staining in tumor cells, and all stromal cells, were regarded as non-evaluable and were not included in the analyses. In another small subset of TMA cores, staining was very weak in intensity in both the tumor cells and stromal cells and these were also considered equivocal in terms of staining quality and were not included. Together, this poor quality staining and uninterpretable staining in weak cases comprised ~ 1% of tissue cores with carcinoma.

Scanned TMA core images were evaluated for the presence of carcinoma by two pathologists with expertise in prostate cancer (I.V. and A.M.D.). Each TMA core containing carcinoma was evaluated for GSTP1 staining positivity using a two-tiered scoring strategy in which any tumor cell staining above background levels (nuclear, cytoplasmic, or both) were considered positive. All others were considered negative. Cases in which there was positive cancer cell staining for GSTP1 protein were categorized in two ways: any patient who had 1) any TMA spot with some positive tumor cell staining or 2) all TMA spots with some positive tumor cells staining; all other cases were considered GSTP1-negative.

In a subset of cases we also assessed ERG and PTEN to determine co-occurrence of GSTP1 positivity with ERG positivity and PTEN loss. ERG was considered positive for a given patient’s tumor if any cancer cells on any TMA cores with cancer were positive and PTEN was considered lost if any TMA cores contained any tumor cells with complete PTEN absence of staining.

In situ hybridization for GSTP1 mRNA

We developed a novel in situ hybridization assay for GSTP1 mRNA using ACD RNAscope, which we analytically validated (Probe-Hs-GSTP1 Cat No. 453221, kit version 2.5 manual assay according to manufacturer’s recommendations) using cell lines with known GSTP1 expression status for positive and negative controls as described () [8,28]. Briefly, FFPE tissue slides were baked at 60°C for 1 hour followed by deparaffinization in 100% xylene twice for 5 minutes each and two changes of 100% alcohol. The slides were treated with endogenous peroxidase blocking pretreatment reagent and then incubated for 15 minutes in a boiling 1× Pretreat 2 reagent (ACD) and then treated with protease digestion buffer (III, Cat. 322337) for 30 minutes at 40°C. The slides were incubated with a RNAscope target probe as follows: for 2 hours at 40°C, followed by signal amplification. 3,3′-Diaminobenzidine (DAB) was used for colorimetric detection for 10 minutes at room temperature. LNCaP, PC3 and DU145 prostate cancer cells were maintained as described [30]. MDA-PCA-2b cells were maintained in F-12K media containing 20% non heat-inactivated FBS, 25 ng/ml cholera toxin (Sigma cat. No. C8052), 10 ng/ml mouse Epidermal Growth Factor (Corning cat. No. 354010), 0.005 mM phosphoethanolamine (Sigma cat. No. P0503), 100 pg/ml hydrocortisone (Sigma cat. No. H0135), 45 nM sodium selenite (Sigma cat# 9133) and 0.005 mg/ml human recombinant insulin (Life Technologies cat# 12585–014) at 37°C and 5% CO2. All cell lines were obtained from ATCC (Manassas, VA). All cell lines were authenticated using short tandem repeat profiling by the Genetic Resources Core Facility at The Johns Hopkins University School of Medicine.

Statistical analysis

Data were tabulated and statistical tests were performed using Stata 15 for Mac OS. The primary analysis for comparisons between groups used GSTP1 protein positivity status for each patient, either any TMA core positive or all cores positive (Tables ). Tests for differences in proportions for any TMA core or all TMA cores positive for GSTP1 protein between Black and White men, between ERG positive and negative men, and between PTEN absent and PTEN present men were performed with the Pearson Chi2 test. Tests for trends for the proportion of men who were GSTP1 protein positive across Gleason grade groups and pathological stages were performed using the Cuzick’s nonparametric trend test across ordered groups.

Results

We applied an analytically validated automated IHC assay to evaluate GSTP1 protein expression [8] in a series of TMAs constructed from patients with clinically localized primary prostatic adenocarcinomas. A total of 5,757 TMA cores containing prostatic adenocarcinoma from 1,673 patients were evaluable for GSTP1 protein scoring by IHC. These consisted of 1,197 cores from 336 patients that self-identified as Black (mean age = 58.4; SD 6.6) and 4,560 TMA cores from 1,337 patients that self-identified as White (mean age = 59.3; SD 6.5) ().

