- Email: [email protected]
Clonal alteration of breast cancer receptors between primary ductal carcinoma in situ (DCIS) and corresponding local events
European Journal of Cancer (2014) 50, 517– 524
Available at www.sciencedirect.com
ScienceDirect journal homepage: www.ejcancer.com
Clonal alteration of breast cancer receptors between primary ductal carcinoma in situ (DCIS) and corresponding local events E. Karlsson a,b,⇑, K. Sandelin c, J. Appelgren d, W. Zhou e, K. Jirstro¨m f, J. Bergh a, F. Wa¨rnberg g a
Department of Oncology–Pathology, Radiumhemmet, Cancer Center Karolinska, Karolinska Institutet and University Hospital, Stockholm, Sweden Department of Oncology, Central Hospital Karlstad, Karlstad, Sweden c Department of Surgery, Karolinska University Hospital, Stockholm, Sweden d Department of Economics and Statistics, Karlstad University, Karlstad, Sweden e Department of Surgery, Uppsala University, Uppsala, Sweden f Division of Pathology, Department of Clinical Sciences, Lund University, Lund, Sweden g Department of Surgery, Uppsala University Hospital, Uppsala, Sweden b
Available online 22 November 2013
KEYWORDS Ductal carcinoma in situ DCIS Breast cancer Hormonal receptor ER Oestrogen receptor PR Progesterone receptor Human epidermal growth factor receptor 2 HER2
Abstract Background: Emerging data propose biomarker alteration due to clonal selection between the primary invasive breast cancer and corresponding metastases. In addition, impact on survival has been demonstrated. The present study investigates the relationship between the oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) between primary ductal carcinoma in situ (DCIS) and intra-individually matched ipsilateral event. Materials and methods: The cohort includes 1504 patients, diagnosed with a primary DCIS between 1986 and 2004. Of the 274 patients who developed a local relapse, 135 developed a new in situ carcinoma and 139 an invasive cancer up to 31st December 2011. ER and PR were identified by immunohistochemistry (IHC) and HER2 by silver-enhanced in situ hybridisation (SISH) as well as IHC. Results: ER (n = 112), PR (n = 113) and HER2 (n = 114) status from both the primary DCIS and the corresponding relapse were assessed and were demonstrated to be discordant in 15.1%, 29.2% and 10.5% respectively. The receptor conversion was both from negative to positive and from positive to negative with no general pattern being seen in spite of sub-dividing into in situ relapse and invasive relapse. However, primary DCIS was HER2 positive in 40.3% whereas in situ and invasive relapses were HER2 positive in 42.9% and 34.5% respectively.
⇑ Corresponding author at: Department of Oncology, Central Hospital Karlstad, 652 30 Karlstad, Sweden.
E-mail address: [email protected] (E. Karlsson). 0959-8049/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejca.2013.10.020
518
E. Karlsson et al. / European Journal of Cancer 50 (2014) 517–524
Conclusions: Receptor conversion for ER, PR and HER2 status occurred between primary DCIS and corresponding local relapse in 10–30%. This study could not confirm that HER2 overexpression in primary DCIS had any impact on tumour progression to invasive cancer which has been proposed. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Primary ductal carcinoma in situ (DCIS) of the breast is a heterogeneous disease with different malignant potential. Altogether, DCIS has a good prognosis [1,2]. However, approximately half of the relapses developed after a primary DCIS will be invasive cancer [3,4]. The expression of oestrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) have been shown to be associated with local relapse [5,6]. However there are conflicting data. Nevertheless, the optimal management of DCIS is controversial due to poor understanding of the natural history of DCIS and the poor understanding of those factors that are involved in tumour progression. Biomarkers such as ER, PR and HER2 help clinicians to optimise and individualise management of patients with primary invasive breast cancer. ER and HER2 are of particular interest since they are both prognostic markers and predictors of treatment response. Moreover, emerging data propose biomarker alteration between the primary invasive breast cancer and the corresponding metastases [7–14] and indeed, some studies, including earlier data from our group have reported impact on survival due to such a change in receptor status [7–9]. In this study we wanted to perform a comparative analysis of ER, PR and HER2 status between primary DCIS and the corresponding local relapse (both in situ and invasive) and to investigate the role of adjuvant radiotherapy on any discordance. 2. Patients and methods 2.1. Study population The study was approved by the Ethical committee at Uppsala University Hospital (Dnr 99 442, Dnr 2005:118) and Umea˚ University (Dnr 05-065M, Dnr 2012-224-32M). The source population includes 1504 patients from two separate cohorts, diagnosed with a primary DCIS between 1986 and 2004. Of these 1504 patients, 458 were identified from a population based cohort diagnosed between 1986 and 2004 in Uppland and Va¨stmanland regions of Sweden. The remaining 1046 were identified from the randomised SweDCIS trial of patients diagnosed between 1987 and 1999 [15]. A total of 274 patients developed a relapse/new cancer in the period up to and including 31st December 2011. Of these 274 patients, 135 (49.3%) patients developed
an in situ relapse and 139 (50.7%) an invasive relapse. From these 274 patients, tissue samples from both the primary DCIS and the corresponding relapse were collected to perform analysis of ER, PR and HER2 status. Thirty patients (2%) out of the total cohort (1504 patients) died due to breast cancer up to the 31st December 2011. 28 of those patients had an invasive relapse as first event and two patients had an in situ relapse as first event (data not shown). 