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Virchows Archiv

, Volume 472, Issue 3, pp 351–359 | Cite as

p63 isoforms in triple-negative breast cancer: ΔNp63 associates with the basal phenotype whereas TAp63 associates with androgen receptor, lack of BRCA mutation, PTEN and improved survival

  • Philip J. Coates
  • Rudolf Nenutil
  • Jitka Holcakova
  • Marta Nekulova
  • Jan Podhorec
  • Marek Svoboda
  • Borivoj Vojtesek
Original Article

Abstract

The TP63 gene encodes two major protein variants that differ in their N-terminal sequences and have opposing effects. In breast, ΔNp63 is expressed by immature stem/progenitor cells and mature myoepithelial/basal cells and is a characteristic feature of basal-like triple-negative breast cancers (TNBCs). The expression and potential role of TAp63 in the mammary gland and breast cancers is less clear, partly due to the lack of studies that employ p63 isoform-specific antibodies. We used immunohistochemistry with ΔNp63-specific or TAp63-specific monoclonal antibodies to investigate p63 isoforms in 236 TNBCs. TAp63, but not ΔNp63, was seen in tumour-associated lymphocytes and other stromal cells. Tumour cells showed nuclear staining for ΔNp63 in 17% of TNBCs compared to 7.3% that were positive for TAp63. Whilst most TAp63+ tumours also contained ΔNp63+ cells, the levels of the two isoforms were independent of each other. ΔNp63 associated with metaplastic and medullary cancers, and with a basal phenotype, whereas TAp63 associated with androgen receptor, BRCA1/2 wild-type status and PTEN positivity. Despite the proposed effects of p63 on proliferation, Ki67 did not correlate with either p63 isoform, nor did they associate with p53 mutation status. ΔNp63 showed no association with patient outcomes, whereas TAp63+ patients showed fewer recurrences and improved overall survival. These findings indicate that both major p63 protein isoforms are expressed in TNBCs with different tumour characteristics, indicating distinct functional activities of p63 variants in breast cancer. Analysis of individual p63 isoforms provides additional information into TNBC biology, with TAp63 expression indicating improved prognosis.

Keywords

Triple-negative breast cancer TAp63 ΔNp63 PTEN BRCA1 BRCA2 Androgen receptor 

Introduction

The human TP63 gene is a member of the TP53 tumour-suppressor gene family and gives rise to at least six protein isoforms due to differential promoter usage and alternative splicing [1, 2, 3]. TAp63 variants have an N-terminal p53-like transactivation domain, while ΔNp63 isoforms lack this domain. TAp63 provides pro-apoptotic and senescence-inducing properties, whereas ΔNp63 isoforms have opposite roles and provide cell survival [1, 2, 3]. In addition to lacking normal limb development and formation of stratified epithelium, Trp63 knockout mice fail to develop mammary glands and a similar phenotype occurs in humans with Limb-Mammary Syndrome and ADULT Syndrome due to inherited TP63 mutations [4, 5], indicating a critical role for p63 in regulating mammary gland development.

In clinical histopathology, p63 is a commonly used marker of terminally differentiated basal/myoepithelial cells and is expressed in some triple-negative breast cancers (TNBCs), being a characteristic feature of the basal-like 2 subtype [6, 7, 8, 9]. In these TNBCs, ΔNp63 regulates cancer stem cell activities, maintains the basal phenotype and influences proliferation, adhesion/migration, EGFR activation and therapy responses [7, 10, 11, 12, 13, 14]. In contrast, TAp63 is reported to promote differentiation along the luminal lineage and to inhibit breast cancer formation in mice [15, 16].

Despite their different activities, most immunohistochemical studies employ antibodies that do not discriminate between TAp63 and ΔNp63 isoforms, and expression profiling array data similarly do not distinguish the separate isoforms. As a consequence, the expression and role of p63 isoforms is largely unknown in human cancers. Here, we used immunohistochemistry with TAp63-specific and ΔNp63-specific monoclonal antibodies to investigate the incidence of these two major p63 isoforms in TNBC. We show that TAp63 isoforms are expressed in a minority of cases and that these patient’s tumours exhibit distinct phenotypic and clinical features.

