Abstract
The coding region of GSTP1 gene is preceded by a large CpG-rich region that is frequently affected by methylation. In many cancer types, GSTP1 is affected by hypermethylation and, as a consequence, it has a low expression. The aim of this review is to give an overview on GSTP1 methylation studies with a special focus on liquid biopsy, thus to summarize methods, results, sample types, different diseases, to have a complete information regarding this promising epigenetic biomarker. We used all the most valuable scientific search engines (PubMed, Medline, Scopus and Web of Science) searching the following keywords: GSTP1, methylation, cancer, urine, serum, plasma and blood. GSTP1 is a largely investigated tissue biomarker in several malignancies such as prostate, breast, lung and hepatocellular carcinoma with good performances especially for diagnostic purposes. As a liquid biopsy biomarker, it has been mainly investigated in prostate cancer (PCa) where it showed a high specificity but a low sensitivity; thus, it is recommended in combination with other biomarkers. Despite the large number of published papers and the promising results, GSTP1 has not yet entered the clinical practice even for PCa diagnosis. For this reason, further large and prospective studies are needed to validate this assay.
Introduction
Glutathione-S transferases (GSTs) are a family of enzymes involved in catalyzing the detoxification of endogenous and exogenous substances by their conjugation with glutathione (GSH) [1]. These proteins are able to interact with several factors, such as regulatory kinases, and to modulate a number of pathways: cell proliferation, differentiation and death. Thanks to their cytoprotective and regulatory functions, GSTs play an important role in cancer cell proliferation and death [2].
GSTP1 belongs to the pi class of these enzymes; its gene is located on chromosome 11q13. The coding region of GSTP1 gene is preceded by a large CpG-rich region that could be prone for cytosine methylation.
In cancer, GSTP1 is often affected by alterations such as gene polymorphisms [3] or hypermethylation [3] with consequences on gene expression [4], [5]. It is commonly considered as a tumor suppressor gene due to its frequent loss of expression [4], but in many tumor types such as breast, colon or lung, it is overexpressed and unmethylated [6]. Thus, it has maybe a dual role.
It is from more than 20 years that researchers publish papers regarding methylation of GSTP1 [7] and the argument still seems to be of great scientific interest [8].
GSTP1 methylation has undoubtedly been the most studied epigenetic marker in prostate cancer (PCa) [9], [10], but it is also associated with tumor development or poor prognosis in other tumors such as hepatocellular carcinoma (HCC) [11] and breast cancer [12]. In most of these studies, GSTP1 has been evaluated as a component of a methylation panel, together with other genes of interest [13], [14].
Alterations in the GSTP1 methylation status have been associated with environmental factors such as viral infections, exposure to chemicals and inflammation [15], [16], [17], [18], [19], [20] (Figure 1).
Representation of: GSTP1 methylation sequence.
Representation of GSTP1 methylation sequence (NCBI reference sequence: NG_012075.1, 4900–5450), GSTP1 normal functions and GSTP1 methylation phenomenon in cancer and liquid biopsy.
Many studies conducted on PCa tissue samples have demonstrated that methylation of GSTP1 can be found since the earliest grades and stages, meaning that it represents an early event in the carcinogenesis process [4], [21]. For this reason, it has been proposed as a diagnostic marker to be used for the evaluation of PCa biopsies with the aim to avoid unnecessary rebiopsies [22].
GSTP1 methylation has been extensively studied on tissue samples, but interestingly, it has been proposed as a biomarker related to “liquid biopsy”, to be evaluated in plasma, serum and urine in a non-invasive manner [23].
Despite the large number of published papers, GSTP1 methylation has not yet entered the clinical practice, even for PCa diagnosis, for which promising results are available for tissue samples but also for blood and urine samples [24].
For this reason, we decided to write a critical review of the studies conducted on GSTP1 methylation, with a special focus on liquid biopsy, thus to summarize methods, obtained results, sample types and different diseases to have complete information regarding this promising epigenetic biomarker.
We used PubMed, Medline, Scopus and Web of Science as scientific engines using the following keywords: GSTP1, methylation, cancer, urine, serum, plasma and blood. We selected original research studies and reviews with an impact factor ≥1 on the basis of their statistical power and biological or clinical relevance.
Methods for studying GSTP1 methylation
Bisulfite-based detection
GSTP1 methylation status could be studied using different analysis methods. The most used is methylation-specific polymerase chain reaction (MS-PCR) because it is rapid, easy to perform and inexpensive [25], [26], [27]. In brief, it consists of two parts: DNA is chemically converted using sodium bisulfite and then real-time PCR is performed with specific primers for methylated/unmethylated sequences. The approaches based on bisulfite conversion have the problem that sometimes some sequences are preferentially amplified, resulting in PCR bias and inaccurate estimate of methylation [28]. Indeed Warnecke and colleagues found that unmethylated DNA is preferentially amplified compared to highly methylated DNA. The authors supposed that it could be due to a higher melting temperature of highly methylated DNA resulting in a lower PCR efficiency compared to unmethylated sequences.
Jeronimo et al. [29] analyzed GSTP1 hypermethylation both with conventional and real-time quantitative MS-PCR, finding that conventional MS-PCR detected GSTP1 hypermethylation in a larger number of samples than quantitative MS-PCR. Conventional real-time MS-PCR is a real-time SYBR Green approach in which the percentage of methylation is not quantified, whereas real-time quantitative MS-PCR does quantify.
