PPM1D gene amplification and overexpression in breast cancer: a qRT-PCR and chromogenic in situ hybridization study

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Abstract

PPM1D (protein phosphatase magnesium-dependent 1δ) maps to the 17q23.2 amplicon and is amplified in 8% of breast cancers. The PPM1D gene encodes a serine threonine phosphatase, which is involved in the regulation of several tumour suppressor pathways, including the p53 pathway. Along with others, we have recently shown that PPM1D is one of the drivers of the 17q23.2 amplicon and a promising therapeutic target. Here we investigate whether PPM1D is overexpressed when amplified in breast cancers and the correlations between PPM1D overexpression and amplification with clinicopathological features and survival of breast cancer patients from a cohort of 245 patients with invasive breast cancer treated with therapeutic surgery followed by adjuvant anthracycline-based chemotherapy. mRNA was extracted from representative sections of tumours containing >50% of tumour cells and subjected to TaqMan quantitative real-time PCR using primers for PPM1D and for two housekeeping genes. PPM1D overexpression was defined as the top quartile of expression levels. Chromogenic in situ hybridization with in-house-generated probes for PPM1D was performed. Amplification was defined as >50% of cancer cells with >5 signals per nucleus/large gene clusters. PPM1D overexpression and amplification were found in 25 and 6% of breast cancers, respectively. All cases harbouring PPM1D amplification displayed PPM1D overexpression. PPM1D overexpression was inversely correlated with expression of TOP2A, EGFR and cytokeratins 5/6 and 17. PPM1D amplification was significantly associated with HER2 overexpression, and HER2, TOP2A and CCND1 amplification. No association between PPM1D gene amplification and PPM1D mRNA overexpression with survival was observed. In conclusion, PPM1D is consistently overexpressed when amplified; however, PPM1D overexpression is more pervasive than gene amplification. PPM1D overexpression and amplification are associated with tumours displaying luminal or HER2 phenotypes. Co-amplification of PPM1D and HER2/TOP2A and CCND1 are not random events and may suggest the presence of a ‘firestorm’ genetic profile.

Main

PPM1D (protein phosphatase magnesium-dependent 1δ), also known as WIP1 (wild-type p53-induced phosphatase 1), is a member of the nuclear type 2C protein phosphatase family.1 The PPM1D gene maps to 17q23.2, a genomic region recurrently amplified in several types of tumours including medulloblastomas, neuroblastomas, pancreatic adenocarcinomas, ovarian clear cell carcinomas and breast cancers.2, 3, 4, 5, 6, 7, 8, 9, 10 We have recently shown in a series of 95 grade III invasive breast cancers that PPM1D is amplified in 7% of luminal and 20% of HER2 amplified cancers and that PPM1D is consistently overexpressed at the mRNA level in cases harbouring PPM1D gene amplification.9

There are several lines of evidence suggesting that PPM1D may constitute a driver of the 17q23.2 amplicon8, 9, 10 and that it may have oncogenic properties when amplified.11 PPM1D is induced by p53 upon DNA damage and has a pivotal role in the negative regulation of p53 itself and also a series of additional proteins involved in the response to cellular stress such as ATM, Chk2, p38 mitogen-activated protein kinase, INK4, ARF, MDM2 and H2AX.12, 13, 14, 15, 16, 17 In particular, PPM1D provides a negative feedback mechanism in the p38/p53 signalling cascade by inhibiting irradiation-induced activation of p38, thus eliciting suppression of p53 activation.3, 18 In addition to its role in the regulation of the p53 pathway, PPM1D is also involved in the negative regulation of G1-S and G2/M checkpoints in response to DNA damage,19 DNA repair mechanisms,11, 20 progesterone receptor signalling21 and apoptosis inhibition.11 PPM1D also seems to be involved in the regulation of the nuclear factor-κB (NF-κB) pathway, as there is evidence to suggest that PPM1D acts as a negative regulator of NF-κB signalling;22, 23 however, NF-κB is a transcriptional enhancer of PPM1D expression.24

In previous studies, we have shown that PPM1D expression and phosphatase activity are required for the survival of cancer cells derived from breast9, 25 and ovarian clear cell carcinomas8 harbouring amplification of 17q23.2. Our previous results provide direct evidence that PPM1D may be one of the drivers of this amplicon8, 9, 10 and that PPM1D may constitute a therapeutic target for a subgroup of breast and ovarian cancers harbouring PPM1D gene amplification.