2.2. Tissue sample and biomarker measurement Tissue microarray (TMA)-blocks were constructed from both primary DCIS and relapses. The detail of this process is described in an earlier publication [16]. ER and PR status were assessed by immunohistochemical (IHC) methods (DakoAutostainer), whereby tumours with >10% positive cells were classified as receptor positive. The antibodies used were NCL-6F11 (Novocastra) for ER and PgR NCL-1A6 (Novocastra) for PR analysis, respectively. Fixation and staining procedures were performed according to manufacturer’s instructions. Positive and negative controls were included in all staining runs. HER2 status was assessed by using IHC analysis with antibody c-erb 2 poly rabbit, A0485 (DAKO) and Hercept-kit, which was confirmed by silverenhanced in situ hybridisation (SISH). Using the Hercept-kit, tumours were classified as positive at the 3+ protein level. For HER2 status priority was given for SISH and if not available IHC was used for evaluation of HER2 levels. HER2 SISH was carried out in an automated instrument (Ventana Benchmark, Ventana Medical system, Tucson, AZ). Evaluation of HER2 gene amplification was performed according to the American Society of Clinical Oncology/College of American Pathologists guideline and Australian HER2 Advisory Board criteria for single HER2 probe testing. Both IHC methods and SISH are described in detail in an earlier publication [16]. Nuclear grade (NG) was evaluated in the primary DCIS prior to the TMA construction by one pathologist (K.J.). Tumour markers were scored by one single observer (W.Z.) and thus, we did not have a problem with inter-laboratory differences or intraobserver variability. 2.3. Statistical methods Fisher’s test was applied for comparison of ER, PR and HER2 status with baseline primary tumour
E. Karlsson et al. / European Journal of Cancer 50 (2014) 517–524
characteristics and between in situ and invasive recurrence groups. For ER, PR and HER2 status in relapse situation, the earliest diagnosed relapse with assessed tumour marker was used. Data analyses were conducted using IBM SPSS Statistics (version 20). 3. Results 3.1. Patient population Fig. 1 presents a flow chart of the total cohort of patients. In summary, out of 1504 patients with a primary DCIS, 274 developed a local relapse. Out of those, 135 patients developed an in situ relapse and 139 patients developed an invasive relapse. 3.2. Primary tumour (DCIS) Baseline primary tumour (DCIS) characteristics for the 274 patients with a local relapse are presented in Table 1. No patient in the study received adjuvant chemotherapy or endocrine therapy. Seventy-eight of 264 (29.5%) patients treated with breast conserving surgery received postoperative radiotherapy, according to local guidelines. In Table 2, distribution of ER, PR and HER2 status in the patients’ primary tumour was stratified into two groups, i.e. those who later developed an in situ relapse and those who later developed an invasive relapse. As can be seen, in all patients with a primary DCIS ER was positive in 78.8% of cases. Whereas, when stratified into groups, ER positivity was seen in 71.9% of primary DCIS for the patients who later developed an in situ relapse. This compares to 85.6% (p = 0.023) of those who later developed an invasive relapse. Furthermore, 58.5% of all patients were PR positive in primary DCIS. In addition, HER2 overexpression was seen more frequently in the group later developing an in situ
519
Table 1 Baseline characteristics in 274 women with a primary ductal carcinoma in situ (DCIS) and a new ipsilateral event. Number
Percent
Age at diagnosis 645 46–60 >60
49 123 102
17.9 44.9 37.2
Mode of detection Screening Clinically Missing
200 73 1
73.3 26.7
Type of surgery Breast conserving surgery Mastectomy
264 10
96.4 3.6
Postoperative radiotherapy Yes No
78 196
28.5 71.5
Tumour size 615 mm >15 mm or multifocal Missing
131 99 44
57.0 43.0
Free margins Yes No or doubtful
215 59
78.5 21.5
Nuclear grade 1 2 3 Missing
21 90 145 18
8.2 35.2 56.6
relapse compared to those who developed an invasive relapse, 48.3% versus 29.8% (p = 0.014). 3.3. Biomarker discordances ER, PR and HER2 status in both the primary DCIS and the corresponding relapse had been assessed in 112, 113 and 114 patients respectively (Fig. 1). Discordance
Total cohort of patient 1504 patients diagnosed with DCIS between 1986-2004 * Patients with local relapse reported up to 2011 N=274 (100%)
DCIS relapse
Invasive breast cancer relapse
N=135 (49,3%)
N=139 (50,7%)
Assessment of ER status
Assessment of PR status
Assessment of HER2 status
Both primary tumor and relapse
Both primary tumor and relapse
Both primary tumor and relapse
N=112
N=113
N=114
* 458 women from a population based cohort diagnosed between 1986-2004 in Uppland and Västmanland regions of Sweden and 1046 women diagnosed with DCIS between 1987-1999 from the SweDICS trial
Fig. 1. Flow chart.
520
E. Karlsson et al. / European Journal of Cancer 50 (2014) 517–524
Table 2 Tumour marker characteristics in 274 primary ductal carcinoma in situ (DCIS) with a subsequent new ipsilateral event. Primary DCIS (n = 274)
Primary DCIS with a subsequent new in situ event (n = 135)
Primary DCIS with a subsequent invasive cancer (n = 139)
Number
Percent
Number
Percent
Number
Percent
Primary ER^ status Positive 152 Negative 41
78.8 21.2
69 27
71.9 28.1
83 14
85.6 14.4
Primary PR^ status Positive 113 Negative 80
58.5 41.5
53 41
56.4 43.6
60 39
60.6 39.4
Primary HER2h status Positive 70 Negative 111
38.7 61.3
42 45
48.3 51.7
28 66
29.8 70.2
p Value*
0.023
0.562
0.014
Abbreviations: ER = oestrogen receptor, PR = progesterone receptor, HER2 = human epidermal growth factor receptor 2. Comparison between in situ and invasive relapse groups using Fisher’s test. ^ Cut-off value >10% for immunohistochemical (IHC) methods. h Analysed using silver-enhanced in situ hybridisation (SISH) directly, if not available IHC 3+ was used as positive. *
Table 3 Intrapatient discordances in ER, PR and HER2 status in primary ductal carcinoma in situ (DCIS) and corresponding new event, presented for in situ- and invasive relapses separately. Primary DCIS/all relapses
Primary DCIS/in situ relapse
Primary DCIS/invasive relapse
Number
Percent
Number
Percent
Number
Percent
68.8 8 7.1 16.1
34 4 4 12
63 7.4 7.4 22.2
43 5 4 6
74.1 8.6 6.9 10.3
100
54
100
58
100
43.4 16.8 12.4 27.4
20 13 8 14
36.4 23.6 14.5 25.5
29 6 6 17
50 10.3 10.3 29.3
100
55
100
58
100
34.2 6.1 4.4 55.3
22 5 2 27
39.3 8.9 3.6 48.2
17 2 3 36
29.3 3.4 5.2 62.1
100
56
100
58
100
ER^ status primary tumour and relapse Primary pos/relapse pos 77 Primary pos/relapse neg 9 Primary neg/relapse pos 8 Primary neg/relapse neg 18 112 ^
PR status primary tumour and relapse Primary pos/relapse pos 49 Primary pos/relapse neg 19 Primary neg/relapse pos 14 Primary neg/relapse neg 31 113 HER2h status primary tumour Primary pos/relapse pos Primary pos/relapse neg Primary neg/relapse pos Primary neg/relapse neg
and relapse 39 7 5 63 114
Abbreviations: ER = oestrogen receptor, PR = progesterone receptor, HER2 = human epidermal growth factor receptor 2. Cut-off value >10% for immunohistochemical (IHC) methods. h Analysed using silver-enhanced in situ hybridisation (SISH) directly, if not available IHC 3+ was used as positive.
