MYC amplification and TERT expression in breast tumor progression

Cancer Genetics and Cytogenetics 176 (2007) 93e99

MYC amplification and TERT expression in breast tumor progression Sigrı´ður K. Bodvarsdo´ttira,b, Margre´t Steinarsdo´ttirc, Ho´lmfrı´ður Hilmarsdo´ttira,b, Jo´n G. Jo´nassona,b,c, Jorunn E. Eyfjo¨rda,b,* a

Department of Medicine, University of Iceland, Vatnsmy´rarvegi 16, 101 Reykjavik, Iceland b Icelandic Cancer Society, Sko´garhlı´ð 8, 105 Reykjavik, Iceland c Landspitali University Hospital, Hringbraut, 101 Reykjavik, Iceland

Received 31 January 2007; received in revised form 30 March 2007; accepted 2 April 2007

Abstract

The complex roles of genomic instability, MYC oncogene amplification, activation of telomerase, and p53 function still remain to be fully described in breast tumors. MYC stimulates the telomerase catalytic subunit, TERT, which interacts with p53. Oncogene MYC amplification analysis was performed on 27 paraffin-embedded breast tumor samples by fluorescence in situ hybridization, selected on the basis of chromosomal instability. TERT immunostaining was performed on a larger group of breast tumor sections. All tumor samples were analyzed for TP53 mutation, genomic index, S-phase fraction, and pathological stages. Amplification of MYC was detected in 16 of 27 tumors (59%) and found to be associated with TNM stages I and II (P 5 0.018), genomic index O 1.5 (P 5 0.033), and S-phase fraction O 5% (P 5 0.020). No association was found between MYC amplification and TERT immunostaining or TP53 mutations. Analysis of TERT in 103 primary breast tumors showed O50% nuclei immunostaining in 58% of cases. High TERT immunostaining associated with genomic index O 1.5 (P 5 0.017), high S-phase fraction (P 5 0.056), and TP53 mutations (P 5 0.030). No association was found between TERT staining and TNM stages. This study supports early involvement of MYC amplification in breast tumor progression. Both MYC amplification and TERT expression appear to be associated with high genomic instability and proliferation. TERT association with TP53 mutations indicates that TERT activity is downregulated by functional p53 protein in breast tumors. Ó 2007 Elsevier Inc. All rights reserved.

1. Introduction Breast carcinoma arises as a result of multiple changes in the genome of normal mammary epithelial cells. These changes include deletions, insertions and translocations, and gains and losses of entire or parts of chromosomes and chromosome arms, and eventually even gross changes in chromosome number [1,2]. Chromosomal instability seems to be driven by chromosomal rearrangements and oncogene amplifications that take place before aneuploidization [3]. These observations are contradicted by observations of aneuploidy being commonly present in early breast lesions, such as in ductal carcinoma in situ [4e6]. A number of studies have shown that chromosomal instability in breast tumors is associated with TP53 mutations [7e9]. Amplification and overexpression of the MYC * Corresponding author. Tel.: þ354-540-1900; fax: þ354-540-1905. E-mail address: [email protected] (J.E. Eyfjo¨rd). 0165-4608/07/$ e see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2007.04.002

oncogene is common in breast cancer [10]. Amplification of MYC has been strongly linked to genomic instability and high histological grade in breast tumors [11e14]. MYC directly activate telomerase expression by binding to the human telomere reverse-transcriptase (TERT ) promoter [15]. Telomerase is activated in 75e90% of breast tumors. Progressive increase in the mean telomerase activity has been detected with increased severity of histopathological change from benign breast disease to ductal carcinoma in situ lesions and invasive ductal carcinoma [16]. Increased TERT expression levels in breast tumors have been associated with worse prognosis and lower overall survival [17]. The association between telomerase activity and genetic alterations reflects complex effects of telomerase activation upon tumor progression in breast carcinomas [18]. This has been supported by a relationship between telomerase activity levels and genetic changes in which low telomerase activity was detected in breast tumors with above-average genomic instability but high telomerase activity was

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detected in breast tumors with below-average genomic instability [19]. Telomerase activation in turn leads to enhanced genomic stability and DNA repair [20]. Recently, it has been shown that telomere dysfunction, in the absence of the cell-cycle control proteins p53 and p21 and of active telomerase, leads to alterations in chromosome numbers as are found in many solid tumors [21]. As such, p53 and TERT have been shown to be interactive proteins, p53 as a powerful inhibitor of the TERT promoter activity [22] and TERT in downregulation of p53-induced apoptosis [23]. Our objective was to examine the complex role of MYC oncogene amplification, genomic instability, and TERT activation in primary breast tumors. Breast tumor samples were selected based on complex karyotype and were analyzed for TNM pathological stage, S-phase fraction, and TP53 mutation. MYC amplification analysis and TERT immunostaining was performed on breast tumor sections. Statistical analysis was performed to establish any correlation between molecular and pathological factors.