The aims of this study were to investigate whether PPM1D is significantly overexpressed when amplified and to define the correlations between PPM1D gene amplification and mRNA overexpression with clinicopathological characteristics of primary breast cancers. As a secondary end point, we sought to define whether PPM1D gene amplification or mRNA overexpression are associated with the outcome of breast cancer patients uniformly treated with therapeutic surgery followed by anthracycline-based chemotherapy alone or with endocrine therapy. As part of this study, we also assess the potential of using chromogenic in situ hybridization as a method to assess PPM1D gene status (ie, amplification).

Materials and methods

Tissue Microarrays

A cohort of 245 patients with invasive breast cancer (185 invasive ductal carcinomas, 27 invasive lobular carcinomas, 25 invasive mixed carcinomas and 8 invasive breast carcinomas of other special types) treated with therapeutic surgery followed by adjuvant anthracycline-based chemotherapy were included in this study. All patients were diagnosed and managed at the Royal Marsden Hospital, London, UK, between 1994 and 2000. All patients were primarily treated with surgery (69 mastectomy and 156 wide local excision) followed by anthracycline-based chemotherapy. Adjuvant endocrine therapy was prescribed for patients with oestrogen receptor-positive tumours (tamoxifen alone in 96% of the patients for the available follow-up period). Complete follow-up was available for 244 patients, ranging from 0.5 to 125 months (median 67 months, mean 67 months). Tumours were graded according to a modified Bloom–Richardson scoring system26 and size was categorized according to the TNM staging.27 The study was approved by the Royal Marsden Hospital ethics committee.

Chromogenic In Situ Hybridization

Chromogenic in situ hybridization for PPM1D gene amplification was performed on 2-μm-thick tissue microarray sections mounted on polylysine-coated slides, using an in-house-generated probe as previously described.28 This probe comprises three bacterial artificial chromosome contigs (RP11-15E18, CTD-2327L2 and RP11-67D12), which map to the region 58.54–58.83 Mb on chromosome 17q23.2 (http://www.ensembl.org) and encompass the PPM1D gene. Heat pretreatment of deparaffinized sections consisted of incubation for 15 min at 98°C in CISH pretreatment buffer (SPOT-Light tissue pretreatment kit; Invitrogen) and digested with pepsin for 5.5 min at room temperature according to the manufacturer's instructions. Slides were hybridized and developed as previously described.28 Appropriate PPM1D gene-amplified breast cancers (n=2), as defined by microarray-based comparative genomic hybridization analysis, were used as controls to determine the sensitivity of the probe and included in the slide run. Negative controls comprised cases defined as lacking PPM1D gene amplification by microarray-based comparative genomic hybridization analysis (n=2). Chromogenic in situ hybridization experiments were analysed by three of the authors (FCG, MAL-G and ML-T) on a multi-headed microscope. Only unequivocal signals were counted. Signals were evaluated at × 400 and × 630 magnification and 30 morphologically unequivocal neoplastic cells in each core were assessed for the presence of the PPM1D gene signals. Amplification was defined as >50% of the neoplastic cells harbouring either >5 copies of the gene or large gene clusters. Chromogenic in situ hybridization analysis was performed with observers blinded to clinicopathological parameters, patients’ survival and results of the quantitative real-time reverse-transcriptase PCR (qRT-PCR) analysis. In addition to probes for PPM1D gene, the tissue microarray was hybridized with SPOT-Light probes (Invitrogen) for CCND1, MYC, HER2, TOP2A, and chromosome 8 centromere. Results not in relation to PPM1D amplification and/or expression were reported elsewhere.29, 30