^
in ER, PR or HER2 status from primary DCIS to relapse was 15.1%, 29.2% and 10.5%, respectively (both in situ and invasive relapses included). Loss of PR or HER2 between primary DCIS and relapse was greater in the in situ relapse site compared to the invasive relapse, 23.6% versus 10.3% and 8.9% versus 3.4%, respectively. Gain of ER, PR or HER2 between primary DCIS to relapse were 7.1%, 12.4% and 4.4%, respectively (both in situ and invasive relapse included). In total, in situ and invasive relapses were ER positive in 70.4% and 81% of cases respectively. For PR, in situ
and invasive relapses were PR positive in 50.9% and 60.3% of cases respectively. Finally, primary DCIS was HER2 positive in 40.3% of instances whereas in situ and invasive relapses were HER2 positive in 42.9% and 34.5% of instances, respectively (Table 3). 3.4. Nuclear grade and changes in biomarker status The proportion of patients with discordant biomarkers was higher amongst the primary DCIS with nuclear grade 3 compared to those with nuclear grade 1 and 2.
E. Karlsson et al. / European Journal of Cancer 50 (2014) 517–524
521
Table 4 Intrapatient discordances in ER, PR and HER2 status in primary ductal carcinoma in situ (DCIS) and corresponding relapse stratified for adjuvant radiotherapy or no treatment. Tumour marker
Radiotherapy Number
ER^ status primary tumour and relapse Primary pos/relapse pos Primary neg/relapse neg Discordant ER primary/relapse Total PR^ status primary tumour and relapse Primary pos/relapse pos Primary neg/relapse neg Discordant PR primary/relapse Total HER2h status primary tumour and relapse Primary pos/relapse pos Primary neg/relapse neg Discordant HER2 primary/relapse Total
p Value*
No radiotherapy Percent
Number
Percent 0.8
19 5 3
70.4 18.5 11.1
58 13 14
68.2 15.3 16.5
27
100.0
85
100.0
12 9 10
38.7 29.0 32.3
37 22 23
45.1 26.8 28.0
82
100.0
0.8
31
100
0.3 12 16 1
41.4 55.2 3.4
27 47 11
31.8 55.3 13.0
29
100.0
85
100.0
Abbreviations: ER = oestrogen receptor, PR = progesterone receptor, HER2 = human epidermal growth factor receptor 2. * Comparison between radiotherapy treatment group and no radiotherapy treatment group using Fisher’s test. ^ Cut-off value >10% for immunohistochemical (IHC) methods. h Analysed using silver-enhanced in situ hybridisation (SISH) directly, if not available IHC 3+ was used as positive.
ER, PR and HER2 status were discordant in 11/55 (20.0%), 20/56 (35.7%) and 8/60 (13.3%) in DCIS nuclear grade 3 and 4/42 (9.6%), 10/41 (24.4%) and 2/42 (4.8%) in DCIS nuclear grade 2 and finally, 0/7, 1/7 and 0/6 in DCIS nuclear grade 1. (p 6 0.001 for Fisher’s test, comparison between DCIS nuclear grade 1–3). 3.5. The effect of adjuvant therapy on change in biomarker status ER, PR and HER2 discordances were analysed for two groups separately, i.e. postoperative radiotherapy or no therapy (Table 4). The proportion of patients with changed ER and HER2 status was larger in the group with no therapy compared to the group which received postoperative radiotherapy; 16.5% (n = 14) versus 11.1% (n = 3) (p = 0.8) and 13% (n = 11) versus 3.4% (n = 1) (p = 0.3), respectively. The proportion of patients with changed PR status in the group with no therapy compared to the group which received radiotherapy was 28.0% (n = 23) versus 32.3% (n = 10) (p = 0.8). 4. Discussion In the present study we investigated the relationship between ER, PR and HER2 status in the primary lesion and the corresponding ipsilateral event in a cohort with primary DCIS and a known relapse.
Our study demonstrates receptor conversion for ER, PR and HER2 being 15.1%, 29.2% and 10.5%, respectively. The receptor conversion was both from negative to positive and from positive to negative and we could not find any general tendency for either loss or gain of receptors for the different biomarkers, not even when stratified for either in situ relapse or invasive relapse. Previous studies have demonstrated discordances of receptor status between the primary invasive breast cancer and the corresponding relapse for ER, PR and HER2, in the range of 10–37%, 24–48% and 3–30%, respectively [7–14]. In addition, two prospective studies have indicated that the treating clinician changed the patient management in approximately 15% of cases as a result of biomarker change [12,13]. Moreover, impact on survival due to biomarker change between the primary invasive breast cancer and the corresponding relapse has been shown in a few studies including earlier data from our group [7–9]. In this study we did not look at survival as the prognosis for women with a DCIS was so good with a low number of breast cancer deaths. The majority of biomarker change were due to the loss of receptors between the primary invasive breast cancer to relapse, although a small proportion of those patients also gained receptors [8]. As can be seen, the primary DCIS both gains and loses a large amount of receptors (Table 3). There are several possible explanations for our findings of tumour markers changing during tumour progression. Both methodological issues and pure
522
E. Karlsson et al. / European Journal of Cancer 50 (2014) 517–524
biological explanations have been discussed [17,18]. The use of various tissues processing in laboratories and sampling techniques (fine needle aspirates, core biopsies or surgical excisions) can potentially lead to false discordance in tumour marker [17]. In the present study, we used TMA, and it is worth noting that the documentation of reliability for the evaluating of TMA material in the assessment of ER, PR and HER2 in tumour samples has been documented [19–21]. The described concordance, between TMA and corresponding whole sections slides for ER, PR and HER2 was approximately 85–95%, 81–88% and 90–100%, respectively. As can be seen, a lower correlation for PR was seen and a possible explanation for this might be a tendency towards a more heterogeneous expression of PR within the tumour compared to ER [20,22]. Also, we cannot exclude the possibility that an assumed local relapse of breast cancer actually represents a new primary breast cancer, thus influencing our results. Biological explanations that have been discussed concerning receptor change are tumour heterogeneity and clonal selection [23,24]. Moreover, several reports have shown that contributors of the stroma and the white adipose tissue can harbour important breast cancer functions. Bone marrow-derived mesenchymal stem cells and endothelial progenitor cells may have the capacity to influence tumour progression [25–28]. In addition, gene expression profiles have shown different quantity of certain genes, in particular of those genes relating to the extracellular matrix which were seen overexpressed in the invasive breast cancer compared to DCIS as well as in their adjacent stroma. These findings possibly indicate a step by step movement from in situ cancer to invasive cancer but also instability of tumour biology throughout tumour progression [29]. We used the 10% cut off for ER and PR positivity as this is the clinically used cut off in Sweden. Recently, the 1% cut off has been proposed for invasive breast cancer at the St Gallen International Expert Consensus [30,31]. We used the 1% cut off in a separate analysis and of course, some lesions that were classified as negative with the 10% cut off were reclassified as positive with the 1% cut off (six primaries and five recurrences for ER and eight and five for PR, respectively). But also, we found it very difficult to use this 1% cut off in DCIS biopsies in the TMAs. Actually, we do not know what true ER or PR positivity is. Today, we use a cut off based on IHC and a clinical assumption that this cut off is related to response to endocrine therapy. Instead, maybe in the future we should use the gene expression based molecular subtypes when looking for conversion between the primary tumour and the corresponding relapse. DCIS can be classified into the same molecular subtypes as invasive breast cancer [32] but still, we have little knowledge of its importance in DCIS.