Materials and methods

Patient tissues and immunohistochemistry

Tissue microarrays (TMAs) were prepared from excess formalin-fixed and paraffin-embedded histological tissue blocks of TNBCs from patients prior to treatment as described previously [12, 17]. The samples were collected from unselected consecutive cases of triple-negative breast carcinoma treated at MMCI between 2004 and 2009. TMAs contained two tissue cores of 1.5 mm diameter for each cancer. After dewaxing and blocking endogenous peroxidase, sections were subjected to antigen retrieval and stained using the antibodies and conditions listed in Table 1. Primary antibodies were applied for 90 min at room temperature, and EnVision+ HRP reagents with DAB+ (Dako) were used for visualisation. Positive controls included reactive lymph node for TAp63 and non-neoplastic breast tissue for ΔNp63 staining. Negative controls were performed by omitting the primary antibody.
Table 1

Antibody details

Antigen

Supplier

Clone

Dilution

Retrieval

Cut-off

ER-alpha

LabVision

SP1

1/4000

EDTA pH 8

< 10%

PgR

LabVision

SP2

1/2000

EDTA pH 8

< 10%

HER2

Dako

HercepTest

1/200

Citrate pH 6

Negative IHC/ISH

ER-beta

Dako

PPG5/10

1/50

EDTA pH 8

10%

AR

Novocastra

NCL-AR-318

1/100

EDTA pH 8

10%

EGFR

Invitrogen

31G7

1/100

EDTA pH 8

2+/3+

CK5/6

Dako

D5/16B4

1/400

EDTA pH 8

2%

CK8/18

Biogenex

5D3

1/200

EDTA pH 8

2%

CK14

Novocastra

LL02

1/100

Citrate pH 6

2%

Alpha-SMA

Sigma

1A4

1/800

EDTA pH 8

10%

Vimentin

Dako

V9

1/400

EDTA pH 8

10%

ΔNp63 (p40)

In house

ΔNp63–1.1

1/200

EDTA pH 8

1%

TAp63

In house

TAp63–4.1

1/6400

EDTA pH 8

1%

p27

Novocastra

NCL-p27

1/100

Citrate pH 6

1%

PTEN

Dako

6H2.1

1/1600

EDTA pH 8

Wt v mutant

p53

In house

DO-1

1/200

EDTA pH 8

Wt v mutant

MDM2

In house

2A9

1/3200

EDTA pH 8

See text

CyclinD1

Novocastra

NCL-L-cyclin D1

1/400

EDTA pH 8

30%

Ki67

Dako

MIB-1

1/200

EDTA pH 8

mean

Patient details including age at diagnosis, grade, lymph node status, response to therapy, relapse, overall survival and disease-free survival were obtained from the clinical records. Histological subtypes were updated from analysis of full-face sections. TNBC status was defined as negativity of all three markers ER-alpha, PgR and HER2 using the standard diagnostic criteria by immunohistochemistry and in situ hybridisation. Testing for the presence of germline mutations of BRCA1 and BRCA2 was performed using genomic DNA isolated from peripheral blood. PCR amplification and analysis of coding regions and splice sites of the individual exons of BRCA1 and BRCA2 employed high melting curve analysis (HRM—high resolution melting, LightScanner, Idaho Technology) or denaturing high-performance liquid chromatography (dHPLC, Wave System 4500, Transgenomic). Fragments with an aberrant profile were sequenced (Genetic Analyser, Applied Biosystems). Examination of intragenous modifications was performed by the multiplex ligation-dependent probe amplification method (MLPA).

