Cell Oncol. (2014) 37:147–154 DOI 10.1007/s13402-014-0170-z
ORIGINAL PAPER
Analysis of gene copy number alterations by multiplex ligation-dependent probe amplification in columnar cell lesions of the breast Anoek H. J. Verschuur-Maes & Cathy B. Moelans & Peter C. de Bruin & Paul J. van Diest
Accepted: 12 March 2014 / Published online: 2 April 2014 # International Society for Cellular Oncology 2014
Abstract Background Columnar cell lesions (CCLs) are possible precursors of breast cancer, but little is known about the role of breast cancer-related genes in the progression of CCL to invasive breast cancer. Methods Gene copy numbers of 17 breast cancer-related genes were analyzed using Multiplex Ligation-dependent Probe Amplification (MLPA) in CCL (N=28), ductal carcinoma in situ (DCIS) grade I likely originating from CCL (N= 5), and paired CCL (N=14/28) with DCIS (N=7) and/or invasive carcinoma (N = 13). The genes included were BIRC5, C11orf30, CCND1, CCNE1, CDH1, CPD, EGFR, ERBB2, ESR1, FGFR1, IKBKB, MAPT, MED1, MTDH, MYC, TOP2A and TRAF4. Results No high level gene amplifications were observed in CCL, but copy number gains were encountered for the C11orf30 (3/28), MYC, CPD, MTDH (2/28), and CCND1, CCNE1, ESR1 and TOP2A genes (1/28). In addition, CDH1 showed loss in 2/28 and TOP2A in 1/28 cases. CCLs with or without atypia exhibited comparable numbers of copy number changes (p=0.312). Overall, the frequency of gene copy number changes increased from CCL towards DCIS and invasive carcinoma (p=0.004). Also in the cases with synchronous lesions, the CCLs exhibited fewer copy number changes than the DCIS/invasive carcinomas.
Electronic supplementary material The online version of this article (doi:10.1007/s13402-014-0170-z) contains supplementary material, which is available to authorized users. A. H. J. Verschuur-Maes : C. B. Moelans : P. J. van Diest (*) Department of Pathology, University Medical Center Utrecht Cancer Center, PO Box 85500, 3508 GA Utrecht, The Netherlands e-mail: [email protected] P. C. de Bruin Department of Pathology, St. Antonius Ziekenhuis, Nieuwegein, The Netherlands
Conclusions CCLs carry copy number changes of several known breast cancer-related genes, thereby substantiating their role in breast carcinogenesis. Among them, CCND1 and ESR1 copy number gains and CDH1 copy number losses are of particular interest. Since the copy number changes observed were more prevalent in DCIS and invasive carcinoma than in CCL, the corresponding gene alterations may represent rather late occurring events in low nuclear grade breast carcinogenesis. Keywords Columnar cell lesions . Flat epithelial atypia . MLPA . Oncogenes . Breast . Multiplex ligation-dependent probe amplification
1 Introduction It has widely been accepted that breast carcinogenesis is a complex multistep process with a sequence of stages in which invasive carcinoma arises from normal breast epithelium via precursor lesions like atypical ductal hyperplasia (ADH), lobular neoplasia and ductal carcinoma in situ (DCIS) [1, 2]. In recent years, it has been recognized that also columnar cell lesions (CCLs) may play a role as possible precursors of (particularly low nuclear grade) breast cancer [3–5]. These lesions are increasingly being diagnosed, since more biopsies are taken because of microcalcifications, which characterize these lesions at mammography [6]. CCLs are cystically dilated enlarged terminal duct lobular units, lined by columnar cell epithelium with either one to two cell layers (columnar cell change, CCC), or more cell layers (columnar cell hyperplasia, CCH). The cells show uniform, ovoid to elongated nuclei oriented along the basement membrane without inconspicuous nucleoli. At the intraluminal site, they often show cytoplasmic snouts and in the lumen secretions are regularly seen, as well as microcalcifications. According to the classification
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proposed by Schnitt et al. [7], the lesions are designated as columnar cell change with atypia or columnar cell hyperplasia with atypia when the cells show cytonuclear or architectural atypia. Together, the lesions with atypia are called CCLs with atypia (CCL-A), also known as flat epithelial atypia according to the World Health Organization, WHO [8]. CCL-A lack the complex architecture seen in ADH and low grade DCIS, although these lesions share many morphological and immunohistochemical features [4, 7, 9]. In the management of CCL a difference is made between CCLs with and without atypia, and many investigators recommend for CCL-A to excise the lesion or remove all microcalcifications by vacuum assisted biopsies or by the BLES procedure [10]. For CCLs without atypia, excision is so far not deemed necessary [11]. Different genetic alterations play a role in the development of breast cancer, including the inactivation of tumour suppressor genes and the activation of oncogenes. In previous studies a progression was found in methylation levels from normal breast epithelium, via CCL to DCIS and invasive carcinoma, indicating that CCLs may act as precursors of breast cancer [12, 13]. Evidence has also been provided, albeit sparse, that CCLs (with atypia) exhibit genetic abnormalities similar to those observed in the accompanying DCIS and invasive carcinoma, as illustrated by studies using comparative genomic hybridization and loss of heterozygosity assays [4, 14–16]. However, as yet little is known about genetic alterations in specific breast cancer-related genes in CCLs that occur during the development of breast cancer. Previously, a set of genes has been selected (and combined in a commercially available Multiplex Ligation-dependent Probe Amplification (MLPA) kit) showing frequent copy number changes in breast cancer and with functions related to carcinogenesis [17]. Our present study is, to our knowledge, the first to investigate copy number changes of a selected set of 17 of these breast cancerrelated genes in normal breast tissue, CCL and (largely synchronous) DCIS and invasive carcinoma by MLPA, to assess the involvement of these genes in the progression of CCL.
2 Methods 2.1 Patient material Tissue samples from cases with only CCL (with or without atypia), as well as from CCL cases with synchronous invasive carcinoma and/or DCIS, were retrieved from paraffin embedded breast resection specimens collected between 1996 and 2009 at the Departments of Pathology of the University Medical Center Utrecht and the St. Antonius Hospital Nieuwegein, The Netherlands, as previously described [13]. Normal breast tissues, also formalin fixed, from seven women undergoing mammoplasty were included as controls. Grading of the CCLs was performed according to the classification
A.H.J. Verschuur-Maes et al.