Quantitative Real-Time Reverse-Transcriptase PCR

Briefly, all cases were reviewed by two pathologists (FCG and ML-T). Representative 8-μm-thick sections were cut from archival blocks and microdissected to ensure at least 50% of tumour cell content. RNA was extracted from microdissected sections using the RNeasy FFPE RNA Isolation Kit (Qiagen) followed by an additional DNase treatment as previously described.29 RNA quantification was performed using the RiboGreen Quant-iT reagent (Invitrogen) and reverse transcription was performed with Superscript III (Invitrogen) using 400 ng of RNA per reaction with triplicate reactions performed for each sample. Quantitative real-time PCR was performed using TaqMan chemistry on the ABI Prism 7900HT (Applied Biosystems), using the standard curve method.29 PPM1D probe was purchased from Applied Biosystems (PPMID ID: Hs00186230_m1-PPM1D). In addition, two reference genes (TFRC ID: Hs00174609_m1-TFRC and MRPL19 ID: Hs00608522_g1-MRPL19) were used, having been previously selected as effectively normalizing for degradation of RNA.29 Target gene expression levels were normalized to the geometric mean of the two reference genes and normalized to a calibrator (pool of tumour cDNAs from the same series). PPM1D overexpression was defined as the top quartile of expression levels.

Immunohistochemistry

The details of the immunohistochemical methods and scoring systems for oestrogen receptor, progesterone receptor, HER2, epidermal growth factor receptor (EGFR), cytokeratin (Ck) 14, Ck5/6 and Ck17, Ki-67, p53, topoisomerase II α (TOP2A), caveolin-1 (CAV1), caveolin-2 (CAV2), FOXA1, E-cadherin, CD44, Bcl2, MDM2, MDM4, nestin and cyclin D1 are described elsewhere31, 32, 33, 34, 35 and summarized in Supplementary Table 1. On the basis of the expression of HER2, oestrogen receptor, Ck 5/6 and EGFR, tumours were classified into basal, HER2 and luminal according to the immunohistochemical panel proposed by Nielsen et al.36

Statistical Analysis

The SPSS statistical software package version 11.5 (SPSS Inc., IBM, Chicago, IL, USA) was used for all statistical analysis. Correlations between categorical variables were performed using the χ2-test and Fisher's exact test. Disease-free survival was expressed as the number of months from diagnosis to the occurrence of distant, local relapse or death (disease-related death). Metastasis-free survival was expressed as the number of months from diagnosis to the occurrence of distant relapse. Overall survival was expressed as the number of months from diagnosis to the occurrence of breast cancer-related death. Cumulative survival probabilities were calculated using the Kaplan–Meier method. Differences between survival rates were tested with the log-rank test. A P-value of 0.05 was considered as statistically significant.

Results

PPM1D Gene Amplification

PPM1D was found to be amplified in 10 out of 181 interpretable cases (6%, Figures 1, 2, 3 and 4). Table 1 summarizes the correlations between PPM1D amplification and clinicopathological features, immunohistochemical and chromogenic in situ hybridization findings in invasive breast carcinomas. PPM1D gene amplification was significantly associated with HER2 overexpression and HER2, TOPO2A and CCND1 amplification. All but one case harbouring PPM1D amplification displayed oestrogen receptor expression (five were oestrogen receptor+/HER2−, four were oestrogen receptor+/HER2+ and one was oestrogen receptor−/HER2+). As expected, no grade I breast cancer displayed PPM1D gene amplification. PPM1D gene amplification was observed in 4% of luminal cancers, 19% of HER2 tumours and none of basal-like and triple negative breast cancers. A statistically significant association between PPM1D gene amplification and HER2 phenotype as defined by Nielsen et al36 (P=0.04) was found. No inverse association between p53 nuclear expression, a surrogate marker for TP53 mutations and PPM1D gene amplification was identified.

Figure 1
figure1

Representative micrographs of a grade III invasive ductal carcinoma of no special type (a) of HER2 phenotype as defined by Nielsen et al36 criteria, displaying PPM1D gene amplification (b), oestrogen receptor expression (c), focal progesterone receptor expression (d) and HER2 gene amplification (e), and lacking TOP2A gene amplification (f).

Figure 2
figure2

Representative micrographs of a grade III invasive ductal carcinoma of no special type (a) of HER2 phenotype as defined by Nielsen et al36 criteria, displaying PPM1D gene amplification (b), oestrogen receptor expression (c), progesterone receptor expression (d), and HER2 (e) and TOP2A (f) gene co-amplification.