Scoring criteria

The percentages of positively stained cells were recorded, and tumours were considered positive for a given antigen according to the criteria in Table 1. For assessment of p53 status, we used the previously described system [18] where p53 immunostaining is combined with staining for MDM2 as a marker of wild-type p53 activity. Very low or absent p53 staining is indicative of non-sense mutations, high p53 staining in the absence of high MDM2 indicates missense p53 mutation and low/intermediate p53 staining with corresponding MDM2 indicates wild-type p53 [18].

Statistical evaluation

Chi-square tests (Pearson) were used to evaluate correlations of p63, TAp63 and ΔNp63 with tumour characteristics and clinicopathological data, except for comparisons with too few patients in any one group, where two-tailed Fisher exact tests were used (VassarStats website for statistical computation; http://vassarstats.net/). Kaplan-Meier and Mantel-Cox log-rank tests were used for survival data [19].

Results

TAp63 and ΔNp63 show distinct expression patterns in normal breast epithelium and stroma

We used previously characterised mouse monoclonal antibodies that recognise either TAp63 or ΔNp63 [13, 14, 20]. These reagents are specific for each isoform and do not cross-react with p53 or p73, unlike some p63 antibodies [20]. Histologically normal ducts adjacent to tumour tissues were present in eight samples and were initially examined for p63 isoform immunostaining. As previously reported, ΔNp63 was positive in myoepithelial cells lining the ducts and acini (Fig. 1), whereas luminal cells were not stained for ΔNp63. TAp63 was not seen in either myoepithelial or luminal breast epithelial cells. TAp63 was occasionally found in lymphocytes (15 samples; 6.4%) and in stromal cells with the morphological appearance of fibroblasts (5 samples; 2.1%). Lymphocytes and other stromal cells were not stained for ΔNp63 (Fig. 1).
Fig. 1

ΔNp63 and TAp63 staining in non-neoplastic breast epithelium and stroma. a, b Shows a low power view of the same area of tissue with a morphologically normal duct showing strong staining for ΔNp63 and absence of TAp63. c, d Shows higher magnification of non-malignant ducts showing localisation of ΔNp63 to basal cells and lack of TAp63 in the epithelium. A TAp63-positive cell is seen in the stroma (arrow). e, f Shows TAp63-positive cells (arrows) in the stroma surrounding two different TNBCs. The tumour margins are marked by dashed white lines

TAp63 is expressed in a minority of TNBCs

Immunostaining data were available for ΔNp63 in 235 and for TAp63 staining in 234 TNBCs. In all cases, p63 staining was seen in the nucleus, with characteristic nucleolar exclusion. Forty cancers (17%) contained at least one ΔNp63-positive tumour cell, 13 (5.5%) contained 5% or more ΔNp63+ tumour cells and 11 (4.7%) contained 10% or more ΔNp63+ tumour cells, with the highest frequency of 90%. These data are broadly similar to a recent study of ΔNp63 in unselected TNBCs, where 6% of tumours contained 5% or more ΔNp63+ cells [21]. For TAp63, 17 (7.3%) cancers showed at least one TAp63+ cell, six (2.6%) contained 5% or more TAp63+ cells and four (1.7%) contained 10% or more, with the highest frequency of 50% seen in three cancers.

Twenty-five cancers contained ΔNp63+ cells in the absence of TAp63 (from 39 ΔNp63+ cancers with data available for both isoforms; 64.1%), whereas only three of the 17 TAp63+ cancers (17.6%) did not contain ΔNp63. Two of the three tumours with 50% TAp63+ cells contained no ΔNp63+ cells and the other contained only 1%. Similarly, of the 13 TNBCs containing 5% or more ΔNp63+ cells, only two contained any TAp63+ cells (15.4%). Thus, low levels of the two isoforms are often seen in the same tumour, but high levels of either isoform associate with a lack of the other isoform (Wilcoxon signed-rank test p(two-tail) = 0.0074; using TNBCs containing TAp63+ and/or ΔNp63+ cells, n = 42).