described by Schnitt and Vincent-Salomon [7] as either CCLs without atypia (including columnar cell change and columnar cell hyperplasia) or as CCLs with atypia (CCL-A, including columnar cell change with atypia and columnar cell hyperplasia with atypia). Classification and grading of the invasive carcinomas and in situ cancers were carried out by two experienced observers (AVM and PvD), according to the World Health Organization (WHO) classification system [8]. As a refinement, we included a subgroup of DCIS grade I lesions with a still recognizable columnar cell type with apical snouts (“DCIS likely originating from CCL”). Anonymous use of redundant tissue for research purposes is part of the standard treatment agreement with patients in both of our hospitals [18]. 2.2 DNA isolation Guided by marked H&E stained slides, abundant CCL, DCIS and invasive carcinoma lesions of at least 5 mm in size, and at least 5 mm apart from each other, were harvested from serial 8 to 10 μm thick paraffin sections (deparaffinized for 10 min) using a scalpel (mesodissection). In all other cases laser microdissection (PALM Microlaser Technologies AG, Bernried, Germany) was performed on 8 to 10 μm thick paraffin sections, from which the areas of interest were catapulted by laser pressure into a cap moistened with a drop of mineral oil. DNA was isolated by adding 50 or 100 μl direct lysis buffer (consisting of 10 mM Tris–HCl pH 8.3, 0.5 % Tween 0.20, 1 mM EDTA) and 10 or 20 μl proteinase K (10 mg/ml, Roche, Almere, The Netherlands), depending on the amount of material. After 1 h the same amount of proteinase K was added and heated in a 56 °C water bath for another 16 h. The samples were then heat inactivated by boiling for 10 min and centrifuged. The supernatant was stored by 4 °C until use. The DNA yield was measured using a Nanodrop Spectrophotometer (Wilmington, DE, USA). 2.3 Multiplex Ligation-dependent Probe Amplification (MLPA) The isolated DNA samples (100–240 ng) were evaluated by MLPA analysis using the P078-B1 breast kit according to the manufacturers’ instructions (MRC Holland, Amsterdam, The Netherlands), as described before [17]. Blood samples from four healthy volunteers without a history of malignancy were included as controls. The reaction products were separated by capillary electrophoresis using a 3130 DNA Analyzer (Applied Biosystems, Foster City, CA, USA) and gene copy numbers were analyzed using GeneScan Analysis (Applied Biosystems) and Coffalyser (version 9.4, MRC-Holland) software packages. Most samples were tested in duplicate. Normal breast tissue samples, which had undergone the same procedures as the test samples, were used as references in the Coffalyser analyses. In case a gene was covered by more than one probe in the kit, the means of the probe peaks was used in
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the analyses. In Table 1 the studied genes, including the chromosomal locations of all corresponding probes (GRCh36version18), are listed. The ADAM9, CDC6, PRDM14 and AURKA genes showed copy number changes in more than one of the normal breast tissue samples included and were, therefore, excluded from our analyses, resulting in 17 evaluable genes.
combined between the CCL, DCIS and invasive carcinoma cases by Chi-square test. P-values2.0 as high level amplifications, as reported before [17, 19–21]. After dichotomizing, we compared all copy number changes of each gene separately and all genes
In total, 63 lesions derived from 43 patients were studied. Table 2 shows the number of studied lesions per category. Fourteen of the patients had CCL with synchronous DCIS and/or invasive carcinoma (Fig. 1). The 13 invasive carcinomas were of the ductal type, except for one lobular cancer. The majority of DCIS (4× grade I, 1× grade II, 2× grade III) and invasive cancers (6× grade I, 4× grade II, 3× grade III) were of
Table 1 Genes included in the P078-B1 MLPA kit (MRC Holland, The Netherlands) used for studying copy number changes in low nuclear grade breast carcinogenesis. The chromosomal position, mapview position, number of probes per gene and transcript description is given for each gene Genes
Chrom.
Mapview position
No. of probes
Transcript description
BIRC5 (Survivin)
17q25
3
Adapter molecule involved in signal transduction, cell communication and cell survival
C11orf30
11q13
2
Transcription regulatory protein
CCND1
11q13
2
Cell cycle control protein involved in signal transduction
CCNE1
19q12
2
Cell cycle control protein involved in signal transduction
CDH1
16q22
2
Adhesion molecule associated with signal transduction
CPD EGFR
17q11 7p11
1 2
Carboxypeptidase involved in protein metabolism Receptor tyrosine kinase involved in signal transduction
ERBB2
17q12
4
Receptor tyrosine kinase associated with signal transduction
ESR1
6q25
2
Transcription factor
FGFR1
8p12
2
Receptor tyrosine kinase involved in signal transduction
IKBKB
8p11
2
Serine/threonine kinase associated with signal transduction
MAPT MED1 (PPARBP) MTDH
17q21 17q12 8q22
1 1 2
MYC
8q24
Structural protein involved in cell growth and/or maintenance Transcription regulatory protein involved in signal transduction Metastasis promoting gene involved in chemoresistence and angiogenesis. Transcription factor involved in apoptosis and cell proliferation
TOP2A
17q21
TRAF4
17q11
17-073.722036 17-073.722396 17-073.724340 11-075.902087 11-075.926543 11-069.167779 11-069.175089 19-035.000150 19-035.005214 16-067.328716 16-067.404826 17-025.795018 07-055.191055 07-055.233957 17-035-118101 17-035.127183 17-035.133169 17-035.136344 06-152.423838 06-152.457215 08-038.391533 08-038.434092 08-042.292902 08-042.302676 17-041.423085 17-034.840858 08-098.742504 08-098.788082 08-128.821796 08-128.822001 08-128.822151 17-035.812698 17-035.816651 17-035.818297 17-024.098403
3
3
DNA topoisomerase protein involved in regulation of the topological status of DNA
1
Adaptor molecule involved in signal transduction, cell proliferation and apoptosis
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Table 2 Overview of the breast lesions studied for the presence of copy number changes in established cancer-related genes Samples
Patients
Pure lesions - Normal breast tissue - CCL - CCL without atypia
10 14 4
- CCL-A - DCIS grade I originated in CCL-A Synchronous lesions - CCL+DCIS+ICa - CCL+DCISb - CCL+ICc a
10 5 6 1 7
Both DCIS and IC: 3× grade I, 1× grade II, 2× grade III
b
DCIS: grade I
c
IC: 2× grade I, 3× grade II, 1× grade III, 1× lobular
CCL columnar cell lesions; DCIS ductal carcinoma in situ; IC invasive carcinoma
low/intermediate grade. Also five DCIS grade I likely originating from a CCL were included. Most patients with CCL and synchronous DCIS and/or invasive carcinoma showed atypia (4× columnar cell change, CCC; 1× columnar cell hyperplasia, CCH; 9× columnar cell lesions with atypia, CCL-A). The mean age at the time of breast surgery of patients with CCL only was 53.0 years, with CCL and DCIS 55.4 years and with CCL and invasive carcinoma 50.6 years, with an overall mean age of 53.0 years (range 32–82 years). 3.2 Occurrence of gene copy number changes in CCL In Table 3, the copy number changes detected for all genes tested are shown, and in the Supplementary Table all results
can be viewed. In the CCLs no high level amplifications were observed for any of the genes tested. In 3/28 (10.7 %) CCL cases copy number gains were observed for the C11orf30 gene, in 2/28 (7.1 %) cases for the CPD, MTDH or MYC genes, and in 1/28 (3.6 %) cases for the CCND1, CCNE1, ESR1 or TOP2A genes. The CDH1 gene showed copy number loss in 2/28 (7.1 %) cases and the TOP2A gene in 1/28 (3.6 %) cases. 3.3 Comparison of gene copy number changes between CCL, DCIS and invasive carcinoma High level amplifications of the CPD, ERBB2, MED1, TOP2A and TRAF4 genes were found to be more prevalent in invasive carcinoma (2/13 cases) and DCIS (5/12 cases) than in CCL (0/28 cases) (p=0.004). Copy number gains of the C11orf30, MTDH, CCND1 and MYC genes showed similar frequencies over the three classes (CCL 13/28 cases, DCIS 6/12 cases, invasive carcinoma 10/13 cases; p=0.444). Also, when high level amplifications and copy number gains were taken together, no significant differences were observed between the three classes (p=0.122). Losses were most frequently observed for the CDH1 and FGFR1 genes, with in total more losses in invasive carcinoma (8/13 cases) and DCIS (5/12 cases) than in CCL (3/28 cases) (p=0.015). FGFR1 was the only gene that showed a significant difference, with more losses in invasive carcinoma than in CCL and DCIS (p= 0.010). In general, CCLs with or without atypia exhibited comparable numbers of gene copy number changes (9/19 CCL-A cases and 7/9 cases, respectively; p=0.312). Overall, the prevalence of gene copy number changes differed significantly between CCL, DCIS and invasive carcinoma, with a lower number of mean copy number changes in CCL (0.57) than in DCIS (1.33) or IC (1.54) (p=0.004). In paired analyses, also more gene copy number changes were present in invasive carcinoma than in CCL (p=0.002). CCL/ DCIS (p=0.11) and DCIS/invasive carcinoma (p=0.655) paired analyses also revealed differences in frequencies of gene copy number changes, but these differences were not significant. 3.4 Comparison of gene copy number changes in synchronous lesions
Fig. 1 Columnar cell change lesion without atypia (left) and invasive carcinoma lesion (right) studied by MLPA. The lesions were microdissected before DNA extraction
Six cases with synchronous CCL, DCIS and invasive carcinoma showed differences in gene copy number changes (N= 1, N=8 and N=9 changes, respectively; listed in Table 4). In seven cases with synchronous CCL/invasive carcinoma and one case with synchronous CCL/DCIS, CCL exhibited slightly fewer gene copy number changes (N=6) compared to the more advanced lesions (N=12; Table 5). In some patients, copy number changes of several genes were present simultaneously in the synchronous lesions. In one patient with CCL,
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Table 3 Frequencies of gains, high level amplifications and losses of 17 genes in columnar cell lesions (CCL), ductal carcinoma in situ (DCIS) and invasive carcinoma (IC), including P-values (Chisquare) CCL, N=28 Genes
DCIS, N=12
IC, N=13
Chromo-some Amplif. Gain
Loss
Amplif.