As expected, carcinoma cells in most prostatic adenocarcinomas were completely negative for GSTP1 protein (92.3%; ). In a subset of cases, however, some or all cancer cells stained positively for GSTP1. While some cases were uniformly positive for GSTP1, in other cases containing GSTP1 positive tumor cells, we often observed heterogeneity in staining in terms of both cell number and staining intensity. This variability occurred among cancer cells at times within a given TMA core, between a man’s TMA cores, and between men. demonstrates representative images of these patterns of staining. Overall, 7.7% of cases contained GSTP1 positive cancer cells in a subset of TMA cores, and 4.5% had GSTP1 positive cancer cells in all TMA cores.

Patterns of GSTP1 protein staining.

Medium power views of TMA spots containing adenocarcinoma with examples of all tumor cells being GSTP1-negative (A), all tumor cells being GSTP1-positive (B), and a spot with heterogeneous mosaic staining with some cells staining positively and some negatively (C). In all examples, there is abundant stromal cell staining that is most apparent in (A) in between tumor nests. Original magnification x 100. (D) Higher power view of boxed regions in (C).

The percentage of adenocarcinomas from Black patients who had any GSTP1 positive cancer-containing TMA cores was 14.9%, which was 2.5 times higher than the percentage of cases with any GSTP1 positive cancer-containing cores from White patients (5.9%; P < 0.001) (). Further, the percentages of tumors from Black patients who had all TMA spots with cancer cells staining positive for GSTP1 (9.5%) was 3-fold higher than the percentage of tumors from White patients (3.2%; P<0.001) (). We confirmed that the number of evaluable TMA cores with carcinoma was similar among tumors from Black and White men, and also ran sensitivity analysis restricted to men with the same number of evaluable cores and similar results were obtained.

Cases with any or all TMA cores with cancer cells staining positive for GSTP1 occurred across all Gleason grade groups and stages, and the higher percentage of cases from Black patients positive for GSTP1 was apparent among all grade groups except in grade group 5 () and except in stage group 3 (). In Black but not White patients, the percentage of cases with any core positive was non-significantly decreased with increasing Gleason grade group ( and significantly decreased with increasing pathological stage (

A subset of TMAs (N = 16 TMA blocks) were stained for ERG protein by IHC, which tightly correlates with TMPRSS2-ERG gene fusions [35,36]. TMPRSS2-ERG gene fusions are the most common form of ETS gene family member fusions in prostate cancer and are mutually exclusive with other ETS family member fusions and a number of other somatic molecular alterations in prostate cancer, such as SPOP mutations or SPINK overexpression [37]. Therefore, prostate cancers harboring ERG gene fusions are considered a distinct molecular subtype of prostate cancer. In this TMA subset, the percentage of tumors from Black patients with any TMA core positive for ERG was significantly lower than for tumors from White patients, consistent with prior reports [19] ().

When not considering race, the percentage of cases with any TMA spot or all TMA spots with cancer cells staining positive for GSTP1 was lower in those who were ERG+ than ERG- (). This same pattern was observed when examining White patients only (, any TMA spot positive). In Black patients, the opposite pattern was observed; the percentage of any TMA spot positive for GSTP1 expressing cancer was higher in men who were ERG positive than ERG negative. The percentage of patients with ERG positive cancers that were also GSTP1 positive was 23.1% in Black men, but only 5.1% in White men, a difference that was statistically significant (, P = 0.001). In contrast, the percentage of patients with ERG negative cancers that were GSTP1 positive was more similar in Black (12.9%) and White (9.4%) patients ( P = 0.38).

A subset of TMAs (N = 16) was also stained by IHC for PTEN, which is a known tumor suppressor in prostate cancer, whose loss is associated with disease progression [23]. Prior work has also found a lower fraction of prostatic carcinomas from Black patients with PTEN loss than carcinomas from White patients [20,21], which we observed here (22.5% PTEN loss for Black patients and 34.9% PTEN loss for White patients). When not considering race, the percentage of patients with any GSTP1-positive TMA spot appeared to be lower in men with any PTEN loss than with PTEN intact (6.6% compared with 10.3%), but this difference was not statistically significant (). However, stratifying by race, the percentage of any TMA spot with cancer cells staining positive for GSTP1 was comparable in those with PTEN loss and PTEN intact in Black men and in White men (). The percentage of patients who had both PTEN loss and were GSTP1 positive was 12.0% in Black men, but only 5.6% in White men; this difference was not statistically significant (, P = 0.24). These percentages were similar to those in those who had both PTEN intact and were GSTP1 positive, with 16.5% in Black men and 8.1% in White men; this difference was statistically significant (, P = 0.029).