Another method based on bisulfite conversion is the bisulfite genomic sequencing that consists of selective deamination of unmethylated cytosine to uracile after treatment with sodium bisulfite, usually followed by PCR amplification and sequencing of the regions of interest [23], [30].
Lan et al. [31] developed a methylation-specific dot blot assay for improving sensitivity and specificity of simultaneous methylation analysis of multiple genes. It consists of the design of two pairs of primers from a high CpG-rich region that map in the inner location of the MS-PCR primers and overlapped each other. Each primer pair is firstly amplified by PCR and then cloned into a vector, so the recombinant plasmids are used for the preparation of biotin or labeled probes. MS-PCR products are dotted on a nylon membrane and fixed by ultraviolet, then the membranes are hybridized and color spots are detected by enzymatic color reaction. This technique allows quantitative analysis of one methylation marker; it is easy to perform but it considers MS-PCR primer designed in a region without CpG [31].
Vasiljevic et al. [32] investigated the methylation status of GSTP1 within a panel of genes using pyrosequencing method that is a sensitive and reproducible method, but much more expensive than others. It is a quantitative method that interrogates different part of the gene in different CG sites and uses internal controls to evaluate bisulfite conversion. Indeed, it consists of DNA bisulfite conversion, and biotin-labeled primers are used to purify PCR product that is subsequently bound to the beads, and then pyrosequencing primers are annealed to the purified PCR products and pyrosequencing is performed [32], [33].
Together with pyrosequencing, MassArray is one of the most valid quantitative DNA methylation analysis that utilizes matrix-assisted laser desorption/ionization time of flight mass spectrometry (MS) and base-specific cleavage to interrogate DNA methylation patterns in sodium bisulfite-converted DNA [34]. Radpour et al. used EpiTYPER assay to evaluate DNA methylation of a panel of tumor suppressor genes, including GSTP1 in breast cancer patients. It makes use of tagged primers for the candidate genes; selected amplicons were mostly located in the promoter regions, or started from the promoter and partially covered the first exon [35].
Payne et al. [36] used HeavyMethyl qPCR technology on the LightCycler 2.0 (Roche), which is a real-time PCR method using oligonucleotide blockers specific to the unmethylated product.
The ConfirmMDx (quantitative methylation assay) is a tissue-based test that evaluates the methylation status of GSTP1, APC and RASSF1 with a multiplex quantitative methylation-specific polymerase chain reaction in residual cancer negative prostate biopsy. In the MATLOC study and the DOCUMENT study, the assay was defined to accurately predict the presence of PCa in a negative biopsy [22], [37].
Next-generation sequencing technology is investigated by Li et al. [38] using microfluidic PCR-based target enrichment and next-generation bisulfite sequencing to assess the methylation status of 48 candidate genes in breast cancer. It is a complete analysis although it could not be considered convenient for the analysis of a single marker.
Wijetunga et al. [39] used the Illumina Infinium assay with the HumanMethylation27 Analysis BeadChip to assess the methylation status of several genes, among which GSTP1 methylation, to be associated with cervical intraepithelial neoplasia progression to cervical cancer. It investigates 27,578 CpG sites, which are selected predominantly from the promoter regions of 14,475 genes. DNA methylation levels at individual CpG loci were identified measuring the fraction of methylated signal in genomic DNA samples. Bisulfite conversion is carried out and individual BeadChip controls confirmed efficient bisulfite conversion, hybridization specificity, base extension and target removal for genomic DNA samples [39], [40].
The scarcity of tumor-specific circulating DNA represents an obstacle for the analysis of methylated DNA. For this reason, Keeley et al. [41] introduced a new technique, defined as “methylation on beads” that allows to DNA extraction and bisulfite conversion for up to 2 mL of plasma or serum.
Detection without using bisulfite treatment
There are several approaches able to avoid the use of the chemical conversion of DNA using bisulfite treatment; these methods give robust and often reproducible results comparable to the bisulfite-based.
Wijetunga et al. [39] used HELP-tagging (HpaII tiny fragment Enrichment by Ligation-mediated PCR Assay), a more comprehensive and quantitative assay than array-based methods to validate their results with Illumina Infinium assay. Briefly, this approach uses enzyme digestion of methylated sequences.
Another well-studied method, based on enzyme digestion, is methylation-specific multiple ligation probe amplification (MS-MLPA) (MRC-Holland, Amsterdam, The Netherlands), which is a semiquantitative method that analyzes the methylation status of different genes at the same time [42], [43]. In brief, DNA is denatured and an incubation at 60 °C for 16–18 h allows the hybridization. The next steps are ligation and digestion by HhaI enzyme, followed by amplification of the samples by PCR. Only if DNA sequences are methylated an amplification product is generated because DNA-probe hybrids are protected against HhaI enzyme digestion. This type of analysis showed a strong concordance, greater than 80%, with conventional MS-PCR as demonstrated by Gurioli et al. [44], although it considers only a single CpG site for GSTP1.
Another method that targets the non-bisulfite converted DNA sequence by methylation-sensitive/insensitive endonuclease is methylation-sensitive restriction endonuclease quantitative PCR (MSRE-qPCR). It is capable of detecting a single CpG island allele, through the digestion of a restriction enzyme and a subsequent PCR reaction [45], [46].