Gain
Loss
BIRC5
17q25
0
0
0
0
1 (8.3 %)
1 (8.3 %) 0
C11orf30 CCND1 CCNE1 CDH1 CPD EGFR ERBB2 ESR1 FGFR1 IKBKB MAPT MED1 MTDH MYC TOP2A TRAF4 Total
11q13 11q13 19q12 16q22 17q11 7p11 17q12 6q25 8p12 8p11 17q21 17q12 8q22 8q24 17q21 17q11
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
3 (10.7 %) 1 (3.6 %) 1 (3.6 %) 0 2 (7.1 %) 0 0 1 (3.6 %) 0 0 0 0 2 (7.1 %) 2 (7.1 %) 1 (3.6 %) 0 13
0 0 2 (16.7 %) 0 0 2 (16.7 %) 0 0 0 2 (7.1 %) 0 0 0 1 (8.3 %) 0 0 0 0 0 1 (8.3 %) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 (8.3 %) 0 0 0 1 (8.3 %) 0 0 0 1 (3.6 %) 1 (8.3 %) 0 0 1 (8.3 %) 0 3 5 6
a
Gain
Loss
0
1 (7.7 %)
0 1 (7.7 %) 0 0 1 (7.7 %) 0 0 0 0 0 1 (7.7 %) 2 (15.4 %) 1 (7.7 %) 0 0 0 0 0 0 0 0 0 0 0 0 0 3 (23.1 %) 0 0 1 (7.7 %) 0 0 1 (7.7 %) 0 0 0 0 3 (23.1 %) 0 0 2 (15.4 %) 0 0 1 (7.7 %) 0 1 (7.7 %) 1 (7.7 %) 0 2 10 8
P-valuea 0.119 0.792 0.383 0.640 0.284 0.992 – 0.181 0.640 0.010 0.214 0.324 0.181 0.345 0.372 0.992 0.141 0.004
P-value calculated by adding all copy number changes and comparing CCL, DCIS and invasive carcinoma
DCIS and invasive carcinoma, high level amplifications of the TRAF4 and CPD genes, copy number gain of the C11orf30 gene and loss of the MAPT gene in both DCIS and invasive carcinoma were observed (without changes in the CCL). In Table 4 Frequencies of copy number changes (including gains, high level amplifications, and losses) in six cases with synchronous CCL, DCIS and invasive carcinoma
a
0 0 0 3 (25 %) 0 0 0 0 0 0 1 (8.3 %) 0 0 0 0 0 5
Amplif.
Copy number changes all present in one patient
addition, a gain of the CCND1 gene was observed in the DCIS and a loss of the TOP2A gene in the CCL. The other five patients with CCL, DCIS and invasive carcinoma showed sporadically a gain or a loss in one of the three lesions (no
CCL N=6 (%)
DCIS N=6 (%)
Invasive carcinoma N=6 (%)
BIRC5 (Survivin) C11orf30 CCND1 CCNE1 CDH1 CPD EGFR ERBB2 ESR1 FGFR1 IKBKB MAPT
0 0 0 0 0 0 0 0 0 0 0 0
0 1 (16.7 1 (16.7 0 2 (33.3 1 (16.7 0 0 0 0 0 1 (16.7
%)a
0 1 (16.7 0 0 2 (33.3 1 (16.7 0 0 0 0 0 1 (16.7
MED1 (PPARBP) MTDH MYC TOP2A TRAF4 Total
0 0 0 1 (16.7 %)a 0 1
0 1 (16.7 %) 0 0 1 (16.7 %)a 8
0 1 (16.7 1 (16.7 1 (16.7 1 (16.7 9
%)a %)a %) %)a
%)a
%) %)a
%)a %) %) %) %)a
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A.H.J. Verschuur-Maes et al.
Table 5 Frequencies of copy number changes (including amplification, gain or loss) in seven cases with synchronous CCL/invasive carcinoma and one case with synchronous CCL/DCIS CCL N=8 (%)
Invasive carcinoma N=7 and DCIS N=1 (%)
BIRC5 (Survivin) C11orf30 CCND1 CCNE1 CDH1 CPD EGFR ERBB2 ESR1 FGFR1 IKBKB MAPT MED1 (PPARBP) MTDH
0 2 (25 %) 1 (12.5 %)a 1 (12.5 %) 2 (25 %)a 0 0 0 0 0 0 0 0 0
1 (12.5 %) 0 2 (25 %)a 0 1 (12.5 %) 0 0 0 0 3 (37.5 %) 1 (12.5 %) 0 0 2 (25 %)
MYC TOP2A TRAF4 Total
0 0 0 6
1 (12.5 %) 0 1 (12.5 %) 12
a
Copy number changes present in one patient with CCL and synchronous DCIS
high level amplifications), without synchronous aberrations. In the seven patients with synchronous CCL/invasive carcinoma and one patient with synchronous CCL/DCIS, one patient with CCL and DCIS (Table 5) showed in both lesions a gain of the CCND1 gene. In one patient, losses of the FGFR1 and IKBKB genes were observed in the invasive carcinoma, with a tendency to loss of the same genes in the synchronous CCL (values of 0.71 and 0.72, respectively). 3.5 Gene co-amplifications in different patient groups We also observed co-amplifications of various genes tested. High level co-amplification of the ERBB2, TOP2A and MED1 genes and copy number gain of the BIRC5 gene were e.g. seen in one patient with DCIS grade I originating from CCL. These alterations were all located on chromosome 17q. As mentioned before, one patient showed co-amplification of the TRAF4, CPD, C11orf30, CCND1 genes and losses of the MAPT and TOP2A genes, alterations located on chromosomes 11q and 17q. Also, a patient with CCH (without synchronous lesions) showed copy number gains of the C11orf30, CPD and TOP2A genes, whereas another CCH case showed copy number gains of the CPD and ESR1 genes. Copy number gains of both the MTDH and MYC genes (chromosome 8q) were seen in two CCL cases and two invasive carcinomas. A patient with
DCIS grade I originating from CCL showed losses of both the CDH1 and BIRC5 genes, and one case with invasive carcinoma showed a loss of the BIRC5 gene (chromosome 17q) and a gain of the CDH1 gene (chromosome 16q).