2. Materials and methods 2.1. Breast tumor selection Sample selection of 27 primary breast tumors was based on chromosomal changes [1] and DNA index O 1 in flow cytometry analysis [24]. In our previous comparative genomic hybridization analysis (unpublished data), chromosome bands 8q24 and 20q13, where the oncogenes MYC and AURKA (alias Aurora-A) are located, were found to be amplified in 70% of cases. Fluorescence in situ hybridization (FISH) analysis was performed on these samples using localized probes for the candidate genes. These 27 samples and an additional 76 primary breast tumor samples were analyzed for TERT immunostaining. 2.2. Tumor tissue and histopathological examination Fresh and paraffin-embedded breast tumor tissue samples and information on histopathological parameters, DNA index, and S-phase fraction were obtained from the Department of Pathology, Landspitali University Hospital. The breast tumors were diagnosed from 1990 to 2001. Average age of patients at surgery was 60 years (range, 27e92 years). TNM staging was determined according to American Joint Commission on Cancer [25]. Permission from the National Bioethics Committee and the Data Protection Authority was obtained (99/041V2S1; 99/111V1S1), as well as informed consent. 2.3. Chromosome harvest and banding analysis Tumor tissue obtained directly from surgery was minced finely in a drop of culture medium and processed for chromosomal analysis. Chromosomes were harvested directly

from the minced tumor tissue or after 3e11 of days short-term culture, as previously described [1]. Mitotic cells were collected under standard colchicine treatment. Chromosomes were G-banded with Wright’s stain [26]. The number of cells analyzed depended on the material and ranged from 5 to 300 cells per sample. Karyotypes were described according to ISCN 1995 [27]. 2.4. Fluorescence in situ hybridization Site-specific FISH analysis was performed on 27 breast tumor sections selected based on complex karyotype [1] and DNA index O 1 in flow cytometry analysis [24]. Genomic index was based on flow cytometry DNA index if available; otherwise, on karyotype index [28]. Paraffinembedded breast cancer tissue was sliced in 4-mm sections for FISH analysis, which was performed using two different probes simultaneously. For the detection of MYC amplification the clone dJ944B18 was used, labeled with SpectrumOrange-dUTP (Abbott-Vysis, Des Plaines, IL), and clone pZ8.4 for the centromere 8 as ploidy control, labeled with fluorescein-12-dUTP (Enzo-Roche; Roche Molecular Diagnostics, Penzberg, Germany). Both clones were courtesy of Dr. Mariano Rocchi’s Web-based Resources for Molecular Cytogenetics (http://www.biologia.uniba.it/rmc/). FISH on MYC was performed mainly as reported for analysis of Aurora-A (AURKA) amplification [29]. Briefly, the slides were deparaffinized, boiled in microwave oven and incubated with pepsin. Probes were diluted in tDenHyb-2 hybridization buffer (InSitus Biotechnologies, Albuquerque, NM) as described by producer instruction. Sections and probes were simultaneously denatured followed by overnight hybridization at 37 C. After washes and counterstaining with 40 ,6-diamidino-2-phenylindole, fluorescence signals were scored in each sample by counting the number of single-copy gene and centromeric signals in an average of 120 well-defined nuclei (range, 66e154). The cutoff for amplification was a MYC/centromere 8 ratio of >1.5 or an average MYC copy number O 4. 2.5. TERT protein immunohistochemistry Immunohistochemical staining for the catalytic subunit of the TERT protein was performed as previously described [17] on 103 breast tumor paraffin-embedded sections, with slight modifications. Briefly, polyclonal rabbit anti-human antibody (anti-telomerase, human Ab-2, catalog no. 58005; Calbiochem, San Diego, CA) was used in dilution 1:400 and detected with EnVision System (catalog no. K 4010; Dako, Glostrup, Denmark), with peroxidase as the final enzymatic reaction. The TERT staining was scored as previously described [17]: grade 1, negative or cytoplasmic staining only, without any nuclear staining; grade 2, 1e10% positive nuclei with either homogeneous staining or a speckled/dotted pattern in the nucleus; grade 3, 10e50% positive nuclei with homogeneous staining in the

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95

nucleus; and grade 4, O50% positive nuclei with homogeneous staining in the nucleus. Samples with O50% positive nuclear immunostaining were analyzed as for those with high TERT staining.

majority of samples (74%) had S-phase fraction O 5%, 14 samples (52%) had high TNM stages of III-IV, 14 had high TERT immunostaining of grade 4, and 10 (37%) had a TP53 mutation (Table 1).