Flanagan et al. [47] evaluated the DNA methylation of a panel of genes, including GSTP1 using microarrays. Arrays consist of the hybridization of the unmethylated fraction of genomic DNA to the microarray containing oligonucleotides that represent the genomic region of interest. In brief, they used a cocktail of three methylation-sensitive enzymes to digest individuals genomic DNA and used ligation-mediated PCR to amplify products, which were cleaned using Qiagen PCR cleanup kit labeled with either Cy3 or Cy5 dyes and cohybridized in matched pairs to the custom array. Hybridization intensity correlates with the DNA methylation status at the genomic locus homologous to each oligonucleotide on the array [47].
Topkaya et al. [48] developed an electrochemical DNA biosensor for the detection of the hypermethylation of GSTP1. This sensor analyzes the samples based on guanine oxidation signals before and after hybridization between probe and synthetic target or denatured PCR samples [48].
Several approaches are available to determine methylation of GSTP1; however, none is currently defined as a “gold standard”: some papers demonstrated good concordance between bisulfite-based methods and restriction enzyme-based methods, but what is the best approach still remains an open question. Maybe some larger, multicentric studies using the same method are needed.
In order to shed light on the differences among the multitude of assays used for the study of GSTP1 methylation, Wu et al. [49] published a meta-analysis in which they described the primer sequences mapping in the GSTP1 gene (NCBI Reference Sequence: NG_012075.1, 4900–5450) used by most of the studies, highlighting the CpG sites, as also shown in Figure 1.
Table 1 summarizes the methods described in this section.
Methods for GSTP1 methylation detection.
| Methylation method | Bisulfite treatment (Yes/No) | Sample type | Cancer type | Reference |
|---|---|---|---|---|
| Conventional MS-PCR | Yes | Tissue/plasma/urine | Prostate | Jerònimo et al. [29] |
| Quantitative MS-PCR | Yes | Tissue/plasma/urine | Prostate | Jerònimo et al. [29] |
| Bisulfite genomic sequencing | Yes | Plasma/blood cells/urine | Prostate | Bryzgunova et al. [23] |
| Methylation specific dot blot assay | Yes | FFPE | Prostate | Lan et al. [31] |
| Pyrosequencing | Yes | Fresh frozen tissue | Prostate | Vasiljević et al. [32], Yoon et al. [33] |
| MassArray | Yes | TRAM mouse tissue | Prostate | Mavis et al. [34] |
| HeavyMethyl qPCR | Yes | Plasma/urine | Prostate | Payne et al. [36] |
| ConfirmMDx | Yes | FFPE | Prostate | Stewart et al. [22], Partin et al. [37] |
| Illumina Infinium assay | Yes | Frozen tissue | Cervical/HCC | Wijetunga et al. [39], Yamada et al. [40] |
| NGS | Yes | FFPE | Breast | Li et al. [38] |
| Methylation on beads | Yes (after) | Plasma/serum | NSCLC | Keeley et al. [41] |
| EpiTYPER assay | Yes | Tissue/plasma/serum | Breast cancer | Radpour et al. [35] |
| MSRE-qPCR | No | serum | Prostate | Bastian et al. [45] |
| HELP-tagging | No | Frozen tissue | Cervical | Wijetunga et al. [39] |
| MS-MLPA | No | Cell lines/frozen tissue/Urine/FFPE | Bladder/Ovarian/prostate | Cabello et al. [42], Ho et al. [43], Gurioli et al. [44], Schwarzenbach et al. [50] |
| Microarray | No | Peripheral blood | Breast cancer | Flanagan et al. [47] |
| Electrochemical DNA biosensor | No | Plasma | Prostate | Topkaya et al. [48] |
Methylation in tissue
Prostate cancer
GSTP1 methylation is one of the most studied epigenetic alterations in PCa, and it is described as an early event in PCa carcinogenesis [4], [51], [52]. For the diagnosis of PCa, GSTP1 hypermethylation reaches a sensitivity of 81.8%±8.8% and a specificity of 94.9%±2.4% according to a recent meta-analysis that pooled 35 studies regarding both prostatectomy and biopsy [53]. GSTP1 methylation seems to have a possible clinical implication in the early diagnosis of PCa, and most of the studies did not find an association with clinical parameters or increased risk or survival [26], [33], [54], [55], [56]. However, GSTP1 has a prognostic value if included in a methylation panel in combination with other genes: Ellinger et al. [56] investigated a panel of nine gene loci (including GSTP1), and although GSTP1 methylation alone is not informative about tumor grading or risk assessment, the copresence of two methylated genes correlates with pathologic stage and/or Gleason score; moreover, DNA hypermethylation at more than five genes correlates with PSA recurrence after radical prostatectomy.
Enokida et al. [57] found an association between Gleason score and methylation status of GSTP1, APC and MDR1.
In addition, the combination of GSTP1 with other markers (in particular APC and RARB) not only gives prognostic information but also increases the diagnostic power of the assay [22], [53], [58], [59].