Rare cases of prostate cancer have been reported that are strongly positive for nuclear p63 staining [33,38-40], and these cases tend to express GSTP1, and at times lack GSTP1 GpG island methylation [33]. We asked whether GSTP1 positive cases in the current study were ever positive for p63 nuclear staining. P63 IHC staining was available for 22 of the cases across 6 of the TMA blocks that stained positively for GSTP1 that have also been stained in our laboratory for p63; none of these cases (0/22) were positive for nuclear p63 staining. We conclude that, while many p63-positive cancers are also positive for GSTP1 [33], most GSTP1-positive cancers are not p63-positive.

To test whether samples with GSTP1 positive protein staining also contained GSTP1 mRNA expression, we developed an ACD RNAscope in situ hybridization assay. We tested the hybridization reaction on cell lines and found that, as expected, PC3 cells and DU145 cells were positive and LNCaP cells were negative [2] (). MDA-PCa-2b cells, which were derived from an African American patient, were negative for GSTP1 mRNA () and protein (). In normal-appearing human prostate tissue, GSTP1 protein is consistently highly expressed in basal cells, with much lower and variable expression in normal appearing luminal cells. Furthermore, the expression is highly elevated in many of the intermediate luminal cells present in the atrophic epithelium of proliferative inflammatory atrophy [7,41]. Using standard slides from RRP specimens we found a similar pattern of hybridization signals for the GSTP1 ACD RNAscope probe set. There were also strong signals found in regions of the urethra as well as ejaculatory ducts, which is consistent with prior studies on GSTP1 protein [42] (). Since RNAscope assays perform less robustly on older prostate cancer specimens [28], we performed in situ hybridization for GSTP1 mRNA and IHC using a “recent case” TMA consisting of an array from 31 RRP cases (TMA set 5). We found 2 cases positive for GSTP1 protein by IHC (2/31 = 6.5%) and the same cases were also positive for GSTP1 mRNA; all cases staining negatively for GSTP1 protein were also negative for GSTP1 mRNA, giving a 100% concordance. We also performed IHC and in situ hybridization for GSTP1 mRNA on a number of standard slides from prostatectomy specimens and found an example that was heterogeneous for GSTP1 protein in the tumor. shows a region from this case that shows tight concordance between GSTP1 protein and mRNA expression.

In situ hybridization for GSTP1 mRNA in cell lines (A-D) and benign prostate (E-G). EJ duct lumen indicates ejaculatory duct lumen.

Discussion

In this study, we used a large number of primary untreated prostate cancer cases from prostatectomies (N = 1673) to estimate the percentage of prostatic adenocarcinomas that are positive for GSTP1 protein by IHC. We found an overall rate of any positive staining in cancer foci of 7.7%. Interestingly, we also found that GSTP1-positive carcinomas were often heterogeneous for GSTP1 protein (41%). In situ hybridization for GSTP1 mRNA showed that the increased protein in the GSTP1-positive cases likely occurred through transcriptional upregulation, suggested by the tight correlation between the protein and mRNA levels. For the first time, we report that GSTP1-positive cases are significantly more common in prostatic adenocarcinomas from Black patients compared with White patients (2.5–3 fold).

Given the difference in the percentage of GSTP1 positive cases by race, the current findings raise the question of whether GSTP1 positive cases represent a distinct molecular subtype of this disease. Interestingly, we found that the increase in GSTP1-positive cancers in Black men was greater in ERG-positive cases. This is of interest because prostate adenocarcinomas from Black men harbor ERG gene rearrangements less frequently than those from White men. In terms of PTEN, there was a similar increase in the percentage of GSTP1-positive cases regardless of PTEN status in Black patients.

Genomics studies of prostate cancers over the last several years have revealed the long tail of molecular alterations that can lead to cancer and influence disease progression and drug resistance [43-48]. These long tail events include low frequency germline and/or somatic molecular alterations involved in DNA repair (e.g., MMR defects caused by 1 of 4 different genes, BRCA2, ATM and CDK12). Other low frequency events considered important include FOXA1 mutations, IDH1/2 mutations, and mutations in a number of genes that affect epigenetic chromatin modifications. Given the large breadth of molecular alterations in prostate cancer, additional studies with larger numbers of patients are required to determine whether GSTP1 positive cases represent a distinct molecular subtype of prostate cancer.