4 Discussion In this study we investigated and compared copy number changes of 17 breast cancer-related genes in CCL, DCIS and invasive breast carcinoma, including synchronous lesions in the same patients, by MLPA. In total, significantly more gene copy number changes were found to be present in DCIS and invasive carcinoma than in CCL (p=0.004), with significantly more high level amplifications and losses in DCIS/invasive carcinoma compared to CCL. Since more gene amplifications and gene losses were found to be present in the more advanced lesions, these alterations seemed to occur mostly at a later stage than CCL during the progression from normal breast tissue, CCL(-A), ADH to DCIS and invasive cancer, which is a well-known progression pathway for low nuclear grade carcinomas [22]. No high level gene amplifications were found in CCL. However, whereas the normal breast tissues tested exhibited no gains or losses for the analyzed genes, some CCLs showed copy number gains for some of the genes (C11orf30, MYC, CPD, MTDH, CCND1, CCNE1, ESR1, TOP2A) or copy number losses for some other genes (CDH1, TOP2A). This means that some early alterations in these breast cancer-related genes do occur in the precursor state CCL. It is not yet clear whether these gains play a role in breast carcinogenesis, whereas this has been shown for high level amplifications [23, 24]. Some CCL cases showed copy number gains of more than one gene on the same chromosome, implying that these co-occurring gains are not random. Some of the copy number changes are particularly interesting. Copy number gain of the CCND1 gene seems to fit with the fact that Cyclin D1 may be overexpressed in CCL, although this has thus far only been shown in one CCL case [22]. Copy number gain of the ESR1 gene has been reported to occur in varying frequencies in breast cancer, although the clinical significance of these observations remains to be established [25]. The observed loss of the CDH1 gene (located on 16q) is compatible with a proposed role of 16q loss in low grade breast carcinogenesis in general and in lobular carcinogenesis in particular, in which CCLs are thought to play a role [26–30]. The genes exhibiting the most frequent copy number changes in CCL were C11orf30 (10.7 %), MYC (7.1 %), CDH1 (7.1 %), MTDH (7.1 %) and CPD (7.1 %), in DCIS CDH1 (25 %), C11orf30 (16.7 %) and CCND1 (16.7 %) and in invasive carcinoma FGFR1 (23.1 %) and MTDH (23.1 %), located on chromosomes 8, 11, 16 and 17. The frequencies of the copy number changes observed are in line with, or slightly
Oncogenes in columnar cell lesions of the breast
lower than, those observed in previous studies using the same MLPA kit [17]. Losses were most prevalent in the CDH1 and FGFR1 genes, as reported before [17], with significantly more FGFR1 gene losses in invasive carcinoma compared to CCL and DCIS. Overlap of the same copy number changes with CCL and synchronous DCIS or invasive carcinoma occurred only in one patient with CCL and DCIS. Another patient with invasive carcinoma showed losses of the FGFR1 and IKBKB genes, with a tendency to loss of the same genes in the synchronous CCL. In this last patient, no DCIS could be studied. In one other case (mentioned above) synchronous DCIS and invasive carcinoma showed the same alterations (including high level amplifications), without changes of these genes in the accompanying CCL. The other five patients with synchronous CCL, DCIS and invasive carcinoma showed sporadically a copy number gain or loss in one of the three lesions (no high level amplifications), without synchronous changes. In our study, DCIS appeared at the genetic level about as advanced as invasive carcinoma, which is in conformity with previous reports [31, 32]. Since only a few overlapping copy number changes in CCL were present in the synchronous DCIS and invasive carcinoma lesions, copy number changes in the studied genes may rather be later events in low nuclear grade breast carcinogenesis. Although significantly more high-level amplifications were observed in DCIS and invasive cancer compared to CCL, this notion may be related to two patients: one patient with DCIS grade I originating from CCL with three high level gene amplifications (ERBB2, TOP2A, MED1) on chromosome 17, and a second patient with invasive carcinoma grade II and DCIS grade II with two gene amplifications (TRAF4 and CPD) also on chromosome 17. Co-amplifications were also seen on chromosome 8. The occurrence of several coamplifications has been reported before, including C11orf30 and CCND1 (50 to 70 % of cases with C11orf30 amplifications also showed CCND1 amplifications [17, 33] and ERBB2 and TOP2A (in approximately 40 % of HER2-amplified breast cancer patients) [24, 34, 35]. For chromosome 17, the occurrence of polysomy as the underlying mechanism has been excluded in breast cancer [32, 36]. The genes ADAM9, CDC6, PRDM14 and AURKA were found to be altered in normal breast tissue in more than one sample. Although we cannot exclude that these changes are true (as has been reported for EGFR copy number changes), we decided to exclude these genes from further analysis [37]. The MLPA technique has the advantage that only minute quantities of small DNA fragments are needed, which makes it suitable for studying paraffin embedded tissue with small lesions [17, 38]. The genes included in the MLPA kit used were selected on the basis of showing frequent copy number changes in breast cancer and with functions related to carcinogenesis [17]. In earlier studies using this same kit, more
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carcinomas with relatively more high-grade tumours were included, whereas we only included three high-grade tumours. This may be a reason for the more frequently observed amplifications in DCIS and invasive ductal carcinoma of the TRAF4, TOP2A, MED1, ERBB2, IKBKB and ESR1 genes in the study by Moelans et al. [17]. Although this kit has not been specifically tuned for low grade breast lesions, it does allow assessment of whether some of the genes that frequently play a role in high grade carcinogenesis are also altered in CCL. This could provide a clue to whether at least some CCLs may act as precursors of high grade cancers. Based on the observation that only few of these genes are altered in the (putative precursors of) low grade breast lesions, we suggest the existence of different parallel pathways leading to low and high grade breast cancers. New MLPA kits aimed at the analysis of genes located on chromosome 16q are, nevertheless, eagerly awaited. Other limitations of this study are the relatively small number of samples tested and the limited number of genes studied. Clearly, additional studies are needed to substantiate our results. In conclusion, we found that CCLs display copy number changes of several established breast cancer-related genes, thereby substantiating the putative role of CCL in breast carcinogenesis. Particularly, CCND1 and ESR1 copy number gains and CDH1 copy number losses are of interest. Since the observed copy number changes were more prevalent in DCIS and invasive carcinoma than in CCL, we propose these copy number changes may represent rather late events in low nuclear grade breast carcinogenesis. Acknowledgments This work was supported by The Oncology Centre of St. Antonius Hospital Nieuwegein. Conflict of interest The authors declare that they have no conflict of interest.