HER2 is of particular interest since it is both an important negative prognostic marker and a predictive factor regarding the efficacy of trastuzumab in the management of primary invasive breast cancer patients. Nevertheless, the role of HER2 in DCIS is unclear. For instance, it has been proposed that HER2 overexpression in DCIS is of major importance for tumour progression towards invasive cancer [33–35]. Some studies have demonstrated a more frequent overexpression of HER2 in DCIS compared to invasive breast cancer and particularly in high grade DCIS [36,37]. The findings from our present study cannot confirm such an influence regarding progression. HER2 overexpression was more frequent in the group of women with a primary DCIS who later developed an in situ relapse compared to those who developed an invasive relapse, 48.3% versus 29.8% (p = 0.014). Also, in our cohort of DCIS with a known recurrence HER2 status in the primary was positive in 40.3% of the cases, compared to 42.9% and 34.5% amongst the in situ relapses and invasive relapses, respectively. In line with this, other studies have shown a higher risk of developing a new in situ relapse amongst those women with a HER2 positive and Ki67 positive primary DCIS, while these women did not show a higher risk of developing an invasive relapse [38,39]. In a previous study (Lindstro¨m/Karlsson et al. JCO 2012), we have shown that usage of adjuvant therapy seemed to be driving clonal selection regarding hormonal receptor status between the primary invasive breast cancers and corresponding recurrences (e.g. the proportion of patients losing ER positivity was biggest in the groups treated with endocrine therapy). Moreover, a study from Niikura et al. demonstrate that loss of HER2 overexpression between primary invasive breast cancer and corresponding relapses was associated with chemotherapy but not with trastuzumab therapy [9]. In the present study, no patient with a primary DCIS received adjuvant endocrine therapy or chemotherapy. However, about a third of all patients received postoperative radiotherapy to the remaining breast. A lower proportion of patients who did receive radiotherapy demonstrated a change in ER and HER2 status from primary DCIS to relapse compared with the patients who received no treatment, although no statistically significant difference was seen between the groups. The decision to give radiotherapy was based on clinical data including NG and this can influence our results to some extent. Interestingly, in this study the proportion of patients with discordant biomarkers was higher in the group with high NG compared with the groups with lower NG. NG has been shown to be a risk factor for local recurrence [40] and in this cohort with DCIS with a known recurrence the proportion of lesions with NG 3
E. Karlsson et al. / European Journal of Cancer 50 (2014) 517–524
was larger than 50%. In comparison, a previous study with a cohort of DCIS selected by size and NG to form a low risk group was used to the Oncotype DX with a special DCIS score [18]. In this study, no difference in risk of local recurrence was seen in relation to NG but the gene expression data did predict local recurrence independently from classical risk factors. It would be highly interesting to test the receptor conversion in our cohort in relation to gene expression data, especially taking radiotherapy into consideration. In conclusion, we addressed the issue of biomarker alteration after the identification/treatment of the primary DCIS and during tumour progression. This study demonstrates receptor conversion for ER, PR and HER2 status between primary DCIS and corresponding local relapse in 10–30% of instances. However, no general pattern for the conversion was seen. And no pattern was seen when stratifying for either in situ or invasive relapse. Finally, this study does not support the premise that HER2 overexpression in primary DCIS is of any major importance for tumour progression towards invasive cancer which has been proposed. However, more studies are needed to evaluate the impact of biomarker discordances after the identification/treatment of the primary DCIS, with the aim of explaining the crucial step: the progression of in situ cancer to invasive cancer. Conflict of interest statement Professor Jonas Bergh has received research grants for molecular marker studies, functional imaging and unrestricted research grants for clinical studies from Amgen, Astra Zeneca, Bayer, Merck, Roche and Sanofi Aventis. No payments to any individuals; payments to Karolinska University Hospital and/or Karolinska Institutet. All remaining authors have declared no conflicts of interest. Acknowledgements ¨ rebro Regional Research Council, FOU Uppsala-O Va¨rmlands La¨ns Landsting, The Swedish Breast Cancer Association (BRO), The Swedish Cancer Society, The Stockholm Cancer Society, The King Gustav V Jubilee Fund, The Swedish Research Council, The Stockholm City Council, Karolinska Institutet and Stockholm County Council Research Strategy Committee, the Karolinska Institutet Research Funds, AstraZeneca and Ma¨rit and Hans Rausing. We thank Anita Ringberg, Harald Anderson, Lars-Gunnar Arnesson, Stefan Emdin, Hans Garmo, Lars Holmberg, Per Karlsson, Hans Nordgren and Arne Wallgren in the Swedish Breast Cancer Group for conducting the SweDCIS study.