In terms of disease aggressiveness, the percentage of men whose tumors were GSTP1- positive tended to decrease with increasing grade and stage in Black men, but not in White men (Tables ). Thus, the increased frequency of GSTP1-positive cancers in Black versus White men occurred in low and intermediate grade and stage cancers only (Tables ).

In terms of patient outcomes, in a series of 640 cases stained by IHC, there was no prognostic significance in univariate analysis for GSTP1 status [16]. Additional studies are underway to determine whether GSTP1 positive cases differ in terms of rates of biochemical recurrence, metastatic disease and deaths due to prostate cancer. Further, it will be of interest to determine whether GSTP1 status is related to response to therapies such as androgen deprivation, radiation, or chemotherapy and whether there are differences in the percentage of cases that are GSTP1 positive in metastatic castration sensitive and castration resistant prostate cancers. A recent study reported an association of BMI1 expression to the propensity of metastatic disease in prostate cancer in African-American men [49] and it will be interesting to correlate GSTP1 expression with that of BMI1 in future studies, including how each may contribute to the development of metastatic disease.

In terms of prostate cancer etiology, major risk factors are ancestry, family history and age. In terms of environmental exposures, at present, the fraction of prostate cancers attributable to known exposures remains quite low [50]. A limitation of our current study is that we have not determined whether men with prostate cancer that are GSTP1 positive, whether they be from Black or White men, have had different exposures than men with prostate cancers that are GSTP1 negative. Additional studies from population-based cohort studies with extensive pre-diagnosis exposure information are required to address this important issue and the potential racial disparity in prostate cancer burden. Also, a further limitation is that in our study we used self-identified race, which is not a biological measure; thus, future studies to include genotyping measures of percent African ancestry could perhaps help in determining whether GSTP1 expression status may be different as a result of genetic differences and/or environmental exposures or both.

To determine whether there is a different etiology for prostate cancers developing in Black versus White patients, gene expression studies can be instructive. A number of studies [21,51-53], albeit not all [54], have reported increases in inflammatory cytokine signaling in tumors from men of African descent. While inflammatory signaling and altered inflammatory cell infiltrates in the tumor microenvironment may occur during any and all temporal phases of cancer development and progression, we have previously postulated that chronic long standing inflammation may lead to prostate cancer most likely via lesions we termed proliferative inflammatory atrophy [41,55,56]. Interestingly, in regions of PIA, there appears to be an induction of GSTP1 in luminal cells, which we refer to as intermediate luminal cells [41,56,57]; with progression to PIN or adenocarcinoma accompanied by CpG island hypermethylation and loss of expression [7]. In terms of GSTP1 positive prostate cancer, it is possible that some cases arise after induction of GSTP1 in luminal cells and without silencing the gene. Martignano et al. found a strong correlation with GSTP1 CpG island hypermethylation and GSTP1 protein status by IHC [17]. Another limitation of our study is that at present we do not know whether the expression of GSTP1 protein in the positive cases correlates with, or is driven by, a lack of GSTP1 CpG island hypermethylation. Additional studies are underway in our laboratory to determine the CpG methylation status of GSTP1 alleles in cases with positive staining.

Finally, the finding that GSTP1-positive prostate cancer subset is substantially over-represented among prostate cancers from Black compared to White men could be of importance from the standpoint of treatment of lethal prostate cancer, providing a potential biological underpinning, at least in part, for the observed disparate outcomes for Black men. In breast cancer, the presence or absence of GSTP1 expression predicts response to cytotoxic chemotherapy, particularly to the taxanes docetaxel and paclitaxel [58-61]. Forced expression of the enzyme in GSTP1-negative breast cancer cells confers docetaxel resistance [58]. In addition, an MCF-7 clone selected for taxane-resistance exhibited activation of GSTP1 expression [62]. Thus, the over-representation of GSTP1-positive prostate cancer in Black men may render them less responsive to taxane chemotherapy, which is the preferred chemotherapy for advanced, lethal metastatic castration resistant prostate cancer [5].

Conclusion

In summary, we systematically examined GSTP1 protein expression in a large number of primary prostatic adenocarcinomas and found an overall rate of positivity of 7.7%, and a higher percentage of cases staining positive in Black men compared with cases from White men (2.5–3 fold). A significant difference in the percentages of GSTP1 positive cases in Black men was present only in ERG-positive cases, as compared with ERG-negative cases. These findings should stimulate additional studies regarding whether GSTP1 positive cases are a unique molecular and clinico-pathological subtype of human prostate cancer, with potential implications for disease etiology, racial disparities and therapeutic resistance mechanisms.