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ORIGINAL PAPER
Analysis of gene copy number alterations by multiplex ligation-dependent probe amplification in columnar cell lesions of the breast Anoek H. J. Verschuur-Maes & Cathy B. Moelans & Peter C. de Bruin & Paul J. van Diest
Accepted: 12 March 2014 / Published online: 2 April 2014 # International Society for Cellular Oncology 2014
Abstract Background Columnar cell lesions (CCLs) are possible precursors of breast cancer, but little is known about the role of breast cancer-related genes in the progression of CCL to invasive breast cancer. Methods Gene copy numbers of 17 breast cancer-related genes were analyzed using Multiplex Ligation-dependent Probe Amplification (MLPA) in CCL (N=28), ductal carcinoma in situ (DCIS) grade I likely originating from CCL (N= 5), and paired CCL (N=14/28) with DCIS (N=7) and/or invasive carcinoma (N = 13). The genes included were BIRC5, C11orf30, CCND1, CCNE1, CDH1, CPD, EGFR, ERBB2, ESR1, FGFR1, IKBKB, MAPT, MED1, MTDH, MYC, TOP2A and TRAF4. Results No high level gene amplifications were observed in CCL, but copy number gains were encountered for the C11orf30 (3/28), MYC, CPD, MTDH (2/28), and CCND1, CCNE1, ESR1 and TOP2A genes (1/28). In addition, CDH1 showed loss in 2/28 and TOP2A in 1/28 cases. CCLs with or without atypia exhibited comparable numbers of copy number changes (p=0.312). Overall, the frequency of gene copy number changes increased from CCL towards DCIS and invasive carcinoma (p=0.004). Also in the cases with synchronous lesions, the CCLs exhibited fewer copy number changes than the DCIS/invasive carcinomas.
Electronic supplementary material The online version of this article (doi:10.1007/s13402-014-0170-z) contains supplementary material, which is available to authorized users. A. H. J. Verschuur-Maes : C. B. Moelans : P. J. van Diest (*) Department of Pathology, University Medical Center Utrecht Cancer Center, PO Box 85500, 3508 GA Utrecht, The Netherlands e-mail: [email protected] P. C. de Bruin Department of Pathology, St. Antonius Ziekenhuis, Nieuwegein, The Netherlands
Conclusions CCLs carry copy number changes of several known breast cancer-related genes, thereby substantiating their role in breast carcinogenesis. Among them, CCND1 and ESR1 copy number gains and CDH1 copy number losses are of particular interest. Since the copy number changes observed were more prevalent in DCIS and invasive carcinoma than in CCL, the corresponding gene alterations may represent rather late occurring events in low nuclear grade breast carcinogenesis. Keywords Columnar cell lesions . Flat epithelial atypia . MLPA . Oncogenes . Breast . Multiplex ligation-dependent probe amplification
1 Introduction It has widely been accepted that breast carcinogenesis is a complex multistep process with a sequence of stages in which invasive carcinoma arises from normal breast epithelium via precursor lesions like atypical ductal hyperplasia (ADH), lobular neoplasia and ductal carcinoma in situ (DCIS) [1, 2]. In recent years, it has been recognized that also columnar cell lesions (CCLs) may play a role as possible precursors of (particularly low nuclear grade) breast cancer [3–5]. These lesions are increasingly being diagnosed, since more biopsies are taken because of microcalcifications, which characterize these lesions at mammography [6]. CCLs are cystically dilated enlarged terminal duct lobular units, lined by columnar cell epithelium with either one to two cell layers (columnar cell change, CCC), or more cell layers (columnar cell hyperplasia, CCH). The cells show uniform, ovoid to elongated nuclei oriented along the basement membrane without inconspicuous nucleoli. At the intraluminal site, they often show cytoplasmic snouts and in the lumen secretions are regularly seen, as well as microcalcifications. According to the classification
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proposed by Schnitt et al. [7], the lesions are designated as columnar cell change with atypia or columnar cell hyperplasia with atypia when the cells show cytonuclear or architectural atypia. Together, the lesions with atypia are called CCLs with atypia (CCL-A), also known as flat epithelial atypia according to the World Health Organization, WHO [8]. CCL-A lack the complex architecture seen in ADH and low grade DCIS, although these lesions share many morphological and immunohistochemical features [4, 7, 9]. In the management of CCL a difference is made between CCLs with and without atypia, and many investigators recommend for CCL-A to excise the lesion or remove all microcalcifications by vacuum assisted biopsies or by the BLES procedure [10]. For CCLs without atypia, excision is so far not deemed necessary [11]. Different genetic alterations play a role in the development of breast cancer, including the inactivation of tumour suppressor genes and the activation of oncogenes. In previous studies a progression was found in methylation levels from normal breast epithelium, via CCL to DCIS and invasive carcinoma, indicating that CCLs may act as precursors of breast cancer [12, 13]. Evidence has also been provided, albeit sparse, that CCLs (with atypia) exhibit genetic abnormalities similar to those observed in the accompanying DCIS and invasive carcinoma, as illustrated by studies using comparative genomic hybridization and loss of heterozygosity assays [4, 14–16]. However, as yet little is known about genetic alterations in specific breast cancer-related genes in CCLs that occur during the development of breast cancer. Previously, a set of genes has been selected (and combined in a commercially available Multiplex Ligation-dependent Probe Amplification (MLPA) kit) showing frequent copy number changes in breast cancer and with functions related to carcinogenesis [17]. Our present study is, to our knowledge, the first to investigate copy number changes of a selected set of 17 of these breast cancerrelated genes in normal breast tissue, CCL and (largely synchronous) DCIS and invasive carcinoma by MLPA, to assess the involvement of these genes in the progression of CCL.
2 Methods 2.1 Patient material Tissue samples from cases with only CCL (with or without atypia), as well as from CCL cases with synchronous invasive carcinoma and/or DCIS, were retrieved from paraffin embedded breast resection specimens collected between 1996 and 2009 at the Departments of Pathology of the University Medical Center Utrecht and the St. Antonius Hospital Nieuwegein, The Netherlands, as previously described [13]. Normal breast tissues, also formalin fixed, from seven women undergoing mammoplasty were included as controls. Grading of the CCLs was performed according to the classification
A.H.J. Verschuur-Maes et al.