2.6. TP53 mutation analysis

3.2. TERT protein pathology

TP53 mutation analysis was performed with constant denaturant gel electrophoresis on exons 5e8. Mutations were confirmed by DNA sequencing. The constant denaturant gel electrophoresis and sequencing conditions were as previously described [30].

Samples from 103 breast tumors were immunostained for the telomerase active TERT subunit (Table 2). Almost 90% of the samples showed positive nuclear staining for the active unit of the telomerase. High TERT nuclear staining O 50% (grade 4) was detected in 60 tumor samples (58.2%) (Fig. 2). No nuclear staining was detected in 11 samples (10.7%). Among samples immunostained for TERT, 48 (46.6%) had genomic index O 1.5. S-phase fraction O 5% was found in 51 tumors (49.5%), and TP53 mutations were detected in 23 tumors (22.3%) (Table 2).

2.7. Statistics Tables of absolute and relative frequencies are presented in the Results section. Associations between categorical variables were examined using Fisher’s exact test. All P-values are two-sided. The statistical package GraphPad InStat version 3 (GraphPad Software, San Diego, CA) was used.

3. Results 3.1. MYC oncogene amplification Amplification of the MYC oncogene was detected by site-specific FISH analysis in 16 of 27 (59%) breast tumor samples with a MYC/centromere ratio O 1.5, MYC mean copy number O 4, or both (Fig. 1). Nineteen tumors (70%) had genomic index O 1.5. Genomic index ranged from 1.10 to 2.74, with a median value of 1.69 (average 1.72). Most of the samples had complex karyotypic changes and variable chromosome number (Table 1). A

3.3. Correlation with pathological factors Correlation between MYC amplification and TERT immunostaining and possible association with other molecular and pathological factors was tested using a two-sided Fisher’s exact test. Among the 27 breast tumors tested for MYC amplification, a significant association was found with low TNM pathological stages I and II (P 5 0.018; odds ratio OR 5 9.90; 95% confidence interval CI 5 1.54e63.72), genomic index O 1.5 (P 5 0.033; OR 5 8.40; 95% CI 5 1.26e56.09), and S-phase fraction O 5% (P 5 0.020; OR 5 20.08; 95% CI 5 0.94e430.54) (Table 3). No correlation was found between MYC amplification and TP53 mutations or high nuclear TERT staining. Among the 103 breast tumor samples tested for TERT immunostaining, high immunostaining with >50% nuclear staining (grade 4) was associated with high genomic instability (P 5 0.015; OR 5 3.03; 95% CI 5 1.25e7.35), S-phase fraction O 5% (P 5 0.056; OR 5 3.42; 95% CI 5 1.02e6.96), and TP53 mutations (P 5 0.030; OR 5 3.42; 95% CI 5 1.15e10.13). No association was found between TERT and TNM stages (Table 3).

4. Discussion

Fig. 1. MYC amplification detection in breast tumor section. MYC amplification detected by site-specific interphase FISH on nuclei from a paraffin-embedded breast tumor sample. MYC amplification is detected as red signal and compared to centromere 8 detected as green signal. This sample show clear MYC amplification.

Amplification of MYC, detected by site-specific FISH, was found to be significantly associated with low TNM stages, high genomic instability, and high S-phase fraction in a group of breast tumor samples selected for complex chromosomal changes (Table 3). These results implicate MYC amplification as an early event in carcinogenesis, in early tumors with high genomic instability and high proliferation. To our knowledge, the present study is the first to associate MYC amplification with low TNM stages in breast tumors. Other studies have shown association of MYC amplification with high genomic instability and proliferation [11,12,14,31]. Involvement of MYC in early genomic instability is supported by a study in which MYC amplification

96

Table 1 MYC analysis, with genetic and karyotypic changes in 27 breast tumor samples TP53 TERT mutation grade in exons 5e8

TNM stage

Genomic index

S-phase Karyotype fraction

1 2

4.39 3.42

5.22 3.94

no 1 exon 5 mut 4

I IIb

1.91 1.57

n.d. 11.5

3 4

2.65 2.36

5.78 6.79

no no

4 1

II IIIa

1.73 2.74

23.1 18.7

5 6 7 8 9 10

1.86 1.7 1.68 1.67 1.61 1.54

4.28 4.18 5.48 2.8 4.22 4.07

no exon 7 mut exon 8 mut exon 5 mut no no

2 4 4 4 4 4

I IIIa I IIIa I IIb

1.63 1.23 2.73 1.73 1.68 1.39

16.8 59.3 12.9 15.1 13.7 n.d.