523
References [1] Wai ES, Lesperance ML, Alexander CS, Truong PT, Moccia P, Culp M, et al. Predictors of local recurrence in a population-based cohort of women with ductal carcinoma in situ treated with breast conserving surgery alone. Ann Surg Oncol 2011;18(1):119–24 [Epub 2010/07/21]. [2] Bijker N, Meijnen P, Peterse JL, Bogaerts J, Van Hoorebeeck I, Julien JP, et al. Breast-conserving treatment with or without radiotherapy in ductal carcinoma-in-situ: ten-year results of European Organisation for Research and Treatment of Cancer randomized phase III trial 10853 – a study by the EORTC Breast Cancer Cooperative Group and EORTC Radiotherapy Group. J Clin Oncol 2006;24(21):3381–7 [Epub 2006/06/28]. [3] Emdin SO, Granstrand B, Ringberg A, Sandelin K, Arnesson LG, Nordgren H, et al. SweDCIS: Radiotherapy after sector resection for ductal carcinoma in situ of the breast. Results of a randomised trial in a population offered mammography screening. Acta Oncol 2006;45(5):536–43 [Epub 2006/07/26]. [4] Julien JP, Bijker N, Fentiman IS, Peterse JL, Delledonne V, Rouanet P, et al. Radiotherapy in breast-conserving treatment for ductal carcinoma in situ: first results of the EORTC randomised phase III trial 10853. EORTC Breast Cancer Cooperative Group and EORTC Radiotherapy Group. Lancet 2000;355(9203): 528–33 [Epub 2000/02/22]. [5] Provenzano E, Hopper JL, Giles GG, Marr G, Venter DJ, Armes JE. Biological markers that predict clinical recurrence in ductal carcinoma in situ of the breast. Eur J Cancer 2003;39(5):622–30 [Epub 2003/03/12]. [6] Ringberg A, Anagnostaki L, Anderson H, Idvall I, Ferno M. Cell biological factors in ductal carcinoma in situ (DCIS) of the breast-relationship to ipsilateral local recurrence and histopathological characteristics. Eur J Cancer 2001;37(12):1514–22 [Epub 2001/08/17]. [7] Liedtke C, Broglio K, Moulder S, Hsu L, Kau SW, Symmans WF, et al. Prognostic impact of discordance between triplereceptor measurements in primary and recurrent breast cancer. Ann Oncol 2009;20(12):1953–8 [Epub 2009/07/15]. [8] Lindstrom LS, Karlsson E, Wilking UM, Johansson U, Hartman J, Lidbrink EK, et al. Clinically used breast cancer markers such as estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 are unstable throughout tumor progression. J Clin Oncol 2012;30(21):2601–8 [Epub 2012/06/20]. [9] Niikura N, Liu J, Hayashi N, Mittendorf EA, Gong Y, Palla SL, et al. Loss of human epidermal growth factor receptor 2 (HER2) expression in metastatic sites of HER2-overexpressing primary breast tumors. J Clin Oncol 2012;30(6):593–9 [Epub 2011/11/30]. [10] Kamby C, Rasmussen BB, Kristensen B. Oestrogen receptor status of primary breast carcinomas and their metastases. Relation to pattern of spread and survival after recurrence. Br J Cancer 1989;60(2):252–7 [Epub 1989/08/01]. [11] Curigliano G, Bagnardi V, Viale G, Fumagalli L, Rotmensz N, Aurilio G, et al. Should liver metastases of breast cancer be biopsied to improve treatment choice? Ann Oncol 2011;22(10):2227–33 [Epub 2011/02/24]. [12] Thompson AM, Jordan LB, Quinlan P, Anderson E, Skene A, Dewar JA, et al. Prospective comparison of switches in biomarker status between primary and recurrent breast cancer: the Breast Recurrence In Tissues Study (BRITS). Breast Cancer Res 2010;12(6):R92 [Epub 2010/11/10]. [13] Amir E, Miller N, Geddie W, Freedman O, Kassam F, Simmons C, et al. Prospective study evaluating the impact of tissue confirmation of metastatic disease in patients with breast cancer. J Clin Oncol 2012;30(6):587–92 [Epub 2011/11/30]. [14] Wilking U, Karlsson E, Skoog L, Hatschek T, Lidbrink E, Elmberger G, et al. HER2 status in a population-derived breast
524
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
E. Karlsson et al. / European Journal of Cancer 50 (2014) 517–524 cancer cohort: discordances during tumor progression. Breast Cancer Res Treat 2011;125(2):553–61 [Epub 2010/07/16]. Holmberg L, Garmo H, Granstrand B, Ringberg A, Arnesson LG, Sandelin K, et al. Absolute risk reductions for local recurrence after postoperative radiotherapy after sector resection for ductal carcinoma in situ of the breast. J Clin Oncol 2008;26(8):1247–52 [Epub 2008/02/06]. Zhou W, Jirstrom K, Johansson C, Amini RM, Blomqvist C, Agbaje O, et al. Long-term survival of women with basal-like ductal carcinoma in situ of the breast: a population-based cohort study. BMC Cancer 2010;10:653 [Epub 2010/12/02]. Foukakis T, Astrom G, Lindstrom L, Hatschek T, Bergh J. When to order a biopsy to characterise a metastatic relapse in breast cancer. Ann Oncol 2012;23(Suppl. 10):x349–53 [Epub 2012/09/ 26]. Pusztai L, Viale G, Kelly CM, Hudis CA. Estrogen and HER-2 receptor discordance between primary breast cancer and metastasis. Oncologist 2010;15(11):1164–8 [Epub 2010/11/03]. Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S, et al. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med 1998;4(7):844–7 [Epub 1998/07/14]. Torhorst J, Bucher C, Kononen J, Haas P, Zuber M, Kochli OR, et al. Tissue microarrays for rapid linking of molecular changes to clinical endpoints. Am J Pathol 2001;159(6):2249–56 [Epub 2001/ 12/06]. Warnberg F, Amini RM, Goldman M, Jirstrom K. Quality aspects of the tissue microarray technique in a population-based cohort with ductal carcinoma in situ of the breast. Histopathology 2008;53(6):642–9 [Epub 2008/12/17]. Zidan A, Christie Brown JS, Peston D, Shousha S. Oestrogen and progesterone receptor assessment in core biopsy specimens of breast carcinoma. J Clin Pathol 1997;50(1):27–9 [Epub 1997/01/ 01]. Fidler IJ, Kripke ML. Metastasis results from preexisting variant cells within a malignant tumor. Science 1977;197(4306):893–5 [Epub 1977/08/26]. Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 2012;366(10):883–92 [Epub 2012/03/09]. Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, Bell GW, et al. Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature 2007;449(7162):557–63 [Epub 2007/10/05]. Farmer P, Bonnefoi H, Anderle P, Cameron D, Wirapati P, Becette V, et al. A stroma-related gene signature predicts resistance to neoadjuvant chemotherapy in breast cancer. Nat Med 2009;15(1):68–74 [Epub 2009/01/06]. Finak G, Bertos N, Pepin F, Sadekova S, Souleimanova M, Zhao H, et al. Stromal gene expression predicts clinical outcome in breast cancer. Nat Med 2008;14(5):518–27 [Epub 2008/04/29]. Martin-Padura I, Gregato G, Marighetti P, Mancuso P, Calleri A, Corsini C, et al. The white adipose tissue used in lipotransfer procedures is a rich reservoir of CD34+ progenitors able to promote cancer progression. Cancer Res 2012;72(1):325–34 [Epub 2011/11/05].