Figure 3
figure3

Representative micrographs of a grade III invasive ductal carcinoma of no special type (a) of luminal phenotype as defined by Nielsen et al36 criteria, displaying PPM1D gene amplification (b), oestrogen receptor expression (c) and focal progesterone receptor expression (d), and lacking HER2 (e) and TOP2A (f) gene amplification.

Figure 4
figure4

Representative micrographs of a grade III invasive ductal carcinoma of no special type (a) of basal-like phenotype as defined by Nielsen et al36 criteria, lacking PPM1D gene amplification (b), oestrogen receptor expression (c), progesterone receptor expression (d), and HER2 (e) and TOP2A (f) gene amplification.

Table 1 Correlations between PPM1D gene amplification as defined by chromogenic in situ hybridization and clinicopathological parameters and immunohistochemical markers in 181 invasive breast carcinomas

It should be noted that although an association between PPM1D gene amplification and amplification of HER2 and TOP2A was observed, PPM1D was not necessarily co-amplified with these genes. In fact, out of the 10 cases harbouring PPM1D gene amplification, only 5 and 3 displayed HER2 and TOP2A gene amplification, respectively (Table 1).

PPM1D Gene Expression

PPM1D overexpression was more pervasive than amplification as it was found in 45 (25%) out of 181 assessable cases that yielded sufficient amounts of optimal quality mRNA for qRT-PCR assay. PPM1D overexpression was inversely correlated with the expression of ‘basal’ markers, including EGFR, Ck5/6, Ck14 and Ck17, and with TOP2A expression. There was no significant association between HER2 expression and PPM1D expression; however, one-third of the cases that showed HER2 amplification or overexpression also showed overexpression of PPM1D. There was no association between the overexpression of PPM1D with TOP2A, HER2 or CCND1 gene amplification. The correlations between PPM1D expression and clinicopathological features and immunohistochemical findings in breast carcinomas are summarized in Table 2 .

Table 2 Correlations between PPM1D mRNA overexpression as defined by qRT-PCR and clinicopathological parameters and immunohistochemical markers in 181 invasive breast carcinomas

Results for both status of PPM1D amplification and mRNA expression were available in 142 cases. PPM1D amplification was found to be strongly associated with PPM1D mRNA expression levels (Mann–Whitney U-test, P<0.00001, Figure 5). In fact, all cases harbouring PPM1D gene amplification displayed PPM1D mRNA overexpression; however, PPM1D mRNA overexpression was more pervasive than gene amplification.

Figure 5
figure5

Box and Whisker plot showing distribution of PPM1D mRNA levels assessed by qRT-PCR in breast cancers according to PPM1D gene amplification status. Cases with amplification (n=8) are labelled as 1, whereas cases without amplification (n=134) are labelled as 0. Extremes and outliers are identified by ‘o’ and ‘*’, respectively.

Survival Analysis

Univariate survival analysis failed to show any association between PPM1D gene amplification and PPM1D overexpression, and disease-free, metastasis-free or overall survival. Exploratory subgroup analysis of the impact of PPM1D gene amplification and mRNA overexpression on the disease-free, metastasis-free and overall survival of patients with ER-positive, ER-negative, HER2-positive, HER2-negative, lymph node-positive and lymph node-negative breast cancer failed to identify any statistically significant associations (data not shown).

Discussion

Here we show that PPM1D gene amplification is present in 6% of cases in a consecutive cohort of 181 breast cancers. PPM1D amplification was significantly more prevalent in tumours of HER2 phenotype (19%) than in luminal cancers (4%, Fisher's exact test P-value=0.007), whereas it was absent in tumours of basal-like and triple-negative phenotype. Furthermore, we confirmed by chromogenic in situ hybridization and qRT-PCR that PPM1D is significantly overexpressed when amplified expanding to previous observations derived from microarray-based comparative genomic hybridization and expression array analysis.9