GSTP1 IHC in prostate cancer cell lines.

IHC was performed in the cell lines indicated. PC3 and DU145 cells are positive and LNCaP and MDA-PCa-2b are negative.

(TIF)

Click here for additional data file.

22 Mar 2021

PONE-D-20-36882

GSTP1 positive prostatic adenocarcinomas are more common in Black than White men in the United States

PLOS ONE

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The reviewers have requested several amendments to the manuscript and to include checking the grammar and spellings.

Any information on the methylation status of GSTP1 gene would be helpful to include and a suggestion was made to include African American cell lines for an in vitro model as important to conclude this study.

Please could you ensure that all the points raised by both reviewers have been fully addressed - this manuscript will be looked at again by the original reviewers.

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Reviewer #2: Yes

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Reviewer #1: The paper entitled "GSTP1 positive prostatic adenocarcinomas are more common in Black than Whitemen in the United States" is interesting. However, the authors need to check and present data a little more seriously. There are many typos in the Manuscript, such as "GSPT1" as "GSP1" or White men as Whte men, etc. A little serious presentation of their findings will significantly increase the quality of the paper. I have the following comments

Comments:

1. The term "Black" and "Whitemen" are not preferable in scientific journals. I suggest the authors use the term African American and Caucasian in the "Title" and the Manuscript's main body.

2. Expand the term "TMA" in the abstract section.

3. Recently another publication observed that BMI1 expressed prominently among African Americans (PMID=30087142). How do the authors compare their findings with this publication?

4. The data on the methylation status of the GSTP1 gene will surely make the paper more valuable for the readers. Authors may include this data.

5. The number of patients between black and white patients is significantly different. Do the authors think that their difference can influence the final result of their observation?

6. Figure 2: Authors should include one or two cell lines representative of the black population. As the study emphasizes the black population, this population in vitro model is a must for this figure.

7. Figure 3: Authors must explain the background of the "representative case" in the legend. Is it from black or white patients"?

Reviewer #2: In this manuscript the authors have analyzed a large collection of prostate adenocarcinoma tissue specimen for the expression of the Glutathione-S-transferase family member GSTP1 by immunohistochemistry. Technically the analysis of the tissue micro arrays is well-performed and statistical analysis adequate. A significant higher number of GSTP1+ cases among Black men compared to White was found. However, while this finding is new and interesting, large parts of this descriptive-only study confirm already published data for GSTP1+ expression in a subset of prostate cancer patients. The authors speculate whether GSTP1 positive cancer might be a distinct molecular subtype of this disease. Molecular subtypes should be defined by genomic aberrations and/or gene expression signatures, expression of a single gene (GSTP1) is – in my opinion – not sufficient to define a molecular subtype. In addition, GSTP1+ cases were found in ERG+ and ERG- cases representing two recognized molecular subtypes of prostate cancer. To get more robust data for the definition of a molecular subtype a gene expression study could be performed on the “recent case” TMA used for RNA in situ hybridization. This would give a first indication whether GSTP1+ cases have a different gene expression signature compared to GSTP1- cases.

Minor points:

1) The GSTP1 antibody was well-controlled prior staining the TMAs. However, results of stainings in the cell lines and knock-out tissue should be added to supplementary material.

2) Which GSTP1 antibody was used? Please indicate clone, company and dilution for every antibody used.

3) Methods section: LNCaP instead of LnCAP

4) Higher magnifications of images in Figure 1 should be shown. In Fig 1C it is difficult to interprete whether the area marked with “tumor cells negative” is indeed malignant tissue?

5) Please include a table for the most important clinical parameters (age, BMI, etc) for all patients in this study (differences in mean between Black and White populations?)

**********

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Reviewer #1: Yes: Hifzur R Siddique

Reviewer #2: No

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20 May 2021

Reviewer #1: The paper entitled "GSTP1 positive prostatic adenocarcinomas are more common in Black than Whitemen in the United States" is interesting. However, the authors need to check and present data a little more seriously. There are many typos in the Manuscript, such as "GSPT1" as "GSP1" or White men as Whte men, etc. A little serious presentation of their findings will significantly increase the quality of the paper. I have the following comments

Response: We appreciate the reviewer’s positive comment that the paper is interesting. We acknowledge that there are some typos in the manuscript and we appreciate those being pointed out and have now corrected them.