11 12 13

1.5 1.5 1.39

4.52 4.52 2.24

exon 8 mut 4 exon 5 mut 3 no 3

IIIa I IIIa

1.75 1.77 1.12

10.5 11.1 1.4

14

1.32

2.6

no

IV

1.87

16.4

15

1.29

3.14

exon 8 mut 3

IIIa

2.56

27.9

16 17 18 19 20 21 22 23 24

1.28 1.27 1.25 1.25 1.19 1.18 1.17 1.11 1.06

2.75 2.17 6.21 2.25 4.32 2.12 2.89 1.69 5.26

no exon 5 mut no no no no no no no

4 4 4 4 4 3 2 4 4

IIb IIIa IIIa I I IV IIIa IIIa I

1.67 1.38 1.82 1.8 1.69 1.45 1.48 1.1 1.84

n.d. 10.5 11.5 5.1 9 10.3 3 2.3 35.4

25

1.06

2.31

exon 8 mut 4

IIIa

1.46

12.6

26 27

0.93 0.93

5.3 2.2

no 4 exon 6 mut 3

I IIIa

1.59 1.75

7.6 20

4

43~44,XX,8,8,add(21)(p12)[5]/88,idem2[1] 42,X,X,del(1)(p13),del(1)(p22),del(5)(p13),add(12)(q24),add(17)(p11),inc[cp2]/63~74,idem2,add(12)(p13), inc[cp27]/128~131,idem4,add(12)(p13)2,add(12)(q24),inc[cp4] 60~64,inc[4] 107~115,!5nO,XXXXX,del(1)(p22)2,þt(1;8)(p12;p11.21)2,þadd(1)(p11),2,del(2)(p14),add(2)(p11),þ3, add(3)(p11),del(3)(p12p24),4,þ5,6,add(6)(q26),(i6)(p11),8,del(9)(q21)2,10,add(11)(?p11),add(12)(p13)2,13, 13,2,add(13)(p11),iso(14)(q10),15,17,?17,18,20,21,hsr(21)(p11),22,add(22)(p11),þ15~26mar,inc[15] 67~88,cx,inc[cp3] 73~83,XX,inc 48~50,XX,þ12[cp2] 65~69,XXXX,þdel(1)(p22)2,add(6)(p25),add(11)(p14),add(15)(p11),cx,inc[cp3] 63~69,add(7)(q?),i(11)(q10),hsr(17)2,inc[cp5] 57~67,X,del(1)(p22),þdel(1)(q11),þt(1;?;14)(q10;?;q12),þt(1;8)(q?;q22~23),t(2;17)(q?33;q21),t(2;5)(q10;q10), add(3)(q11),der(3;19)(q10;q10),5,del(5)(q?),add(6)(p10),7,t(8;9)(p23;q11),9,10,t(11;13)(q12;q10),þadd(12)(p13), 14,i(15)(q10),der(13;15)(q10;q10),del(16)(p13),17,17,17,18,der(19)t(X;19)(q13;q13),þder(19)t(5;19)(q?;q13), 20,21,21,21,22,22,der(22)t(2;22)(q12;p11),þ1~3r,þ7~10mar,inc[cp12] 80,X?,?t(1;19)(q11;p13),hsr(5),der(15)t(15;17)(p11;12),inc[2]/47,XX,þ8[2] 75~82,XXX,þ1,þ6,i(6)(p10),þ7,i(17)(q10),þ18,þ20[12] 45~46,XX,del(1)(p34),add(2)(p23),der(1;16)(q10;p10),add(19)(q13)[4]/43~46,XX,inv(2)(p21q11),del(2)(p25)[3]/ 45~46,XX,add(19)(q13),del(20)(q11)[2]/49,XXþ10,der(1;16)(q10;p10),þ2xder(1;16)(q10;p10)[2]/46,XX[107] 75~84,!3nO,X,X,del(X)(q21),del(1)(p33),add(1)(q4?),add(1)(p23)del(q21q24),þadd(1)(p12)(q43), 2,del(3)(q12~13),þinv?(3)(p21q11),þadd(4)(p13),i(5)(q10),add(6)(q26),del(6)(q21),þdel(6)(q16), 7,del(7)(q11),add(7)(q32),8,add(9)(p22)2,10,del(11)(p11),þder(11)t(11;?15)(p11~13;q12~14),þadd(11)(p?),13, 14,15,þadd(16)(q?),17,18,þ19,þadd(19)(p13),þdel(22)(q12)2,der(?)t(?;3)(?;q11),þ13~20mar,inc[cp6] 106~112,XX,del(X)(q11),del(1)(p22),del(1)(q21),add(2)(p13),del(3)(p13),del(3)(q23),add(5)(q35),add(6)(q15), der(6)dup(6)(p21p25)add(6)(q27),dup(6)(p21p25),del(7)(q22),dic(10;20)(q26;p13),del(10)(q24),add(13)(p13), der(13)t(?2;13)(p11;p11),dic(13;?)(p11;?),add(15)(p11),add(17)(q11),add(18)(q23),add(19)(p13), dic(19;?)(p13;?),inc[cp33] 76~79,der(2;16),inc[cp2] 57~64,X,add(X)(p22),del(1)(p13),add(3)(p13),add(11)(p13),add(17)(p11),þr,inc[cp4] 64~71,?XXX,i(1)(q10),add(16)(p13),inc[cp5]/45,XX,19[3] 45~53,XX,þ2,þ11,þ20,inc[cp4] 73~76,inc[cp2] 50~63,XX,add(7)(p22),add(9)(p?),add(13)(q?),add(21)(p?),add(22)(q?),inc[12] 60,i(1)(q10),inc[4] 44~48,XX,1,i(1)(q10),add(3)(q29),þ5,þ7,add(11)(q23),der(16)t(16;17),17,þ20,þmar[42]/45~50,XX,þX,inc[3] 76~87,XXXX,del(1)(p22),þi(1)(q10),2,3,del(3)(q11),der(6)t(3;6;16)(p11;q11;q24),6,6,7,del(7)(q22),8,8,9, 10,add(11)(p12),14,15,15,del(16)(q21)2,der(16)t(3;6;16)(p11;q11;q24),add(17)(13)2,þ5~7mar[cp18] 58~62,!3nO,XX,X,del(1)(p22),2,3,5,6,i(7)(p10),8,9,add(11)(q21)2,13,13,13,add(14)(p11),15, 15,þ16,17,18,add(19)(q13),add(19)(?p/q?)2,21,21,22,þ4~7mar,1~3dmin[cp10] 59~65,XX,del(1)(q12),del(3)(p12),add(11)(q23),add(15)(p11),inc[cp14] 53~57,inc[1]