Given that up to 30% PCa patients are negative at initial biopsy, GSTP1 has been proposed as a biomarker able to avoid unnecessary rebiopsies [53]. This is the aim of an extensive retrospective study (MATLOC) that included 498 subjects with histopathologically negative prostate biopsies that repeated the exam within 1 month. This study demonstrated that a combined methylation assay, including GSTP1, APC and RASSF1, is a good predictor of patient outcome (OR=3.17) reaching a negative predictive value of 90% [22].
According to Aubry et al. [60], the use of this epigenetic assay for the management of initial biopsy-negative patients is also able to consistently reduce the costs related to PCa screening.
Breast cancer
Similarly to PCa, GSTP1 hypermethylation was described as an early event in breast cancer [61].
GSTP1 was found to be methylated in 25%–45% of breast tumors, without striking differences between ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). The absence of methylation was observed in healthy tissue [61], [62], [63], [64], [65].
Lee et al. found a lower frequency of methylation also in usual ductal hyperplasia (UDH) (16.7%) and described a progressive increase of methylation during breast carcinogenesis. Thus, they suggested that investigating GSTP1 in precursor lesions such as UDH may be useful for cancer chemoprevention purposes [61].
Although in PCa there is not a clear correlation between GSTP1 status and clinical-pathological features, in breast cancer GSTP1 methylation is associated with a more aggressive phenotype of estrogen receptor (ER) positive breast cancer [66]. Arai et al. [67] found a significant correlation with large tumor size, lymph node metastases and poor relapse-free survival, even if the frequency of GSTP1 methylation in the overall case series was lower than other studies (14%). Furthermore, GSTP1 methylation was also associated with positivity to ER as demonstrated by a number of studies [64], [68], [69]. Shinozaki et al. [70] demonstrated that GSTP1 and RARB methylation frequencies were significantly higher in patients with macroscopical sentinel lymph node metastasis, compared with patients with microscopical metastasis or without metastasis.
Tserga et al. [71] observed that GSTP1 hypermethylation is associated with advanced T-category and that a panel of four genes (GSTP1, RASSF1, DAPK1 and MGMT) may be associated with the development of a more aggressive breast cancer.
On the other hand, in the Dejeux et al. [72] study, GSTP1 hypermethylation was associated with better response to doxorubicin treatment, which is consistent with data reporting a better survival of patients with lower GSTP1 expression [73].
Miyake et al. investigated pretreatment biopsies of patients treated with paclitaxel followed by 5-fluorouracil/epirubicin/cyclophosphamide (P-FEC). In ER-negative tumors, the response rate was significantly higher in GSTP1-negative tumors (80.0%) than GSTP1-positive tumors (30.6%) [74].
Lung cancer
Regarding lung cancer, there is not a striking consensus among studies: GSTP1 methylation frequency in cancerous tissue of non-small cell lung cancer (NSCLC) patients ranges from 0% to 25%, whereas no methylation was found in adjacent benign tissue [75], [76], [77], [78], [79], [80].
However, other studies reported a methylation status of GSTP1 also in noncancerous adjacent tissue: with lower frequency (5% vs. 15% in cancerous tissue) [81], but even with comparable frequency (33% vs. 46% in cancerous tissue [82], and 15% vs. 18% in cancerous tissue [83]).
Lastly, two studies observed an association with histology: Harden et al. [84] correlated GSTP1 hypermethylation with nonsquamous histology, whereas Haroun et al. [79] correlated it with large cell lung cancer. In conclusion, due to these high heterogeneous results, more studies are needed to clearly understand the role of this epigenetic marker in lung cancer.
Hepatocellular carcinoma
In HCC, GSTP1 resulted methylated in about 50% of cancerous tissues, with remarkably lower (or often absent) methylation in benign adjacent tissue and no methylation in normal liver [16], [17], [18], [40], [85], [86], [87], [88], [89], [90], [91], [92], [93], with the exception of two studies that reported high methylation rates also in noncancerous adjacent tissues [94], [95].
Various studies proposed GSTP1 methylation as a diagnostic tool for HCC reporting a sensitivity of 50%–75% and a specificity of 70%–91% [85], [86], [87].
However, GSTP1 was found to be better performing if combined with other genes (such as APC or RASSF1) for a methylation-based diagnostic assay [85], [86].
Regarding the prognostic role of GSTP1, the results are more heterogeneous: in the study conducted by Zhen et al., GSTP1 was found to be more frequently methylated in tumors with capsular invasion, and consequently with invasion and metastasis [90]; GSTP1 methylation was also associated with poor histopathologic grading, multiple tumors number [88] and poorer survival [91].
Li et al. [89] did not find a correlation with clinicopathological features, and according to Anzola et al. [96], GSTP1 methylation was more frequent in well differentiated tumors without a significant prognostic meaning.
GSTP1 was also associated with HBV infection [17], [18], cirrhosis [87], [91], [94] and aflatoxin-b exposure [16], which is not surprising due to the high involvement of such environmental factors in HCC.
Other tumors
GSTP1 methylation in bladder and urothelial cancer does not seem to be a frequent event, with a frequency ranging from 1% to 15% [13], [97], [98]. Two studies reported high frequencies. Sacristan et al. [99] found a correlation between GSTP1 methylation and tumor stage in bladder cancer: GSTP1 was methylated in 62.2% pTa, 55.6% pT1 low-grade and 19.8% pT1 high-grade tumors with an overall frequency of 44%. Casadio et al. [13] reported GSTP1 methylation in non-recurrent bladder cancer patients (26%) compared with recurrent patients (5%).