Comments:

1. The term "Black" and "Whitemen" are not preferable in scientific journals. I suggest the authors use the term African American and Caucasian in the "Title" and the Manuscript's main body.

Response: We have communicated separately with the editor about this issue, who responded to us after they communicated with the reviewer about this. Since the NCI SEER program currently prefers the terminology we used, it was agreed we would keep these designations as we have them.

2. Expand the term "TMA" in the abstract section.

Response: We thank the reviewer for pointing that out. We have expanded the acronym TMA in the abstract.

3. Recently another publication observed that BMI1 expressed prominently among African Americans (PMID=30087142). How do the authors compare their findings with this publication?

Response: We would like to thank the reviewer for pointing out this paper and have included a brief discussion of it in the revised submission in the discussion.

4. The data on the methylation status of the GSTP1 gene will surely make the paper more valuable for the readers. Authors may include this data.

Response: While we very much appreciate the importance of this question, we have recently begun a relatively large study of this question and plan to publish it separately as it has enough scope and complexity to present separately. The results are currently not available and are beyond the scope of the current manuscript.

5. The number of patients between black and white patients is significantly different. Do the authors think that their difference can influence the final result of their observation?

Response: At Johns Hopkins our fraction of patients over the last several decades that are Black that undergo prostatectomy overall is 9.5% and the percentage that is White is 85.5%. In the current study we augmented these numbers by increasing the percentage of Black patients by including TMAs that were enriched for Black patients, such that our percentage of Black men in this study was 20% (see methods section for TMA dataset descriptions). We submit that our estimate found in this study of the percentage of GSTP1-positive cancers in Black men represents a highly statistically significant difference between Black and White men, and a reasonable estimate of the overall prevalence of this phenotype, since we included a relatively large number (N= 336) of Black men. To better refine our estimates, we are currently working toward increasing our overall number of both Black and White men in additional studies from patients both within our institution and beyond, although this will take more than one year at least to complete, and we feel that publishing the current findings are important to allow other researchers to also follow up on these studies

6. Figure 2: Authors should include one or two cell lines representative of the black population. As the study emphasizes the black population, this population in vitro model is a must for this figure.

Response: There are relatively few prostate cancer cell line models over all in the field (compared to that for other common cancer types), and even fewer cell lines from African American subjects. Nonetheless, we agree this is an important point and have now included the results of GSTP1 in situ hybridization from MDA-PCa-2 cell line, which was derived from an African American (PMID:9815652). We found this cell line to be negative for both GSTP1 mRNA and protein. We have revised the updated Figure 2 to include these results.

7. Figure 3: Authors must explain the background of the "representative case" in the legend. Is it from black or white patients"?

Response: We appreciate the reviewer for pointing out that it would be helpful if provided additional information about this case. We have now added details about this case. The specimen was from a 73 year old Black man with a Gleason score of 4+5=9, and a pathological stage of T2N0Mx with margins negative. This information is now included in the manuscript.

Reviewer #2: In this manuscript the authors have analyzed a large collection of prostate adenocarcinoma tissue specimen for the expression of the Glutathione-S-transferase family member GSTP1 by immunohistochemistry. Technically the analysis of the tissue micro arrays is well-performed and statistical analysis adequate. A significant higher number of GSTP1+ cases among Black men compared to White was found. However, while this finding is new and interesting, large parts of this descriptive-only study confirm already published data for GSTP1+ expression in a subset of prostate cancer patients. The authors speculate whether GSTP1 positive cancer might be a distinct molecular subtype of this disease. Molecular subtypes should be defined by genomic aberrations and/or gene expression signatures, expression of a single gene (GSTP1) is – in my opinion – not sufficient to define a molecular subtype. In addition, GSTP1+ cases were found in ERG+ and ERG- cases representing two recognized molecular subtypes of prostate cancer. To get more robust data for the definition of a molecular subtype a gene expression study could be performed on the “recent case” TMA used for RNA in situ hybridization. This would give a first indication whether GSTP1+ cases have a different gene expression signature compared to GSTP1- cases.

Response: We appreciate the positive feedback regarding the performance of GSTP1 IHC and analysis on a large collection of adenocarcinoma specimens.

In terms of defining a new molecular subtype, we agree with the Reviewer that the expression of a single gene, such as GSTP1, is not sufficient to define a molecular subtype. In a number of locations in the manuscript we indicate that GSTP1 status may help define a novel molecular subtype, not that it indeed does define such a subtype. For example, in the abstract we stated: “This observation should prompt additional studies to determine whether GSTP1 positive cases represent a distinct molecular subtype of prostate cancer.” Also, in the discussion we stated: “Given the large breadth of molecular alterations in prostate cancer, additional studies with larger numbers of patients are required to determine whether GSTP1 positive cases represent a distinct molecular subtype of prostate cancer.”