Although PPM1D has been shown to have a pivotal role in the regulation of p53 signalling by dephosphorylating p38 and suppressing activation of downstream targets of p53, PPM1D amplification and overexpression were not mutually exclusive with p53 nuclear expression. Presence of p53 nuclear expression, albeit not a perfect surrogate for TP53 mutations, is strongly associated with TP53 mutation.37 This observation suggests that PPM1D amplification may confer advantages to tumour cells above and beyond p53 pathway inactivation. In fact, PPM1D has been shown to be involved in a series of interactions that could account for non-P53-related functions, including the regulation of progesterone receptor expression, specific DNA repair mechanisms and control of the NF-κB pathway.24 In fact, our previous studies have shown that PPM1D silencing and inhibition of its phosphatase activity are selectively lethal for breast and ovarian clear cell cancer cell lines in a manner that does not completely correlate with TP53 gene status.8, 9

In agreement with our previous observations, we show here that PPM1D overexpression is more prevalent than gene amplification. This is not surprising, as a substantial number of oncogenes, such as MYC, EGFR and CCND1, are more frequently overexpressed than amplified;30, 38, 39 however, in a way akin to these bona fide oncogenes, PPM1D is consistently and significantly overexpressed when amplified in support of the contention that PPM1D may constitute one of the drivers of the 17q23.2 amplicon.

At variance with TOP2A amplifications, which are almost exclusively found in association with HER2 gene amplification (Arriola et al29, Slamon and Press40 and references therein), PPM1D is not necessarily co-amplified with HER2. This is not surprising, as PPM1D maps 20 Mb distal to the smallest region of amplification of the HER2 amplicon on chromosome 17q.29

It should be noted that PPM1D gene amplification was associated with amplification of other loci on chromosome 17 (eg, HER2 (17q12) and TOP2A (17q21.2)) and on other chromosomes (eg, CCND1 on 11q13). This observation suggests that cases harbouring PPM1D gene amplification may display a ‘firestorm’ pattern (ie, tumours whose genome is characterized by multiple, clustered high-level amplifications).9, 41, 42 Re-analysis of the data from Natrajan et al9 revealed that all cases harbouring PPM1D gene amplification (n=8) displayed a ‘firestorm’ profile (Supplementary Figure 1).9 Taken together, it is plausible that amplification of 17q23.2 may stem from a global pattern of genetic instability that favours the acquisition of multiple, high-level gene amplifications throughout the genome.

Although we could not find an association between PPM1D gene amplification and PPM1D mRNA expression with the outcome of breast cancer patients, our findings should be interpreted with caution, as this is a retrospective, single-institution study. Furthermore, the prevalence of cases displaying PPM1D gene amplification is relatively low (6%); therefore, it is plausible that in larger cohorts with longer follow-up, associations with survival may be identified. Further studies testing the prognostic impact of PPM1D gene amplification in larger cohorts of breast cancer patients, in particular in tumours of HER2 and luminal phenotype, are warranted.

Given the increasingly more coherent data to suggest that PPM1D may constitute a therapeutic target in tumours harbouring 17q23.2 amplification and the availability of preclinical compounds that selectively inhibit PPM1D, it is probable that novel drugs inhibiting PPM1D will be developed and tested in vivo in the near future.25, 43 Hence, the availability of molecular diagnostics to identify the group of patients that may benefit from PPM1D inhibitors would facilitate the development of biology-driven clinical trials to test PPM1D as a therapeutic target. Here, we described reagents that can be used for the identification of PPM1D amplification and overexpression in breast cancer archival samples, including a chromogenic in situ hybridization probe for PPM1D that can be readily applied to formalin-fixed paraffin-embedded tissue samples. Given the lack of validated anti-PPM1D antibodies applicable for immunohistochemistry, the TaqMan-based qRT-PCR we described in this paper may constitute an alternative to assess PPM1D expression levels in routinely processed pathological samples, provided that the samples have an excess of 50% of tumour cells.

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Acknowledgements

This study was funded in part by Breakthrough Breast Cancer. KJD is the recipient of a Swiss National Science Foundation (SNF) fellowship.

Author information

Correspondence to Jorge S Reis-Filho.

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The authors declare no conflict of interest.

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Supplementary Information accompanies the paper on Modern Pathology website

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Keywords

  • breast cancer
  • chromogenic in situ hybridization
  • p53
  • prognosis
  • real time reverse transcriptase PCR
  • therapeutic target

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