In gastric cancer, the frequency of GSTP1 methylation ranges from 1.4% to 20.6% of patients [100], [101], [102], [103], [104], [105]. It is reported to be more frequent in diffuse type than in intestinal type gastric carcinomas (28% vs. 8%) [101]. In the Kang et al. [103] study, GSTP1 methylation was present in 16.3% of gastric cancer tissues, whereas it was absent in gastric adenoma, intestinal metaplasia and chronic gastritis; thus, GSTP1 proved to be a high specific marker for cancerous lesions.
Some studies reported also a remarkable higher frequency of GSTP1 methylation in gastric cancer tissues of EBV-positive compared to EBV-negative patients [19], [20].
GSTP1 methylation as a circulating cell free DNA biomarker
The analysis of circulating DNA could serve as a minimally invasive approach for diagnosis, prognosis and monitoring of cancer to be used also for the follow-up of patients during specific treatments [106]. The use of methylated tumor-specific circulating DNA has shown great promise as a cancer biomarker [107]. Many papers demonstrated that the methylation of specific regions in circulating DNA could serve as biomarkers for early diagnosis of cancer, as they are frequently early events in the carcinogenesis.
The majority of papers present in literature regard GSTP1 methylation of prostate and breast cancer and also other tumors present GSTP1 methylation.
Prostate cancer
The role of GSTP1 methylation as a noninvasive biomarker for the early diagnosis of PCa has been widely investigated in circulating cell free DNA from plasma [108]. Chuang et al. identified that 31% of plasma samples presented GSTP1 methylation, with a good concordance with the matched paraffin-embedded tissue, whereas 93% of benign prostate hyperplasia (BPH) samples showed no hypermethylation, as confirmed in serum samples [55], [109].
Dumache et al. [110] identified GSTP1 hypermethylation in 93% of PCa patients and 10% of healthy individuals, giving a predictive accuracy of 93% with a sensitivity and specificity of 95% and 87%, respectively.
Papadopoulou et al. [111] reported that 75% of plasma samples from patients with newly diagnosed PCa and 36.8% of patients being treated for PCa presented GSTP1 promoter hypermethylation using MS-PCR approach, highlighting that it could be also a prognostic marker.
Mahon et al. [112] found that the methylation of GSTP1 in plasma DNA using sensitive MS-PCR could serve as a potential prognostic marker in men with castration resistant PCa. Interestingly, they found that GSTP1 could be not only a prognostic marker, but also a potential therapeutic marker for chemotherapy. These findings must be confirmed in larger case series but pave the way for an additional use of GSTP1, together with PSA, for the follow-up of patients during chemotherapy or other treatments [112].
GSTP1 methylation analysis has also been performed in DNA derived from serum samples.
Patients with localized disease could benefit from GSTP1 methylation detection in serum samples, as Bastian et al. [45] demonstrated. The authors detected GSTP1 methylation in serum samples of patients with localized PCa using a restriction endonuclease quantitative PCR. They suggest an association with a four times greater risk of biochemical recurrence after surgery in the presence of an aggressive disease [45]. A defined panel of methylated genes, among which GSTP1, could be a useful biomarker in men with hormone refractory PCa [113]. On the contrary, Brait et al. [114] tested a panel of epigenetic biomarkers, including GSTP1 in early stage PCa patients and found that, although the frequency of GSTP1 methylation is high in primary PCa, it is not a suitable methylation marker in serum due to low sensitivity.
Sunami et al. [115] found that 13% of serum DNA deriving from PCa patients showed GSTP1 methylated and its methylation was correlated with higher stages of the disease.
A study conducted on plasma, serum, nucleated blood cells, ejaculates, urine after prostate massage and prostate tissue from 33 PCa patients and 26 control patients with BPH revealed that GSTP1 methylation analysis provides a highly specific tool for diagnosis of PCa in body fluids [116].
Moreover, a meta-analysis suggests that the analysis of GSTP1 methylation in plasma, serum or urine samples may complement PSA screening for PCa diagnosis [49]. Payne et al. [36] showed that methylated GSTP1 in urine significantly discriminate PCa from biopsy-negative patients with a greater sensitivity compared to plasma analyses. For this reason, the use of different body fluids at the same time could give more complete information regarding GSTP1 methylation status.
Breast cancer
Regarding breast cancer, Matuschek et al. [117] found hypermethylation of GSTP1 in 18% of breast cancer patients’ serum. Methylated GSTP1 was predominantly found in the serum of patients with large primaries and significantly correlated with positive Her2/neu status. Interestingly, the presence of circulating tumor cells was significantly associated with GSTP1 methylation.
Yamamoto et al. established a new one-step MS-PCR (OS-MSP) for analyzing gene methylation in serum DNA of patients with breast cancer. The sensitivity of this assay was significantly higher than that of the assay involving conventional tumor markers (CEA and/or CA15-3) for stages I (24% vs. 8%) and II (26% vs. 8%) breast cancer and similar to that of the assay involving the conventional tumor markers for stage III (18% vs. 19%) and metastatic breast cancers (55% vs. 59%). The results of the OS-MSP assay and those of the assay involving CEA and/or CA15-3 seemed to compensate for each other because the sensitivity of these assays increased to 78% when used in combination for metastatic breast cancer. As a result, the combination of this assay and the assay involving CEA and/or CA15-3 is promising for enhancing the sensitivity of diagnosis of metastatic breast cancer [118].