We further agree that it is important to obtain more robust data for the identification of a molecular subtype by performing a gene expression study. In fact, we are currently performing such a study on a large number of fresh frozen tissues in which we have GSTP1 expression and RNAseq. The data from that study are still being analyzed and will take some time to complete given the substantial scope of that effort. Thus, that analysis is beyond the scope of the current study and we submit that the current study will also prompt others to perform similar investigations.

Minor points:

1) The GSTP1 antibody was well-controlled prior staining the TMAs. However, results of stainings in the cell lines and knock-out tissue should be added to supplementary material.

Response: We thank the reviewer for this suggestion. We have added a supplemental Figure (supplemental Fig. 1) to show the cell lines for both IHC and in situ hybridization. We have removed the reference to the knockout tissue as we realized those were stained with a different antibody.

2) Which GSTP1 antibody was used? Please indicate clone, company and dilution for every antibody used.

Response: We appreciate pointing out this omission and have added the details for this.

3) Methods section: LNCaP instead of LnCAP

Response: We have fixed this typo.

4) Higher magnifications of images in Figure 1 should be shown. In Fig 1C it is difficult to interprete whether the area marked with “tumor cells negative” is indeed malignant tissue?

Response: We have added a higher power image as a separate panel to the revised Figure 1.

5) Please include a table for the most important clinical parameters (age, BMI, etc) for all patients in this study (differences in mean between Black and White populations?)

Response: At present, although we don't have BMI on all of these patients, we will annotate that information for a followup study when we look at patient outcomes with respect to GSTP1 expression status. We did add the mean and SD of the patient ages by race in the results section, first paragraph in the revised manuscript.

2 Jun 2021

GSTP1 positive prostatic adenocarcinomas are more common in Black than White men in the United States

PONE-D-20-36882R1

Dear Dr. De Marzo,

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Additional Editor Comments (optional):

The authors have fully addressed all the comments raised by both reviewers. The manuscript has been improved by inclusion of additional data and through individual amendments to the main text as suggested by the reviewers.

Reviewers' comments:

17 Jun 2021

PONE-D-20-36882R1

GSTP1 positive prostatic adenocarcinomas are more common in Black than White men in the United States

Dear Dr. De Marzo:

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Table 1

Total number of patients and TMA spots with carcinoma.

Race	No. of patients	No. of evaluable TMA cores with carcinoma
Black	336	1197
White	1337	4560
Total	1673	5757

Table 2

GSTP1 protein staining in cancer cells in Black and White patients.

	Any cores positive				All cores positive
Race	No	Yes	Total	P Value	No	Yes	Total	P Value
Black	286 (85.1%)	50 (14.9%)	336		304 (90.5%)	32 (9.5%)	338
White	1,258 (94.1%)	79 (5.9%)	1,337		1,294 (96.8%)	43 (3.2%)	1,339
Total	1,544	129	1,673	< 0.001	1,598	75	1,673	< 0.001

N in each cell represents number of patients.

Table 3

GSTP1 protein staining by Gleason grade groups in Black and White patients.

	Black				White
	Any cores positive				Any cores positive
Grade group	No	Yes	Total	P Value	No	Yes	Total	P Value
1 (GS 6)	68 (77.3%)	20 (22.7%)	88 (100%)		230 (93.1%)	17 (6.9%)	247 (100%)
2 (GS 3+4 = 7)	97 (86.6%)	15 (13.4%)	112 (100%)		448 (94.7)	25 (5.3%)	473 (100%)
3 (GS 4+3 = 7)	43 (89.6%)	5 (10.4%)	48 (100%)		287 (95.7%)	13 (4.3%)	300 (100%)
4 (GS 8)	24 (82.8%)	5 (17.2%)	29 (100%)		150 (93.2%)	11 (6.8%)	161 (100%)
5 (GS 9–10)	54 (91.5%)	5 (8.5%)	59 (100%)		143 (91.7%)	13 (8.3%)	156 (100%)
Total	286 (84.9%)	50 (14.8%)	336 (100%)	0.039	1,258 (94.0%)	79 (5.9%)	1,337 (100%)	0.50
	All cores positive				All cores positive
Grade group	No	Yes	Total	P Value	No	Yes	Total	P Value
1 (GS 6)	74 (84%)	14 (15.9%)	88 (100%)		238 (96.3%)	9 (3.6%)	247 (100%)
2 (GS 3+4 = 7)	106 (94.6%)	6 (5.4%)	112 (100%)		460 (97.3%)	13 (2.8%)	473 (100%)
3 (GS 4+3 = 7)	44 (91.7%)	4 (8.3%)	48 (100%)		294 (98.0%)	6 (2%)	300 (100%)
4 (GS 8)	24 (82.8%)	5 (17.2%)	29 (100%)		152 (94.4%)	9 (5.6%)	161 (100%)
5 (GS 9–10)	56 (94.9%)	3 (5.1%)	61 (100%)		150 (96.2%)	6 (3.9%)	156 (100%)
Total	304 (90.5%)	32 (9.5%)	338 (100%)	0.21	1294 (96.8%)	43 (3.2%)	1,337 (100%)	0.46