described by Schnitt and Vincent-Salomon [7] as either CCLs without atypia (including columnar cell change and columnar cell hyperplasia) or as CCLs with atypia (CCL-A, including columnar cell change with atypia and columnar cell hyperplasia with atypia). Classification and grading of the invasive carcinomas and in situ cancers were carried out by two experienced observers (AVM and PvD), according to the World Health Organization (WHO) classification system [8]. As a refinement, we included a subgroup of DCIS grade I lesions with a still recognizable columnar cell type with apical snouts (“DCIS likely originating from CCL”). Anonymous use of redundant tissue for research purposes is part of the standard treatment agreement with patients in both of our hospitals [18]. 2.2 DNA isolation Guided by marked H&E stained slides, abundant CCL, DCIS and invasive carcinoma lesions of at least 5 mm in size, and at least 5 mm apart from each other, were harvested from serial 8 to 10 μm thick paraffin sections (deparaffinized for 10 min) using a scalpel (mesodissection). In all other cases laser microdissection (PALM Microlaser Technologies AG, Bernried, Germany) was performed on 8 to 10 μm thick paraffin sections, from which the areas of interest were catapulted by laser pressure into a cap moistened with a drop of mineral oil. DNA was isolated by adding 50 or 100 μl direct lysis buffer (consisting of 10 mM Tris–HCl pH 8.3, 0.5 % Tween 0.20, 1 mM EDTA) and 10 or 20 μl proteinase K (10 mg/ml, Roche, Almere, The Netherlands), depending on the amount of material. After 1 h the same amount of proteinase K was added and heated in a 56 °C water bath for another 16 h. The samples were then heat inactivated by boiling for 10 min and centrifuged. The supernatant was stored by 4 °C until use. The DNA yield was measured using a Nanodrop Spectrophotometer (Wilmington, DE, USA). 2.3 Multiplex Ligation-dependent Probe Amplification (MLPA) The isolated DNA samples (100–240 ng) were evaluated by MLPA analysis using the P078-B1 breast kit according to the manufacturers’ instructions (MRC Holland, Amsterdam, The Netherlands), as described before [17]. Blood samples from four healthy volunteers without a history of malignancy were included as controls. The reaction products were separated by capillary electrophoresis using a 3130 DNA Analyzer (Applied Biosystems, Foster City, CA, USA) and gene copy numbers were analyzed using GeneScan Analysis (Applied Biosystems) and Coffalyser (version 9.4, MRC-Holland) software packages. Most samples were tested in duplicate. Normal breast tissue samples, which had undergone the same procedures as the test samples, were used as references in the Coffalyser analyses. In case a gene was covered by more than one probe in the kit, the means of the probe peaks was used in
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the analyses. In Table 1 the studied genes, including the chromosomal locations of all corresponding probes (GRCh36version18), are listed. The ADAM9, CDC6, PRDM14 and AURKA genes showed copy number changes in more than one of the normal breast tissue samples included and were, therefore, excluded from our analyses, resulting in 17 evaluable genes.
combined between the CCL, DCIS and invasive carcinoma cases by Chi-square test. P-values2.0 as high level amplifications, as reported before [17, 19–21]. After dichotomizing, we compared all copy number changes of each gene separately and all genes
In total, 63 lesions derived from 43 patients were studied. Table 2 shows the number of studied lesions per category. Fourteen of the patients had CCL with synchronous DCIS and/or invasive carcinoma (Fig. 1). The 13 invasive carcinomas were of the ductal type, except for one lobular cancer. The majority of DCIS (4× grade I, 1× grade II, 2× grade III) and invasive cancers (6× grade I, 4× grade II, 3× grade III) were of
Table 1 Genes included in the P078-B1 MLPA kit (MRC Holland, The Netherlands) used for studying copy number changes in low nuclear grade breast carcinogenesis. The chromosomal position, mapview position, number of probes per gene and transcript description is given for each gene Genes
Chrom.
Mapview position
No. of probes
Transcript description
BIRC5 (Survivin)
17q25
3
Adapter molecule involved in signal transduction, cell communication and cell survival
C11orf30
11q13
2
Transcription regulatory protein
CCND1
11q13
2
Cell cycle control protein involved in signal transduction
CCNE1
19q12
2
Cell cycle control protein involved in signal transduction
CDH1
16q22
2
Adhesion molecule associated with signal transduction
CPD EGFR
17q11 7p11
1 2
Carboxypeptidase involved in protein metabolism Receptor tyrosine kinase involved in signal transduction
ERBB2
17q12
4
Receptor tyrosine kinase associated with signal transduction
ESR1
6q25
2
Transcription factor
FGFR1
8p12
2
Receptor tyrosine kinase involved in signal transduction
IKBKB
8p11
2
Serine/threonine kinase associated with signal transduction
MAPT MED1 (PPARBP) MTDH
17q21 17q12 8q22
1 1 2
MYC
8q24
Structural protein involved in cell growth and/or maintenance Transcription regulatory protein involved in signal transduction Metastasis promoting gene involved in chemoresistence and angiogenesis. Transcription factor involved in apoptosis and cell proliferation
TOP2A
17q21
TRAF4
17q11
17-073.722036 17-073.722396 17-073.724340 11-075.902087 11-075.926543 11-069.167779 11-069.175089 19-035.000150 19-035.005214 16-067.328716 16-067.404826 17-025.795018 07-055.191055 07-055.233957 17-035-118101 17-035.127183 17-035.133169 17-035.136344 06-152.423838 06-152.457215 08-038.391533 08-038.434092 08-042.292902 08-042.302676 17-041.423085 17-034.840858 08-098.742504 08-098.788082 08-128.821796 08-128.822001 08-128.822151 17-035.812698 17-035.816651 17-035.818297 17-024.098403
3
3
DNA topoisomerase protein involved in regulation of the topological status of DNA
1
Adaptor molecule involved in signal transduction, cell proliferation and apoptosis
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Table 2 Overview of the breast lesions studied for the presence of copy number changes in established cancer-related genes Samples
Patients
Pure lesions - Normal breast tissue - CCL - CCL without atypia
10 14 4
- CCL-A - DCIS grade I originated in CCL-A Synchronous lesions - CCL+DCIS+ICa - CCL+DCISb - CCL+ICc a
10 5 6 1 7
Both DCIS and IC: 3× grade I, 1× grade II, 2× grade III
b
DCIS: grade I
c
IC: 2× grade I, 3× grade II, 1× grade III, 1× lobular
CCL columnar cell lesions; DCIS ductal carcinoma in situ; IC invasive carcinoma
low/intermediate grade. Also five DCIS grade I likely originating from a CCL were included. Most patients with CCL and synchronous DCIS and/or invasive carcinoma showed atypia (4× columnar cell change, CCC; 1× columnar cell hyperplasia, CCH; 9× columnar cell lesions with atypia, CCL-A). The mean age at the time of breast surgery of patients with CCL only was 53.0 years, with CCL and DCIS 55.4 years and with CCL and invasive carcinoma 50.6 years, with an overall mean age of 53.0 years (range 32–82 years). 3.2 Occurrence of gene copy number changes in CCL In Table 3, the copy number changes detected for all genes tested are shown, and in the Supplementary Table all results
can be viewed. In the CCLs no high level amplifications were observed for any of the genes tested. In 3/28 (10.7 %) CCL cases copy number gains were observed for the C11orf30 gene, in 2/28 (7.1 %) cases for the CPD, MTDH or MYC genes, and in 1/28 (3.6 %) cases for the CCND1, CCNE1, ESR1 or TOP2A genes. The CDH1 gene showed copy number loss in 2/28 (7.1 %) cases and the TOP2A gene in 1/28 (3.6 %) cases. 3.3 Comparison of gene copy number changes between CCL, DCIS and invasive carcinoma High level amplifications of the CPD, ERBB2, MED1, TOP2A and TRAF4 genes were found to be more prevalent in invasive carcinoma (2/13 cases) and DCIS (5/12 cases) than in CCL (0/28 cases) (p=0.004). Copy number gains of the C11orf30, MTDH, CCND1 and MYC genes showed similar frequencies over the three classes (CCL 13/28 cases, DCIS 6/12 cases, invasive carcinoma 10/13 cases; p=0.444). Also, when high level amplifications and copy number gains were taken together, no significant differences were observed between the three classes (p=0.122). Losses were most frequently observed for the CDH1 and FGFR1 genes, with in total more losses in invasive carcinoma (8/13 cases) and DCIS (5/12 cases) than in CCL (3/28 cases) (p=0.015). FGFR1 was the only gene that showed a significant difference, with more losses in invasive carcinoma than in CCL and DCIS (p= 0.010). In general, CCLs with or without atypia exhibited comparable numbers of gene copy number changes (9/19 CCL-A cases and 7/9 cases, respectively; p=0.312). Overall, the prevalence of gene copy number changes differed significantly between CCL, DCIS and invasive carcinoma, with a lower number of mean copy number changes in CCL (0.57) than in DCIS (1.33) or IC (1.54) (p=0.004). In paired analyses, also more gene copy number changes were present in invasive carcinoma than in CCL (p=0.002). CCL/ DCIS (p=0.11) and DCIS/invasive carcinoma (p=0.655) paired analyses also revealed differences in frequencies of gene copy number changes, but these differences were not significant. 3.4 Comparison of gene copy number changes in synchronous lesions
Fig. 1 Columnar cell change lesion without atypia (left) and invasive carcinoma lesion (right) studied by MLPA. The lesions were microdissected before DNA extraction
Six cases with synchronous CCL, DCIS and invasive carcinoma showed differences in gene copy number changes (N= 1, N=8 and N=9 changes, respectively; listed in Table 4). In seven cases with synchronous CCL/invasive carcinoma and one case with synchronous CCL/DCIS, CCL exhibited slightly fewer gene copy number changes (N=6) compared to the more advanced lesions (N=12; Table 5). In some patients, copy number changes of several genes were present simultaneously in the synchronous lesions. In one patient with CCL,
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151
Table 3 Frequencies of gains, high level amplifications and losses of 17 genes in columnar cell lesions (CCL), ductal carcinoma in situ (DCIS) and invasive carcinoma (IC), including P-values (Chisquare) CCL, N=28 Genes
DCIS, N=12
IC, N=13
Chromo-some Amplif. Gain
Loss
Amplif.