Fujita et al. used OS-MSP to detect DNA methylation in serum of breast cancer patients with high sensitivity, analyzing GSTP1, RASSF1A and RARβ2 methylation. They showed that this assay represents a significant and independent prognostic factor [119], [120]. Sharma et al. evaluated the promoter methylation status of GSTP1 with methylation-specific PCR in tumor and circulating DNA of 100 invasive ductal breast carcinoma patients. The frequency of tumor hypermethylation was 25% and correlated with methylation in paired serum DNA, suggesting a potential role of DNA methylation in serum samples as a promising biomarker for diagnosis and prognosis of breast cancer [121]. In another study, Sharma et al. [122] reported that the frequency of circulating BRCA1 and GSTP1 methylated DNA in pretherapeutic DNA for responders was significantly lower than non-responders.
Other tumors
Others tumors, such as testicular cancer or HCC, showed a hypermethylation of GSTP1 as diagnostic marker to be further investigated.
Ellinger et al. [46] reported that GSTP1 and other five genes’ methylation status could be analyzed in cell-free circulating serum DNA in patients with testicular cancer, concluding that this method has the potential to implement the diagnostic accuracy for patients with testicular germ cell cancer. Real-time polymerase chain reaction following methylation-sensitive restriction endonuclease treatment is performed.
The diagnostic ability of plasma GSTP1 methylation analysis between HCC patients and controls revealed a sensitivity of 56% and a specificity of 90% [123], but it could be also useful in HCC monitoring [124].
On the contrary, GSTP1 methylation for NSCLC was found in 0% of patients’ serum [125].
Methylation in urine
Urine represents a precious source of biomarkers for the study of urological pathologies thanks to the shedding of cellular and cell-free material from the uro-genital apparatus directly into this sample type [126], [127], [128], [129].
Urinary DNA-based biomarkers have been largely investigated in the field of urological cancers [128], [129], [130], [131], in particular, the majority of studies focused on PCa: the largerly reported high frequency of GSTP1 methylation in PCa tissues makes it a good candidate for the assessment of a methylation-based urinary diagnostic assay.
Most of the studies investigated urinary sediments in urine collected after digital rectal examination (DRE) [36], [132], [133], [134], [135], [136]. Rogers et al. performed a direct comparison between urine collected after biopsy and collected after DRE, and they reported no significant differences on the detection of methylation status of three genes (GSTP1, APC and EDNRB); even if it is not significant, the frequency of GSTP1 methylation was slightly higher in the post DRE urine (24%) compared with post biopsy urine (18%). Thus, this study demonstrated that post-DRE urine or post biopsy urine are comparable and suitable for methylation analyses [137].
Hoque et al. [138] compared GSTP1 methylation status in primary PCa tissue and in urine and found a 48% of concordance between the two biological sources, which is remarkably higher than the one reported in their previous study (27%) [139].
As stated by the authors, this is possibly due to differences in primer design or case series size between the two studies [138].
Woodson et al. [132] made a comparison between GSTP1 methylation status in biopsies and in urine finding a higher sensitivity in biopsies (91% vs. 75%) but a higher specificity in urine (98% vs. 88%) reporting a higher frequency of GSTP1 methylation in urine of patients with stage III tumors compared with stage II tumors (100% vs. 20%).
Early studies reported a higher frequency of GSTP1 methylation in serum or plasma rather than urine (36.2% and 72% in serum/plasma; 30.4% and 36% in urine) [29], [134].
However, more recently, Payne et al. [36] reported that GSTP1 methylation has a better diagnostic performance in whole urine (AUC=0.69) rather than plasma (AUC=0.55).
In another study, GSTP1 methylation performed only in urine showed a high specificity for PCa (74%), but with a low sensitivity (38.4%) [133].
One study reported a strangely low frequency of GSTP1 methylation in urine (only 1 patient out of 34, 3%), that may be attributable to the unusual method of urine collection: catheterization during prostatectomy, instead of the more frequent post-DRE urine collection [140].
GSTP1 methylation as a PCa urinary biomarker was found to be better performing if used in combination with other methylated genes, so various methylation-based panel were developed: GSTP1, RARB and APC (sensitivity 53%–55%, specificity 76%–80%) [135]; GSTP1, RARB, APC and RASSF1a (sensitivity 86%, accuracy 89%) [136]; GSTP1, MGMT, ARF and p16 (sensitivity 87%, specificity 100%) [138].
Two studies showed that panels including GSTP1 methylation have also a prognostic value: GSTP1 and APC methylation can help discriminating between low-risk and aggressive tumors [141], whereas the combination of GSTP1, APC, CRIP3 and HOXD8 is useful for the reclassification of PCa patients under active surveillance [142].
Regarding bladder and urothelial cancer, Chan et al. [143] found a relatively low frequency of GSTP1 methylation in transitional cell carcinoma (5%), suggesting that GSTP1 could not be suitable for the diagnosis of this malignancy.
Hoque et al. [131] found a high frequency of GSTP1 methylation in urine of bladder cancer patients (43%) with high tumor specificity (100%). They also compared primitive tissue methylation status reporting a perfect match (100%) between results obtained from tissue and urine.