P63 isoforms show specific clinicopathological associations

Examples of tumour cell immunostaining for ΔNp63 and TAp63 are shown in Fig. 2. The histopathological and clinical characteristics of TNBC in association with p63 isoforms are shown in Table 2. In addition, there was no association of either p63 isoform with cyclinD1 or with p27 staining. ΔNp63 showed a strong association with the basal phenotype markers CK5/6 and CK14 but did not associate with the mesenchymal markers vimentin or SMA. TAp63 positivity was associated with CK5/6 but not with CK14. TNBCs with TAp63 staining were more likely to show androgen receptor expression than TAp63 cancers (p = 0.038), showed a trend to be SMA+ (odds ratio 3.62; 95% CI 1.06–12.39; p = 0.054) and were less likely to be BRCA1/2 mutant (p = 0.027). TAp63+ TNBCs rarely showed loss of PTEN immunostaining (p = 0.014).
Fig. 2

ΔNp63 and TAp63 staining in triple-negative breast cancer cells. Examples of staining for ΔNp63 and TAp63 in four different TNBCs. a, b Shows high-level staining for ΔNp63 in the absence of TAp63. c, d Shows high-level TAp63 in the absence of ΔNp63. e, f Shows low-level staining for TAp63 in the absence of ΔNp63. g, h Shows a tumour with both p63 isoforms, with higher percentages of ΔNp63+ than TAp63+ cells

Table 2

Immunohistochemical staining of triple-negative breast cancers for ΔNp63 and TAp63 in relation to tumour phenotype and clinicopathological characteristics

 

ΔNp63-

ΔNp63+

p value

TAp63-

TAp63+

p value

CK5/6−

77

7

0.011

83

2

0.029

CK5/6+

110

30

125

14

CK14−

109

16

0.045

118

6

0.166

CK14+

81

24

95

10

CK8/18−

9

2

1

11

0

1a

CK8/18+

147

30

168

9

AR < 10

130

28

0.56

153

5

0.038a

AR ≥ 10

26

4

26

4

ERβ < 10

117

23

1

133

7

0.465a

ERβ ≥ 10

40

8

47

1

SMA 0–1

168

37

1a

188

13

0.054a

SMA 2–3

14

3

16

4

VIM−

64

12

0.671

72

4

0.718a

VIM+

90

20

106

4

EGFR 0–1

76

12

0.219

81

7

0.842

EGFR 2–3

112

28

129

10

BRCA1/2 wt

43

12

0.144

48

6

0.027a

BRCA1/2 mut

47

6

53

0

PTEN−

68

11

0.292

77

1

0.014

PTEN+

111

27

124

14

Cyclin D1 < 30

83

15

0.34

94

4

0.111

Cyclin D1 > 30

90

23

102

11

Ki67 (mean)

64.07

65.1

0.818b

63.43

72.94

0.146b

Ki67 (S.D.)

24.63

27.93

25.08

25.26

Stage I-II

148

34

0.351

167

14

0.743a

Stage III-IV

35

5

38

2

pN 0

99

19

0.823

112

5

1a

pN 1–3

62

13

71

4

G 1–2

13

3

1a

15

1

1a

G 3

170

36

190

15

Living

121

29

0.34

134

15

0.021

Dead

61

10

70

1

Relapse no

134

30

0.56

149

14

0.256a

Relapse yes

46

8

53

2

p53 wt

40

13

0.115

51

2

0.73a

p53 mut

110

19

122

7

P values are chi2

aPearson or Fisher exact two-tailed

bStudent’s t test is used to compare Ki67 values

Analysis of the impact of p63 on survival showed that TAp63 associated with a better overall survival, with only one of 16 patients with TAp63+ cancers having died at last follow-up (p = 0.021) and only two patients showing a relapse, although this was not significant with the small numbers involved (odds ratio for relapse 0.40; 95% C.I. 0.088–1.83; p = 0.256). Kaplan-Meier analysis showed a similar trend for improved overall survival in relation to TAp63+ cancers (p = 0.075; Cox-Mantel). By Kaplan-Meier estimation, whilst 92.86% TAp63+ patients showed disease-specific survival compared to 76.02% of TAp63 patients (10 year disease-specific data), this was not statistically significantly different (Cox-Mantel log-rank hazard = 0.33; p = 0.255; Fig. 3). No such associations were seen for overall survival or relapse in relation to ΔNp63 (Table 2).
Fig. 3