Gain
Loss
BIRC5
17q25
0
0
0
0
1 (8.3 %)
1 (8.3 %) 0
C11orf30 CCND1 CCNE1 CDH1 CPD EGFR ERBB2 ESR1 FGFR1 IKBKB MAPT MED1 MTDH MYC TOP2A TRAF4 Total
11q13 11q13 19q12 16q22 17q11 7p11 17q12 6q25 8p12 8p11 17q21 17q12 8q22 8q24 17q21 17q11
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
3 (10.7 %) 1 (3.6 %) 1 (3.6 %) 0 2 (7.1 %) 0 0 1 (3.6 %) 0 0 0 0 2 (7.1 %) 2 (7.1 %) 1 (3.6 %) 0 13
0 0 2 (16.7 %) 0 0 2 (16.7 %) 0 0 0 2 (7.1 %) 0 0 0 1 (8.3 %) 0 0 0 0 0 1 (8.3 %) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 (8.3 %) 0 0 0 1 (8.3 %) 0 0 0 1 (3.6 %) 1 (8.3 %) 0 0 1 (8.3 %) 0 3 5 6
a
Gain
Loss
0
1 (7.7 %)
0 1 (7.7 %) 0 0 1 (7.7 %) 0 0 0 0 0 1 (7.7 %) 2 (15.4 %) 1 (7.7 %) 0 0 0 0 0 0 0 0 0 0 0 0 0 3 (23.1 %) 0 0 1 (7.7 %) 0 0 1 (7.7 %) 0 0 0 0 3 (23.1 %) 0 0 2 (15.4 %) 0 0 1 (7.7 %) 0 1 (7.7 %) 1 (7.7 %) 0 2 10 8
P-valuea 0.119 0.792 0.383 0.640 0.284 0.992 – 0.181 0.640 0.010 0.214 0.324 0.181 0.345 0.372 0.992 0.141 0.004
P-value calculated by adding all copy number changes and comparing CCL, DCIS and invasive carcinoma
DCIS and invasive carcinoma, high level amplifications of the TRAF4 and CPD genes, copy number gain of the C11orf30 gene and loss of the MAPT gene in both DCIS and invasive carcinoma were observed (without changes in the CCL). In Table 4 Frequencies of copy number changes (including gains, high level amplifications, and losses) in six cases with synchronous CCL, DCIS and invasive carcinoma
a
0 0 0 3 (25 %) 0 0 0 0 0 0 1 (8.3 %) 0 0 0 0 0 5
Amplif.
Copy number changes all present in one patient
addition, a gain of the CCND1 gene was observed in the DCIS and a loss of the TOP2A gene in the CCL. The other five patients with CCL, DCIS and invasive carcinoma showed sporadically a gain or a loss in one of the three lesions (no
CCL N=6 (%)
DCIS N=6 (%)
Invasive carcinoma N=6 (%)
BIRC5 (Survivin) C11orf30 CCND1 CCNE1 CDH1 CPD EGFR ERBB2 ESR1 FGFR1 IKBKB MAPT
0 0 0 0 0 0 0 0 0 0 0 0
0 1 (16.7 1 (16.7 0 2 (33.3 1 (16.7 0 0 0 0 0 1 (16.7
%)a
0 1 (16.7 0 0 2 (33.3 1 (16.7 0 0 0 0 0 1 (16.7
MED1 (PPARBP) MTDH MYC TOP2A TRAF4 Total
0 0 0 1 (16.7 %)a 0 1
0 1 (16.7 %) 0 0 1 (16.7 %)a 8
0 1 (16.7 1 (16.7 1 (16.7 1 (16.7 9
%)a %)a %) %)a
%)a
%) %)a
%)a %) %) %) %)a
152
A.H.J. Verschuur-Maes et al.
Table 5 Frequencies of copy number changes (including amplification, gain or loss) in seven cases with synchronous CCL/invasive carcinoma and one case with synchronous CCL/DCIS CCL N=8 (%)
Invasive carcinoma N=7 and DCIS N=1 (%)
BIRC5 (Survivin) C11orf30 CCND1 CCNE1 CDH1 CPD EGFR ERBB2 ESR1 FGFR1 IKBKB MAPT MED1 (PPARBP) MTDH
0 2 (25 %) 1 (12.5 %)a 1 (12.5 %) 2 (25 %)a 0 0 0 0 0 0 0 0 0
1 (12.5 %) 0 2 (25 %)a 0 1 (12.5 %) 0 0 0 0 3 (37.5 %) 1 (12.5 %) 0 0 2 (25 %)
MYC TOP2A TRAF4 Total
0 0 0 6
1 (12.5 %) 0 1 (12.5 %) 12
a
Copy number changes present in one patient with CCL and synchronous DCIS
high level amplifications), without synchronous aberrations. In the seven patients with synchronous CCL/invasive carcinoma and one patient with synchronous CCL/DCIS, one patient with CCL and DCIS (Table 5) showed in both lesions a gain of the CCND1 gene. In one patient, losses of the FGFR1 and IKBKB genes were observed in the invasive carcinoma, with a tendency to loss of the same genes in the synchronous CCL (values of 0.71 and 0.72, respectively). 3.5 Gene co-amplifications in different patient groups We also observed co-amplifications of various genes tested. High level co-amplification of the ERBB2, TOP2A and MED1 genes and copy number gain of the BIRC5 gene were e.g. seen in one patient with DCIS grade I originating from CCL. These alterations were all located on chromosome 17q. As mentioned before, one patient showed co-amplification of the TRAF4, CPD, C11orf30, CCND1 genes and losses of the MAPT and TOP2A genes, alterations located on chromosomes 11q and 17q. Also, a patient with CCH (without synchronous lesions) showed copy number gains of the C11orf30, CPD and TOP2A genes, whereas another CCH case showed copy number gains of the CPD and ESR1 genes. Copy number gains of both the MTDH and MYC genes (chromosome 8q) were seen in two CCL cases and two invasive carcinomas. A patient with
DCIS grade I originating from CCL showed losses of both the CDH1 and BIRC5 genes, and one case with invasive carcinoma showed a loss of the BIRC5 gene (chromosome 17q) and a gain of the CDH1 gene (chromosome 16q).