Abbreviations: c8, centromere 8; mut, mutated; n.d., not determined; TNM, tumorenodeemetastasis.

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Case MYC MYC mean /c8 ratio copy number

S.K. Bodvarsdo´ttir et al. / Cancer Genetics and Cytogenetics 176 (2007) 93e99 Table 2 Characteristics of 103 breast tumors included in TERT analysis Characteristic TERT immunostaining Grade 1: no nuclear staining Grade 2: 1e10% nuclear staining Grade 3: 10e50% nuclear staining Grade 4: O50% nuclear staining TNM stages I IIaeIIb IIIaeIIIb IV Genomic index O1.5 !1.5 Not analyzed S-phase fraction O5% !5% Not analyzed TP53 Mutated Wild type Not analyzed

no. (%) 11 10 22 60

(10.7) (9.7) (21.4) (58.2)

25 49 25 4

(24.3) (47.6) (24.3) (3.9)

48 (46.6) 38 (36.9) 17 (16.5) 51 (49.5) 27 (26.2) 25 (24.3) 23 (22.3) 78 (75.7) 2 (1.9)

seemed to be the first identifiable genetic alteration associated with progression from ductal carcinoma in situ to invasive ductal carcinoma [32]. MYC amplification has been known to play a role in development of a more aggressive phenotype of ductal carcinoma in situ through higher proliferative activity [31]. These studies, along with the present findings, support involvement of MYC in early stages of breast carcinogenesis. MYC oncogene activation can induce DNA damage by elevating reactive oxygen species and may also override damage control (e.g. by phosphorylation of p53), thereby accelerating tumor progression via genetic instability [33]. p53 has been shown to repress MYC transcription

Fig. 2. TERT immunohistochemistry on breast tumor section. High-grade TERT immunostaining on paraffin-embedded ductal invasive carcinoma showing definite staining of more than 50% of nuclei. Scale bar: 100 mm.