The impact of p63 isoform expression on survival. Kaplan-Meier plots for overall survival or disease-free survival in TNBCs according to expression of ΔNp63 or TAp63, as indicated. Mantel-Cox log-rank tests were used and p values are shown

Histological subtyping of the tumours showed that TAp63-positive cells were not present in any of the seven metaplastic cancers, contrasting with three (42.6%) of these tumours containing ΔNp63+ tumour cells, although this did not reach significance due to the small numbers involved. One metaplastic cancer showed squamous differentiation, and 80% of the cells in this tumour were ΔNp63+, in keeping with the known role of ΔNp63 in squamous differentiation [1, 2]. We also noted that medullary carcinomas and carcinomas with medullary features were more often positive for ΔNp63. In total, 13/49 (26.5%) of these tumours contained ΔNp63+ cells compared to 26/183 (14.2%) of cancers that did not show a medullary growth pattern (p = 0.04). No such associations were seen for TAp63 (see Table 3 for details).
Table 3

Staining for p63 isoforms in TNBCs of different histological subtypes

Histological type

TAp63+

ΔNp63+

NST (n = 165; 70.8%)

10/164 6.1%

21/164 12.8%

MEDULLARY (n = 29; 12.4%)

3/28 10.7%

9/29 31%

MED-FEAT (n = 20; 8.6%)

2/20 10%

4/20 20%

METAPLASTIC (n = 7; 3.0%)

0/7

0%

3/7 42.9%

LOBUL-PLEIO (n = 5; 2.1%)

0/5

0%

0/5

0%

PAPILLARY (n = 3; 1.3%)

0/3

0%

0/3

0%

MICROPAP (n = 3; 1.3%)

0/3

0%

1/3

33%

ACC (n = 1; 0.4%)

1/1

100%

1/1100%

NST no special type, MED-FEAT with medullary features (formerly termed “atypical medullary”), METAPLASTIC included 5 chondroid (matrix producing), 1 squamous and 1 spindle cell, LOBUL-PLEIO lobular-pleiomorphic, MICROPAP micropapillary, ACC adenoid cystic

Discussion

The human TP63 gene is aberrantly expressed in a variety of human cancers (reviewed in [22]). Despite giving rise to two main variants that act in either similar or opposite manners to p53, immunohistochemical studies of p63 generally employ antibodies that do not distinguish between the TAp63 and ΔNp63 isoforms. The use of antibodies to ΔNp63 (also known as p40) is increasing due to increased discriminatory power for differential diagnosis of various malignancies compared to pan-p63 antibodies [23, 24, 25, 26]. However, this approach does not allow the identification of tumours that express both ΔNp63 and TAp63. In addition, lack of ΔNp63 staining in the presence of p63 does not reliably identify TAp63, because gene fusions that lack the N-terminal sequences of both TAp63 and ΔNp63 have been reported [27, 28]. Therefore, investigations of the two major N-terminal p63 variants require methods that specifically identify each of these isoforms.