4 Discussion In this study we investigated and compared copy number changes of 17 breast cancer-related genes in CCL, DCIS and invasive breast carcinoma, including synchronous lesions in the same patients, by MLPA. In total, significantly more gene copy number changes were found to be present in DCIS and invasive carcinoma than in CCL (p=0.004), with significantly more high level amplifications and losses in DCIS/invasive carcinoma compared to CCL. Since more gene amplifications and gene losses were found to be present in the more advanced lesions, these alterations seemed to occur mostly at a later stage than CCL during the progression from normal breast tissue, CCL(-A), ADH to DCIS and invasive cancer, which is a well-known progression pathway for low nuclear grade carcinomas [22]. No high level gene amplifications were found in CCL. However, whereas the normal breast tissues tested exhibited no gains or losses for the analyzed genes, some CCLs showed copy number gains for some of the genes (C11orf30, MYC, CPD, MTDH, CCND1, CCNE1, ESR1, TOP2A) or copy number losses for some other genes (CDH1, TOP2A). This means that some early alterations in these breast cancer-related genes do occur in the precursor state CCL. It is not yet clear whether these gains play a role in breast carcinogenesis, whereas this has been shown for high level amplifications [23, 24]. Some CCL cases showed copy number gains of more than one gene on the same chromosome, implying that these co-occurring gains are not random. Some of the copy number changes are particularly interesting. Copy number gain of the CCND1 gene seems to fit with the fact that Cyclin D1 may be overexpressed in CCL, although this has thus far only been shown in one CCL case [22]. Copy number gain of the ESR1 gene has been reported to occur in varying frequencies in breast cancer, although the clinical significance of these observations remains to be established [25]. The observed loss of the CDH1 gene (located on 16q) is compatible with a proposed role of 16q loss in low grade breast carcinogenesis in general and in lobular carcinogenesis in particular, in which CCLs are thought to play a role [26–30]. The genes exhibiting the most frequent copy number changes in CCL were C11orf30 (10.7 %), MYC (7.1 %), CDH1 (7.1 %), MTDH (7.1 %) and CPD (7.1 %), in DCIS CDH1 (25 %), C11orf30 (16.7 %) and CCND1 (16.7 %) and in invasive carcinoma FGFR1 (23.1 %) and MTDH (23.1 %), located on chromosomes 8, 11, 16 and 17. The frequencies of the copy number changes observed are in line with, or slightly
Oncogenes in columnar cell lesions of the breast
lower than, those observed in previous studies using the same MLPA kit [17]. Losses were most prevalent in the CDH1 and FGFR1 genes, as reported before [17], with significantly more FGFR1 gene losses in invasive carcinoma compared to CCL and DCIS. Overlap of the same copy number changes with CCL and synchronous DCIS or invasive carcinoma occurred only in one patient with CCL and DCIS. Another patient with invasive carcinoma showed losses of the FGFR1 and IKBKB genes, with a tendency to loss of the same genes in the synchronous CCL. In this last patient, no DCIS could be studied. In one other case (mentioned above) synchronous DCIS and invasive carcinoma showed the same alterations (including high level amplifications), without changes of these genes in the accompanying CCL. The other five patients with synchronous CCL, DCIS and invasive carcinoma showed sporadically a copy number gain or loss in one of the three lesions (no high level amplifications), without synchronous changes. In our study, DCIS appeared at the genetic level about as advanced as invasive carcinoma, which is in conformity with previous reports [31, 32]. Since only a few overlapping copy number changes in CCL were present in the synchronous DCIS and invasive carcinoma lesions, copy number changes in the studied genes may rather be later events in low nuclear grade breast carcinogenesis. Although significantly more high-level amplifications were observed in DCIS and invasive cancer compared to CCL, this notion may be related to two patients: one patient with DCIS grade I originating from CCL with three high level gene amplifications (ERBB2, TOP2A, MED1) on chromosome 17, and a second patient with invasive carcinoma grade II and DCIS grade II with two gene amplifications (TRAF4 and CPD) also on chromosome 17. Co-amplifications were also seen on chromosome 8. The occurrence of several coamplifications has been reported before, including C11orf30 and CCND1 (50 to 70 % of cases with C11orf30 amplifications also showed CCND1 amplifications [17, 33] and ERBB2 and TOP2A (in approximately 40 % of HER2-amplified breast cancer patients) [24, 34, 35]. For chromosome 17, the occurrence of polysomy as the underlying mechanism has been excluded in breast cancer [32, 36]. The genes ADAM9, CDC6, PRDM14 and AURKA were found to be altered in normal breast tissue in more than one sample. Although we cannot exclude that these changes are true (as has been reported for EGFR copy number changes), we decided to exclude these genes from further analysis [37]. The MLPA technique has the advantage that only minute quantities of small DNA fragments are needed, which makes it suitable for studying paraffin embedded tissue with small lesions [17, 38]. The genes included in the MLPA kit used were selected on the basis of showing frequent copy number changes in breast cancer and with functions related to carcinogenesis [17]. In earlier studies using this same kit, more
153
carcinomas with relatively more high-grade tumours were included, whereas we only included three high-grade tumours. This may be a reason for the more frequently observed amplifications in DCIS and invasive ductal carcinoma of the TRAF4, TOP2A, MED1, ERBB2, IKBKB and ESR1 genes in the study by Moelans et al. [17]. Although this kit has not been specifically tuned for low grade breast lesions, it does allow assessment of whether some of the genes that frequently play a role in high grade carcinogenesis are also altered in CCL. This could provide a clue to whether at least some CCLs may act as precursors of high grade cancers. Based on the observation that only few of these genes are altered in the (putative precursors of) low grade breast lesions, we suggest the existence of different parallel pathways leading to low and high grade breast cancers. New MLPA kits aimed at the analysis of genes located on chromosome 16q are, nevertheless, eagerly awaited. Other limitations of this study are the relatively small number of samples tested and the limited number of genes studied. Clearly, additional studies are needed to substantiate our results. In conclusion, we found that CCLs display copy number changes of several established breast cancer-related genes, thereby substantiating the putative role of CCL in breast carcinogenesis. Particularly, CCND1 and ESR1 copy number gains and CDH1 copy number losses are of interest. Since the observed copy number changes were more prevalent in DCIS and invasive carcinoma than in CCL, we propose these copy number changes may represent rather late events in low nuclear grade breast carcinogenesis. Acknowledgments This work was supported by The Oncology Centre of St. Antonius Hospital Nieuwegein. Conflict of interest The authors declare that they have no conflict of interest.
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