[29] Vargas AC, Reed AE, Waddell N, Lane A, Reid LE, Smart CE, et al. Gene expression profiling of tumour epithelial and stromal compartments during breast cancer progression. Breast Cancer Res Treat 2012;135(1):153–65 [Epub 2012/06/22]. [30] Goldhirsch A, Wood WC, Coates AS, Gelber RD, Thurlimann B, Senn HJ. Strategies for subtypes – dealing with the diversity of breast cancer: highlights of the St. Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2011. Ann Oncol 2011;22(8):1736–47 [Epub 2011/06/29]. [31] Goldhirsch A, Winer EP, Coates AS, Gelber RD, PiccartGebhart M, Thurlimann B, et al. Personalizing the treatment of women with early breast cancer: highlights of the St Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2013. Ann Oncol 2013;24(9):2206–23 [Epub 2013/ 08/07]. [32] Muggerud AA, Hallett M, Johnsen H, Kleivi K, Zhou W, Tahmasebpoor S, et al. Molecular diversity in ductal carcinoma in situ (DCIS) and early invasive breast cancer. Mol Oncol 2010;4(4):357–68 [Epub 2010/07/29]. [33] Roses RE, Paulson EC, Sharma A, Schueller JE, Nisenbaum H, Weinstein S, et al. HER-2/neu overexpression as a predictor for the transition from in situ to invasive breast cancer. Cancer Epidemiol Biomarkers Prev 2009;18(5):1386–9 [Epub 2009/04/ 23]. [34] Harada S, Mick R, Roses RE, Graves H, Niu H, Sharma A, et al. The significance of HER-2/neu receptor positivity and immunophenotype in ductal carcinoma in situ with early invasive disease. J Surg Oncol 2011;104(5):458–65 [Epub 2011/05/11]. [35] Liao N, Zhang GC, Liu YH, Li XR, Yao M, Xu FP, et al. HER2positive status is an independent predictor for coexisting invasion of ductal carcinoma in situ of the breast presenting extensive DCIS component. Pathol Res Pract 2011;207(1):1–7 [Epub 2010/ 11/26]. [36] Latta EK, Tjan S, Parkes RK, O’Malley FP. The role of HER2/ neu overexpression/amplification in the progression of ductal carcinoma in situ to invasive carcinoma of the breast. Mod Pathol 2002;15(12):1318–25 [Epub 2002/12/14]. [37] Allred DC, Clark GM, Molina R, Tandon AK, Schnitt SJ, Gilchrist KW, et al. Overexpression of HER-2/neu and its relationship with other prognostic factors change during the progression of in situ to invasive breast cancer. Hum Pathol 1992;23(9):974–9 [Epub 1992/09/01]. [38] Kerlikowske K, Molinaro AM, Gauthier ML, Berman HK, Waldman F, Bennington J, et al. Biomarker expression and risk of subsequent tumors after initial ductal carcinoma in situ diagnosis. J Natl Cancer Inst 2010;102(9):627–37 [Epub 2010/ 04/30]. [39] Rakovitch E, Nofech-Mozes S, Hanna W, Narod S, Thiruchelvam D, Saskin R, et al. HER2/neu and Ki-67 expression predict non-invasive recurrence following breast-conserving therapy for ductal carcinoma in situ. Br J Cancer 2012;106(6):1160–5 [Epub 2012/03/01]. [40] Ringberg A, Nordgren H, Thorstensson S, Idvall I, Garmo H, Granstrand B, et al. Histopathological risk factors for ipsilateral breast events after breast conserving treatment for ductal carcinoma in situ of the breast – results from the Swedish randomised trial. Eur J Cancer 2007;43(2):291–8 [Epub 2006/11/23].
Available at www.sciencedirect.com
ScienceDirect journal homepage: www.ejcancer.com
Clonal alteration of breast cancer receptors between primary ductal carcinoma in situ (DCIS) and corresponding local events E. Karlsson a,b,⇑, K. Sandelin c, J. Appelgren d, W. Zhou e, K. Jirstro¨m f, J. Bergh a, F. Wa¨rnberg g a
Department of Oncology–Pathology, Radiumhemmet, Cancer Center Karolinska, Karolinska Institutet and University Hospital, Stockholm, Sweden Department of Oncology, Central Hospital Karlstad, Karlstad, Sweden c Department of Surgery, Karolinska University Hospital, Stockholm, Sweden d Department of Economics and Statistics, Karlstad University, Karlstad, Sweden e Department of Surgery, Uppsala University, Uppsala, Sweden f Division of Pathology, Department of Clinical Sciences, Lund University, Lund, Sweden g Department of Surgery, Uppsala University Hospital, Uppsala, Sweden b
Available online 22 November 2013
KEYWORDS Ductal carcinoma in situ DCIS Breast cancer Hormonal receptor ER Oestrogen receptor PR Progesterone receptor Human epidermal growth factor receptor 2 HER2
Abstract Background: Emerging data propose biomarker alteration due to clonal selection between the primary invasive breast cancer and corresponding metastases. In addition, impact on survival has been demonstrated. The present study investigates the relationship between the oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) between primary ductal carcinoma in situ (DCIS) and intra-individually matched ipsilateral event. Materials and methods: The cohort includes 1504 patients, diagnosed with a primary DCIS between 1986 and 2004. Of the 274 patients who developed a local relapse, 135 developed a new in situ carcinoma and 139 an invasive cancer up to 31st December 2011. ER and PR were identified by immunohistochemistry (IHC) and HER2 by silver-enhanced in situ hybridisation (SISH) as well as IHC. Results: ER (n = 112), PR (n = 113) and HER2 (n = 114) status from both the primary DCIS and the corresponding relapse were assessed and were demonstrated to be discordant in 15.1%, 29.2% and 10.5% respectively. The receptor conversion was both from negative to positive and from positive to negative with no general pattern being seen in spite of sub-dividing into in situ relapse and invasive relapse. However, primary DCIS was HER2 positive in 40.3% whereas in situ and invasive relapses were HER2 positive in 42.9% and 34.5% respectively.
⇑ Corresponding author at: Department of Oncology, Central Hospital Karlstad, 652 30 Karlstad, Sweden.