Isoform-specific RT-PCR has previously identified p63 isoform mRNAs in breast cancer, including TAp63 and ΔNp63 mRNAs in normal and benign breast lesions and in luminal-type breast cancers, plus additional isoforms that lack exon 4 of the TP63 mRNA in cancers only [29]. That study also used immunohistochemistry with the 4A4 monoclonal antibody which identifies all p63 isoforms [29]. Like all methods based on tissue lysates, the use of RT-PCR has the drawback that the specific cells which express the different mRNA isoforms is unknown and whilst pan-p63 antibodies are able to distinguish p63 in tumour versus stroma or normal mammary epithelium [29], they do not identify specific isoforms in individual tumour cells. This is particularly important when multiple isoforms are identified in the same sample, as seen here and previously, where all 20 tumour samples contained ΔNp63 mRNA, and 18 of these also contained TAp63 mRNA [29]. By immunohistochemistry with 4A4, only six cancers showed p63+ tumour cells [29], indicating that ΔNp63 and TAp63 are commonly expressed in non-malignant cells in human breast cancer tissue samples. Those data can be explained by our findings of ΔNp63 specifically in non-malignant basal cells and of TAp63 specifically in lymphocytes and stromal fibroblast-like cells, and with previous observations of ΔNp63 or TAp63 isoforms in these cell types [20, 30, 31]. Moreover, all six tumour samples in the previous study that were stained with 4A4 contained both ΔNp63 and TAp63 mRNAs [29], so that it remained unclear whether the tumour cells express both isoforms or whether one is present in tumour and the other only in non-neoplastic cells within the samples used for mRNA analysis.

Here, we used immunohistochemistry with isoform-specific TAp63 and ΔNp63 monoclonal antibodies, allowing us to localise each of these specific isoforms to tumour or stromal cells in a large series of TNBCs. In agreement with other studies, p63 is expressed by tumour cells in only a small proportion of TNBCs (varying from 6% using ΔNp63 antibody and a cut-off of 5%; 11% using pan-p63 and a 1% cut-off; or 6% using pan-p63 and a cut-off score of 2–3 versus 0–1 [21, 32, 33]). Using isoform-specific immunohistochemistry, we were also able to show that ΔNp63 is more commonly found in cancer cells in TNBCs than TAp63, but both may be present in an individual cancer. Importantly, the levels of these two variants are significantly different and occasional tumours show high TAp63 in the absence of ΔNp63 and vice versa. These data therefore indicate that the two variants are controlled by different regulatory factors, in keeping with the use of different promoters that direct expression of TAp63 and ΔNp63 isoforms and the findings that they show tissue- and cell type-specific distributions in normal adult tissues [1, 2, 3, 20, 34, 35].

Using genomic approaches, p63 has been associated with the BL2 subtype of TNBCs [6, 8]. Based on immunostaining for basal cytokeratins, this distinction is largely due to ΔNp63 rather than TAp63. In contrast, TAp63 but not ΔNp63 associated with AR, suggesting that TAp63 may be driving a “luminal” type differentiation pathway (the LAR subtype of TNBC [6, 36]), in keeping with the suggested roles of ΔNp63 and TAp63 in inhibiting and promoting luminal differentiation, respectively [14, 15, 37]. We also found associations between TAp63 and BRCA1/2 status, where BRCA mutation was associated with a lack of TAp63. Whilst our findings appear opposite to initial reports that p63 is more commonly expressed in BRCA-mutated cancers [7, 9], those data did not discriminate between p63 isoforms and were based on the assumption that basal-like cancers are more likely to be BRCA1 mutant and are more often p63+ than non-basal cancers [7], or used immunostaining to identify high/low BRCA1 without identifying mutation status specifically, nor did they identify TNBC status [9]. Indeed, our data support another study that specifically investigated p63 and BRCA1 mutation in human breast cancer [38]. Thus, TAp63 is associated with BRCA wild-type status in human TNBCs. The underlying biological reasons for this association will require further studies. We also investigated whether TAp63 or ΔNp63 associate with p53 mutation, based on evidence that these isoforms can bind to and promote mutant p53 pro-oncogenic effects [3, 39, 40, 41]. However, we did not find associations between either ΔNp63 or TAp63 with p53 mutation assessed by imunnohistochemical measurement of p53 functional activity [18], suggesting that the effects of mutant p53 are unrelated to the potential interactions with p63 isoforms.