N in each cell represents number of patients.

Table 4

GSTP1 protein staining by pathological stage in Black and White patients.

	Black				White
	Any cores positive				Any cores positive
Stage	No	Yes	Total	P Value	No	Yes	Total	P Value
1	129 (78.7%)	35 (21.3%)	164		459 (93.1%)	34 (6.9%)	493
2	87 (89.7%)	10 (10.3%)	97		507 (96.0%)	21 (4.0%)	528
3	69 (93.2%)	5 (6.8%)	74		287 (92.3%)	24 (7.7%)	311
Total	285 (85.1%)	50 (14.9%)	335	<0.004	1253 (94%)	79 (5.9%)	1332	0.045
	All cores positive				All cores positive
Stage	No	Yes	Total	P Value	No	Yes	Total	P Value
1	143 (87.2%)	21 (12.8%)	164		476 (96.6%)	17 (3.5%)	493
2	91 (93.8%)	6 (6.2%)	97		515 (97.5%)	13 (2.5%)	528
3	69 (93.2%)	5 (6.8%)	74		298 (95.8%)	13 (4.2%)	311
Total	303 (90.5%)	32 (9.5%)	335	0.139	1289 (96.8%)	43 (3.2%)	1332	0.373

Stage groups: 1 = “Organ Confined”—T2N0; 2 –“Extraprostatic Extension”—T3aN0; 3 = Seminal Vesicle Invasion or Pelvic Lymph Node Metastasis—T3BN0 or any N1 (AJCC 2007 staging criteria). N in each cell represents number of patients.

Table 5

GSTP1 protein staining by ERG status overall.

	ERG expression
GSTP1 any TMA spot positive	Negative	Positive	Total	P Value
No	238 (89.5%)	189 (92.6%)	427
Yes	28 (10.5%)	15 (7.4%)	43	0.24
Total	266	204	470
GSTP1 all TMA spots positive
No	247 (92.9%)	197 (96.6%)	444
Yes	19 (7.1%)	7 (3.4%)	26	0.08
Total	266	204	470

Table 6

GSTP1 protein staining by ERG status in Black and White patients.

	ERG Positive				ERG Negative
	Race				Race
GSTP1 any TMA spot positive	Black	White	Total	P Value*	Black	White	Total	P Value
No	20 (76.9)	169 (94.9%)	189 (92.6)		74 (87.1%)	164 (90.6%)	238(89.5%)
Yes	6 (23.1%)	9 (5.1%)	15 (7.4%)	0.001	11 (12.9%)	17 (9.4%)	28 (10.5%)	0.38
Total	26	178	204		85	181	266

Table 7

GSTP1 protein staining by PTEN loss overall.

	PTEN status
GSTP1 any TMA spot positive	Loss (any)	Intact	Total
No	141 (93.4%)	287 (89.7%)	428
Yes	10 (6.6%)	33 (10.3%)	43
Total	151	320	471

P = 0.19.

Table 8

GSTP1 protein staining by PTEN loss in Black and White patients.

	PTEN Loss (any)					PTEN Intact
	Race					Race
GSTP1 any positive	Black	White	Total	P Value	GSTP1 any positive	Black	White	Total	P Value
No	22 (88%)	119 (94.4%)	141		No	71 (83.5%)	216 (91.9)	287
Yes	3 (12%)	7 (5.6%)	10		Yes	14 (16.5%)	19 (8.1%)	33
Total	25	126	151	0.24	Total	85	235	320	0.029