E-mail address: [email protected] (E. Karlsson). 0959-8049/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejca.2013.10.020
518
E. Karlsson et al. / European Journal of Cancer 50 (2014) 517–524
Conclusions: Receptor conversion for ER, PR and HER2 status occurred between primary DCIS and corresponding local relapse in 10–30%. This study could not confirm that HER2 overexpression in primary DCIS had any impact on tumour progression to invasive cancer which has been proposed. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Primary ductal carcinoma in situ (DCIS) of the breast is a heterogeneous disease with different malignant potential. Altogether, DCIS has a good prognosis [1,2]. However, approximately half of the relapses developed after a primary DCIS will be invasive cancer [3,4]. The expression of oestrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) have been shown to be associated with local relapse [5,6]. However there are conflicting data. Nevertheless, the optimal management of DCIS is controversial due to poor understanding of the natural history of DCIS and the poor understanding of those factors that are involved in tumour progression. Biomarkers such as ER, PR and HER2 help clinicians to optimise and individualise management of patients with primary invasive breast cancer. ER and HER2 are of particular interest since they are both prognostic markers and predictors of treatment response. Moreover, emerging data propose biomarker alteration between the primary invasive breast cancer and the corresponding metastases [7–14] and indeed, some studies, including earlier data from our group have reported impact on survival due to such a change in receptor status [7–9]. In this study we wanted to perform a comparative analysis of ER, PR and HER2 status between primary DCIS and the corresponding local relapse (both in situ and invasive) and to investigate the role of adjuvant radiotherapy on any discordance. 2. Patients and methods 2.1. Study population The study was approved by the Ethical committee at Uppsala University Hospital (Dnr 99 442, Dnr 2005:118) and Umea˚ University (Dnr 05-065M, Dnr 2012-224-32M). The source population includes 1504 patients from two separate cohorts, diagnosed with a primary DCIS between 1986 and 2004. Of these 1504 patients, 458 were identified from a population based cohort diagnosed between 1986 and 2004 in Uppland and Va¨stmanland regions of Sweden. The remaining 1046 were identified from the randomised SweDCIS trial of patients diagnosed between 1987 and 1999 [15]. A total of 274 patients developed a relapse/new cancer in the period up to and including 31st December 2011. Of these 274 patients, 135 (49.3%) patients developed
an in situ relapse and 139 (50.7%) an invasive relapse. From these 274 patients, tissue samples from both the primary DCIS and the corresponding relapse were collected to perform analysis of ER, PR and HER2 status. Thirty patients (2%) out of the total cohort (1504 patients) died due to breast cancer up to the 31st December 2011. 28 of those patients had an invasive relapse as first event and two patients had an in situ relapse as first event (data not shown). 2.2. Tissue sample and biomarker measurement Tissue microarray (TMA)-blocks were constructed from both primary DCIS and relapses. The detail of this process is described in an earlier publication [16]. ER and PR status were assessed by immunohistochemical (IHC) methods (DakoAutostainer), whereby tumours with >10% positive cells were classified as receptor positive. The antibodies used were NCL-6F11 (Novocastra) for ER and PgR NCL-1A6 (Novocastra) for PR analysis, respectively. Fixation and staining procedures were performed according to manufacturer’s instructions. Positive and negative controls were included in all staining runs. HER2 status was assessed by using IHC analysis with antibody c-erb 2 poly rabbit, A0485 (DAKO) and Hercept-kit, which was confirmed by silverenhanced in situ hybridisation (SISH). Using the Hercept-kit, tumours were classified as positive at the 3+ protein level. For HER2 status priority was given for SISH and if not available IHC was used for evaluation of HER2 levels. HER2 SISH was carried out in an automated instrument (Ventana Benchmark, Ventana Medical system, Tucson, AZ). Evaluation of HER2 gene amplification was performed according to the American Society of Clinical Oncology/College of American Pathologists guideline and Australian HER2 Advisory Board criteria for single HER2 probe testing. Both IHC methods and SISH are described in detail in an earlier publication [16]. Nuclear grade (NG) was evaluated in the primary DCIS prior to the TMA construction by one pathologist (K.J.). Tumour markers were scored by one single observer (W.Z.) and thus, we did not have a problem with inter-laboratory differences or intraobserver variability. 2.3. Statistical methods Fisher’s test was applied for comparison of ER, PR and HER2 status with baseline primary tumour
E. Karlsson et al. / European Journal of Cancer 50 (2014) 517–524
characteristics and between in situ and invasive recurrence groups. For ER, PR and HER2 status in relapse situation, the earliest diagnosed relapse with assessed tumour marker was used. Data analyses were conducted using IBM SPSS Statistics (version 20). 3. Results 3.1. Patient population Fig. 1 presents a flow chart of the total cohort of patients. In summary, out of 1504 patients with a primary DCIS, 274 developed a local relapse. Out of those, 135 patients developed an in situ relapse and 139 patients developed an invasive relapse. 3.2. Primary tumour (DCIS) Baseline primary tumour (DCIS) characteristics for the 274 patients with a local relapse are presented in Table 1. No patient in the study received adjuvant chemotherapy or endocrine therapy. Seventy-eight of 264 (29.5%) patients treated with breast conserving surgery received postoperative radiotherapy, according to local guidelines. In Table 2, distribution of ER, PR and HER2 status in the patients’ primary tumour was stratified into two groups, i.e. those who later developed an in situ relapse and those who later developed an invasive relapse. As can be seen, in all patients with a primary DCIS ER was positive in 78.8% of cases. Whereas, when stratified into groups, ER positivity was seen in 71.9% of primary DCIS for the patients who later developed an in situ relapse. This compares to 85.6% (p = 0.023) of those who later developed an invasive relapse. Furthermore, 58.5% of all patients were PR positive in primary DCIS. In addition, HER2 overexpression was seen more frequently in the group later developing an in situ
519
Table 1 Baseline characteristics in 274 women with a primary ductal carcinoma in situ (DCIS) and a new ipsilateral event. Number
Percent
Age at diagnosis 645 46–60 >60
49 123 102
17.9 44.9 37.2
Mode of detection Screening Clinically Missing
200 73 1
73.3 26.7
Type of surgery Breast conserving surgery Mastectomy
264 10
96.4 3.6
Postoperative radiotherapy Yes No
78 196
28.5 71.5
Tumour size 615 mm >15 mm or multifocal Missing
131 99 44
57.0 43.0
Free margins Yes No or doubtful
215 59
78.5 21.5
Nuclear grade 1 2 3 Missing
21 90 145 18
8.2 35.2 56.6
relapse compared to those who developed an invasive relapse, 48.3% versus 29.8% (p = 0.014). 3.3. Biomarker discordances ER, PR and HER2 status in both the primary DCIS and the corresponding relapse had been assessed in 112, 113 and 114 patients respectively (Fig. 1). Discordance
Total cohort of patient 1504 patients diagnosed with DCIS between 1986-2004 * Patients with local relapse reported up to 2011 N=274 (100%)
DCIS relapse
Invasive breast cancer relapse
N=135 (49,3%)
N=139 (50,7%)
Assessment of ER status
Assessment of PR status
Assessment of HER2 status
Both primary tumor and relapse
Both primary tumor and relapse
Both primary tumor and relapse
N=112
N=113
N=114
* 458 women from a population based cohort diagnosed between 1986-2004 in Uppland and Västmanland regions of Sweden and 1046 women diagnosed with DCIS between 1987-1999 from the SweDICS trial
Fig. 1. Flow chart.