GSTP1 was included by Hoque et al. [144] in a panel with CDKN2A, MGMT and ARF reaching a sensitivity of 82% and a specificity of 96%.
Hoque et al. [131] investigated with the same approach also renal cell carcinoma with less exciting results: GSTP1 was methylated only in 2 out of 17 primary tumors, and only one case was confirmed in urine; therefore, an eventual GSTP1 diagnostic application in this pathology is probably excluded.
Table 2 summarizes sensitivity and specificity of GSTP1 methylation detection as liquid biopsy.
Summary of the studies reporting GSTP1 sensitivity and/or specificity in liquid biopsies.
| Sample type | Targets | Method | Cancer type | Cancer patients, n | Controls, n | Sensitivity, % | Specificity, % | Reference |
|---|---|---|---|---|---|---|---|---|
| Plasma | GSTP1 | MSRE-qPCR | Prostate | 36 | 27 | 31 | 93 | Chuang et al. [109] |
| Plasma | GSTP1 | MS-PCR | Prostate | 31 | 44 | 95 | 87 | Dumache et al. [110] |
| Plasma | GSTP1 | MS-PCR | Prostate | 12 | 9 | 75 | 100 | Papadopoulou et al. [111] |
| Plasma | GSTP1 | MS-PCR | Prostate | 64 | 45 | 80 | 82 | Altimari et al. [145] |
| Serum | GSTP1 | MSRE-qPCR | Prostate | 85 | 35 | – | 100 | Bastian et al. [45] |
| Serum | GSTP1, RASSF1, RARB | MS-PCR | Prostate | 83 | 40 | 28 | – | Sunami et al. [115] |
| Serum | GSTP1, TIG1, PTGS2, Reprimo | MSRE-qPCR | Prostate | 168 | 42 BPH | 42.3 | 92.9 | Ellinger et al. [146] |
| 11 Healthy | 47 (panel) | 92.9 (panel) | ||||||
| Blood/urine/ejaculate | GSTP1 | MS-PCR | Prostate | 33 | 26 | – | 100 | Goessl et al. [116] |
| Serum | qMS-PCR | Prostate | 84 | 30 | 1.2 | 100 | Brait et al. [114] | |
| Serum | APC, GSTP1, PTGS2, ARF, p16(INK), RASSF1A | MSRE-qPCR | Testicular | 73 | 35 | 67 | 97 | Ellinger et al. [46] |
| Plasma | GSTP1 | MSRE-qPCR | Hepatocellular | 72 | 37 benign liver diseases and 41 normal controls | 56 | 90 | Huang et al. [123] |
| Serum | GSTP1, APC, PTGS2 | MSRE-qPCR | Kidney | 35 | 54 | 17.1 | 98.1 | Hauser et al. [147] |
| 62.9 (panel) | 87 (panel) | |||||||
| Serum | GSTP1, BRCA1, MGMT | MS-PCR | Breast | 100 | 30 | 22 | 97 | Sharma et al. [121] |
| Serum | GSTP1, RASSF1, RARB2 | OS-MSP | Breast | 101 primary breast cancer 58 metastatic breast cancer | 87 5 | 22 (panel) 55 (panel) | 93 (panel) | Yamamoto et al. [118] |
| Urine (sediments) | GSTP1, APC, EDNRB | MS-PCR | Prostate | 12 | 24 (post DRE urine) 18 (post biopsy urine) | 80 | Rogers et al. [137] | |
| Urine | GSTP1 | qMS-PCR | Prostate | 24 | 69 BPH 8 PIN | 75 | 98 | Woodson et al. [132] |
| Urine (sediments) | GSTP1 | MS-PCR | Prostate | 14 | 52 | 38 | 74 | Dimitriadis et al. [133] |
| Urine (sediments), plasma or serum | GSTP1 | MS-PCR | Prostate | 33 | 26 | 36 (urine) 72 (plasma or serum) | – | Goessl et al. [134] |
| Urine, plasma | GSTP1 | MS-PCR and qMS-PCR | Prostate | 69 | 31 BPH | 30 (urine) 36 (plasma) | 97 | Jerónimo et al. [29] |
| Urine (sediments) | GSTP1, RASSF1A | MS-PCR | Prostate | 34 | 79 | 53.3 (panel) | 45.9 (panel) | Prior et al. [148] |
| Urine (sediments) | GSTP1, p16, ARF, MGMT | qMS-PCR | Prostate | 52 | 91 | 87 (panel) | 100 (panel) | Hoque et al. [138] |
| Urine (sediments) | GSTP1, RARB, APC | MS-PCR | Prostate | 113 | 128 | 33–36 53–55 (panel) | 91–95 76–80 (panel) | Vener et al. [135] |
| Urine (sediments) | GSTP1, RARB, APC, RASSF1A | qMS-PCR | Prostate | 95 | 38 | 83.2 (panel) | 87 | Rouprêt et al. [136] |
| Urine (supernatant), plasma, | GSTP1 | sequencing | Prostate | 5 | 5 healthy 5 BPH | 100 | 100 | Bryzgunova et al. [23] |
| Urine (post-prostatic massage), plasma | GSTP1, RASSF2, HIST1H4K TFAP2E | Heavy Methyl qPCR | Prostate | 91 | 51 biobsy negative patients 50 young asymptomatic males | 64 (GSTP1 and RASSF2) | 61 (GSTP1 and RASSF2) | Payne et al. [36] |
| Urine (Sediments) | GSTP1 | MS-PCR | Prostate | 18 | 21 healthy controls 6 atypia/PIN | 58 | – | Gonzalgo et al. [149] |
| Urine (sediments) | GSTP1, CDKN2A, MGMT, ARF | MS-PCR | Bladder | 160 | 94 | 82 (panel) | 96 (panel) | Hoque et al. [144] |
| Urine (sediments) | GSTP1, RARB, DAPK, E-cadherin, p16, p15, MGMT | MS-PCR | Bladder | 22 | 17 | 5.1 | – | Chan et al. [142] |
| Urine (sediments) | GSTP1,CDH1, APC, MGMT, RASSF1A, p16, RAR-beta2, ARF | qMS-PCR | Renal | 17 | 91 | 5.9 | – | Hoque et al. [131] |
qMS-PCR, methylation specific quantitative polymerase chain reaction; MS-PCR, methylation specific polymerase chain reaction; MSRE-qPCR, methylation-sensitive restriction enzymes-based quantitative PCR; OS-MSP, one-step methylation-specific polymerase chain reaction; BPH, benign prostate hyperplasia; DRE, digital rectal examination; PIN, prostatic intraepithelial.