97

Table 3 Correlation of MYC amplification and TERT immunostaining with molecular and pathological factors MYC amplification, no. (%) Yes Sample size TNM pathological stages I and II III and IV Genomic index O1.5 !1.5 S-phase O5% !5% TP53 mutation TP53 mutation NoneTP53 mutation TERT immunostaining O50% cell nuclei !50% cell nuclei

No

n 5 16 n 5 11

TERT immunostaining, no. (%) P

O50%

!50%

P

n 5 60 n 5 43

11 (69) 2 (18) 0.018 45 (75) 29 (67) N.S. 5 (31) 9 (92) 15 (25) 14 (33) 14 (88) 5 (45) 0.033 33 (67) 15 (41) 0.017 2 (12) 6 (55) 16 (33) 22 (59) 14 (100) 6 (60) 0.020 33 (75) 18 (53) 0.056 0 4 (40) 11 (25) 16 (47) 6 (37) 4 (36) N.S. 18 (31) 5 (12) 0.030 10 (63) 7 (64) 40 (69) 38 (88)

12 (75) 6 (55) N.S. d 4 (25) 5 (35) d

d d

P-values according to Fisher’s exact test. Abbreviations: N.S., not significant at the 0.05 level.

through a mechanism that involves histone deacetylation at the MYC promoter [34]; however, we found no association between MYC amplification and TP53 mutations in tumors. In the present study, MYC amplification was detected in 59% of the breast tumors tested. This is high, compared with the average MYC amplification in sporadic breast cancer, which was found to be ~16% in a meta-analysis of 29 studies based on Southern blotting [35]. Studies using methods based on FISH in sporadic breast cancer have reported MYC amplification ranging from 5% to 45% [11e14,36e38]. MYC amplification was detected in 53% of breast tumors from BRCA1 mutation carriers [37] and in 70% of high-grade sporadic breast tumors [39]. Both of those groups are expected to have high genomic instability, as in the present study, and this may explain the high proportion of breast tumors with MYC amplification. Our data did not show correlation between TERT expression and MYC amplification, even though MYC is known to directly activate TERT [15]. Only one study has found association between TERT and MYC expression [40]; other studies, including ours, did not [41,42]. After TERT activation, MYC amplification seems to trail off, probably due to enhanced genomic stability and DNA repair [20]. TERT protein expression was detected in almost 90% of the tumors by immunostaining. High-grade protein expression pattern (grades 3 and 4) was seen in ~80% of the tumors, and 58% had O50% of nuclei stained (Table 2). This is in agreement with other studies that have found

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telomerase activity to be in 75e93% of breast tumors [19,40,43e46]. Two other studies, based like the present study on TERT immunohistochemistry, detected TERT expression in 59e71% of breast tumors [17,41]. Association of TERT nuclear staining O 50% (grade 4) was found with high genomic index and high S-phase fraction (Table 3). One other study has reported association between telomerase activity and DNA index and high S-phase fraction in breast tumors [46]. We also found association between grade 4 TERT nuclear staining and TP53 mutations (Table 3). One study found association between telomerase activity and p53 protein accumulation in breast tumors, but did not find correlation with TP53 mutations [43]. The present study found no correlation between high TERT expression and TNM stages, but other studies have shown association between increased telomerase activity and late stages [45,47e49]. Here we have reported MYC amplification and TERT expression analysis in a selection of breast tumors based on complex chromosomal changes. Amplification of MYC was shown to be associated with early tumorigenesis factors such as high genomic instability, high S-phase fraction, and low TNM stage. High TERT immunostaining was associated with TP53 mutations, which is in accord with p53triggered inhibition of TERT expression [22]. It is well known that increased telomerase expression is associated with worse disease prognosis and shorter overall survival of breast cancer [17,46]. Amplification of MYC appears to be an early event in breast tumorigenesis, one that diminishes after TERT activation. The correlation between high TERT immunostaining and TP53 mutations implies that functional p53 may be responsible for suppression of TERT transcription during tumor progression. These data indicate that breast tumor progression is dependent on interplay among MYC amplification, genomic instability, and TERT and p53 activity.

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Acknowledgments

[16]

The authors thank the Department of Pathology and the Department of Genetics and Molecular Medicine at Landspitali University Hospital for their collaboration and the Icelandic Cancer Society Biobank for supplying samples. This work was supported by the Icelandic Research Foundations (RANNIS) and the Icelandic Cancer Society.

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