In addition to identifying distinct phenotypic associations of TAp63 and ΔNp63, we also provide evidence for improved prognosis of patients with TAp63+ TNBC. This finding is compatible with TAp63 acting as a p53-like transcription factor [1, 2, 3, 22] and with the abilities of TAp63 to induce senescence [42] and suppress metastasis [43]. On the other hand, although TAp63 can transcriptionally activate growth arrest pathways in experimental situations [1, 2, 3], we did not find an association between TAp63 and lower proliferation, suggesting that neither inhibition of proliferation nor induction of senescence are provided by TAp63 in TNBC. This lack of ability to reduce proliferation of cancer cells in TNBC is similar to previous findings that TAp63+ cervical squamous carcinoma cells proliferate at an equal rate to TAp63 cells in the same tumour [20] and is supported by the findings that TAp63 transgene expression accelerates chemically induced skin cancer formation [44]. Furthermore, experimental evidence that TAp63 promotes adult stem cell maintenance [45] seems incompatible with improved patient prognosis. TAp63 has also been associated with regulating cell metabolism [46], and we identified an inverse relationship with PTEN loss and TAp63. Loss of PTEN in TNBCs correlates with PI3K pathway induction [47], and mutations in PIK3CA and PTEN are mutually exclusive in breast cancers [48]. That TAp63 was rarely seen in cancers with loss of PTEN suggests that TAp63 is either a regulator of this metabolic pathway or is itself negatively regulated by PI3K. In this regard, ΔNp63 is negatively regulated by PI3K in non-transformed basal mammary epithelial cells and luminal breast cancer cells [21, 49]. Further studies are warranted to identify the roles of cancer-associated metabolic pathways in regulating TAp63 and ΔNp63 or the role of p63 isoforms in regulating these pathways.

In conclusion, we have used isoform-specific antibodies to investigate the levels of TAp63 and ΔNp63 in human TNBCs. TAp63 was found in a smaller proportion of these tumours than ΔNp63 and may be detected independently or in combination with ΔNp63. Similar to other clinical and experimental studies, TAp63 is associated with improved prognostic indicators [20, 44, 50] but without influencing tumour cell proliferation. Whilst these data are derived from a small number of patients, our study raises many questions regarding the biological activity of TAp63 in human cancer and demonstrates the scientific and clinical value of analysing the two major p63 variants separately.

Notes

Funding

This work was funded by grants MEYS-NPSI-LO1413 and Award LM15089 from the Ministry of Education, Youth and Sports, Czech Republic; GACR P206/12/G151 from the Grant Agency of the Czech Republic; and MH CZ-DRO (MMCI 00209805) from the Ministry of Health, Czech Republic. The funders did not have a role in the planning, performing or analysing the data, or in manuscript preparation or submission.

Compliance with ethical standards

All patient data were anonymised, and the study was performed retrospectively on redundant excess tissues following ethical approval by the Biobanking and Biomolecular resources Research Infrastructure (BBMRI) at MMCI, in accordance with European Union regulations and the Declaration of Helsinki.

Conflict of interest

BV is a consultant for and RN is a co-owner of Moravian Biotechnology, who produces the p63 isoform-specific antibodies used in this study. The company did not provide financial support or have any influence over the design or execution of the studies. All other authors declare that they have no conflicts of interest.

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Philip J. Coates
    • 1
  • Rudolf Nenutil
    • 1
  • Jitka Holcakova
    • 1
  • Marta Nekulova
    • 1
  • Jan Podhorec
    • 2
  • Marek Svoboda
    • 2
  • Borivoj Vojtesek
    • 1
  1. 1.RECAMO, Masaryk Memorial Cancer CentreBrnoCzech Republic
  2. 2.Department of Comprehensive Cancer CareMasaryk Memorial Cancer InstituteBrnoCzech Republic

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