Discussion
The study of epigenetic alterations in cancer has been intensively pursued for finding diagnostic, prognostic and predictive biomarkers. Methylation at specific CpG sites of tumor suppressor genes promoters is the most studied epigenetic event, and recently it has been proposed also in body fluids as a liquid biopsy approach [150], thanks to the technological advance in methods [151] and to the growing interest in minimally invasive approaches.
Papers regarding GSTP1 methylation were published since 20 years ago [7]; as a consequence, GSTP1 could be considered as a “milestone” methylation biomarker. It represents a typical example of a gene involved in a variety of tumors, and it has been evaluated in many sample types: cell lines, tissues, plasma and urine samples. GSTP1 methylation has different functions depending on the cancer type: it has been ascribed as prognostic, predictive and diagnostic marker [22], [53], [61], [62], [66], [67], [72], [108], [119], [120]. Most papers are published on PCa in which the methylation frequency in tissue samples is higher than 50% [53], rendering it an optimal diagnostic marker; in addition, its methylation status seems to be not correlated with pathological stage and grades and it is present since the earlier phases of cancer development [33], [56]. Another important aspect is that, as demonstrated by Millar and colleagues [51], DNA methylation is not confined to specific CpG sites in the promoter region of the GSTP1 gene but is extensive throughout the CpG islands; thus, a number of different assays can be designed considering different CpG sites [44], [49].
All these characteristics in PCa made GSTP1 methylation an ideal diagnostic marker to be investigated using noninvasive approaches having good sensitivity and specificity characteristics. It can also be considered as an ideal biomarker for its non-invasiveness, the analysis is rapid with standard methods such as MS-PCR, the cost is low and the results are easy to be interpreted. However, despite all these promising aspects, it has not yet entered the clinical practice. This has probably happened for a heterogeneity problem among the studies conducted on blood or urine. An interesting meta-analysis, performed in 2011 by Wu and coworkers [49], has underlined that considering a pooled specificity and sensitivity calculated considering plasma, serum and urine samples from the different studies, GSTP1 had a specificity better than that of PSA, but its sensitivity seemed to be too low to be used as a biomarker alone. The heterogeneity of the studies in terms of methods and also sample types is the reason for the wide range of sensitivity among different studies, so they suggested a potential use of GSTP1 together with PSA for an early diagnostic approach. An interesting issue is that, considering the natural exfoliation of cancer cells, urine can be used as a source of biomarkers and molecular alterations in PCa, in a less invasive manner than blood collection [152].
Regarding non-prostatic tumors, it is of note that GSTP1 has a lower methylation frequency (below 50% in primary tumor tissues) and, as a consequence, it has a different putative role, not ascribable to an early diagnostic marker. Although in PCa GSTP1 can be studied alone or in combination with conventional PSA, for the other diseases, due to its lower methylation frequency, it is better to consider it in combination with other methylation markers, to maximize the accuracy [70], [71], [85], [86], [131], [144]. In breast cancer, the most investigated after PCa, it can be considered as a prognostic/predictive marker as it is correlated with ER expression and clinicopathological features [64], [67], [68], [70].
Despite GSTP1 methylation in breast cancer is associated with a poorer patient’s outcome, individuals with high methylation seem to have a better response to doxorubicin, compared to those without gene methylation. In this specific context, GSTP1 seems to have a role as predictive biomarker [73].
Conclusions
GSTP1 represents a typical example of an ideal epigenetic biomarker, as its methylation is involved in a number of diseases, in particular in PCa.
GSTP1 can be used as a “liquid biopsy biomarker” and it could be detected with good results in circulating cell free DNA and urinary DNA.
For PCa early diagnosis, it has many good features that make it suitable for clinical application. However, further multicentric, large, prospective studies are needed to validate this assay and apply it in clinical practice.
Acknowledgments
The authors thank Francesco Mazza for manuscript revision.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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