520
E. Karlsson et al. / European Journal of Cancer 50 (2014) 517–524
Table 2 Tumour marker characteristics in 274 primary ductal carcinoma in situ (DCIS) with a subsequent new ipsilateral event. Primary DCIS (n = 274)
Primary DCIS with a subsequent new in situ event (n = 135)
Primary DCIS with a subsequent invasive cancer (n = 139)
Number
Percent
Number
Percent
Number
Percent
Primary ER^ status Positive 152 Negative 41
78.8 21.2
69 27
71.9 28.1
83 14
85.6 14.4
Primary PR^ status Positive 113 Negative 80
58.5 41.5
53 41
56.4 43.6
60 39
60.6 39.4
Primary HER2h status Positive 70 Negative 111
38.7 61.3
42 45
48.3 51.7
28 66
29.8 70.2
p Value*
0.023
0.562
0.014
Abbreviations: ER = oestrogen receptor, PR = progesterone receptor, HER2 = human epidermal growth factor receptor 2. Comparison between in situ and invasive relapse groups using Fisher’s test. ^ Cut-off value >10% for immunohistochemical (IHC) methods. h Analysed using silver-enhanced in situ hybridisation (SISH) directly, if not available IHC 3+ was used as positive. *
Table 3 Intrapatient discordances in ER, PR and HER2 status in primary ductal carcinoma in situ (DCIS) and corresponding new event, presented for in situ- and invasive relapses separately. Primary DCIS/all relapses
Primary DCIS/in situ relapse
Primary DCIS/invasive relapse
Number
Percent
Number
Percent
Number
Percent
68.8 8 7.1 16.1
34 4 4 12
63 7.4 7.4 22.2
43 5 4 6
74.1 8.6 6.9 10.3
100
54
100
58
100
43.4 16.8 12.4 27.4
20 13 8 14
36.4 23.6 14.5 25.5
29 6 6 17
50 10.3 10.3 29.3
100
55
100
58
100
34.2 6.1 4.4 55.3
22 5 2 27
39.3 8.9 3.6 48.2
17 2 3 36
29.3 3.4 5.2 62.1
100
56
100
58
100
ER^ status primary tumour and relapse Primary pos/relapse pos 77 Primary pos/relapse neg 9 Primary neg/relapse pos 8 Primary neg/relapse neg 18 112 ^
PR status primary tumour and relapse Primary pos/relapse pos 49 Primary pos/relapse neg 19 Primary neg/relapse pos 14 Primary neg/relapse neg 31 113 HER2h status primary tumour Primary pos/relapse pos Primary pos/relapse neg Primary neg/relapse pos Primary neg/relapse neg
and relapse 39 7 5 63 114
Abbreviations: ER = oestrogen receptor, PR = progesterone receptor, HER2 = human epidermal growth factor receptor 2. Cut-off value >10% for immunohistochemical (IHC) methods. h Analysed using silver-enhanced in situ hybridisation (SISH) directly, if not available IHC 3+ was used as positive.
^
in ER, PR or HER2 status from primary DCIS to relapse was 15.1%, 29.2% and 10.5%, respectively (both in situ and invasive relapses included). Loss of PR or HER2 between primary DCIS and relapse was greater in the in situ relapse site compared to the invasive relapse, 23.6% versus 10.3% and 8.9% versus 3.4%, respectively. Gain of ER, PR or HER2 between primary DCIS to relapse were 7.1%, 12.4% and 4.4%, respectively (both in situ and invasive relapse included). In total, in situ and invasive relapses were ER positive in 70.4% and 81% of cases respectively. For PR, in situ
and invasive relapses were PR positive in 50.9% and 60.3% of cases respectively. Finally, primary DCIS was HER2 positive in 40.3% of instances whereas in situ and invasive relapses were HER2 positive in 42.9% and 34.5% of instances, respectively (Table 3). 3.4. Nuclear grade and changes in biomarker status The proportion of patients with discordant biomarkers was higher amongst the primary DCIS with nuclear grade 3 compared to those with nuclear grade 1 and 2.
E. Karlsson et al. / European Journal of Cancer 50 (2014) 517–524
521
Table 4 Intrapatient discordances in ER, PR and HER2 status in primary ductal carcinoma in situ (DCIS) and corresponding relapse stratified for adjuvant radiotherapy or no treatment. Tumour marker
Radiotherapy Number
ER^ status primary tumour and relapse Primary pos/relapse pos Primary neg/relapse neg Discordant ER primary/relapse Total PR^ status primary tumour and relapse Primary pos/relapse pos Primary neg/relapse neg Discordant PR primary/relapse Total HER2h status primary tumour and relapse Primary pos/relapse pos Primary neg/relapse neg Discordant HER2 primary/relapse Total
p Value*
No radiotherapy Percent
Number
Percent 0.8
19 5 3
70.4 18.5 11.1
58 13 14
68.2 15.3 16.5
27
100.0
85
100.0
12 9 10
38.7 29.0 32.3
37 22 23
45.1 26.8 28.0
82
100.0
0.8
31
100
0.3 12 16 1
41.4 55.2 3.4
27 47 11
31.8 55.3 13.0
29
100.0
85
100.0
Abbreviations: ER = oestrogen receptor, PR = progesterone receptor, HER2 = human epidermal growth factor receptor 2. * Comparison between radiotherapy treatment group and no radiotherapy treatment group using Fisher’s test. ^ Cut-off value >10% for immunohistochemical (IHC) methods. h Analysed using silver-enhanced in situ hybridisation (SISH) directly, if not available IHC 3+ was used as positive.
ER, PR and HER2 status were discordant in 11/55 (20.0%), 20/56 (35.7%) and 8/60 (13.3%) in DCIS nuclear grade 3 and 4/42 (9.6%), 10/41 (24.4%) and 2/42 (4.8%) in DCIS nuclear grade 2 and finally, 0/7, 1/7 and 0/6 in DCIS nuclear grade 1. (p 6 0.001 for Fisher’s test, comparison between DCIS nuclear grade 1–3). 3.5. The effect of adjuvant therapy on change in biomarker status ER, PR and HER2 discordances were analysed for two groups separately, i.e. postoperative radiotherapy or no therapy (Table 4). The proportion of patients with changed ER and HER2 status was larger in the group with no therapy compared to the group which received postoperative radiotherapy; 16.5% (n = 14) versus 11.1% (n = 3) (p = 0.8) and 13% (n = 11) versus 3.4% (n = 1) (p = 0.3), respectively. The proportion of patients with changed PR status in the group with no therapy compared to the group which received radiotherapy was 28.0% (n = 23) versus 32.3% (n = 10) (p = 0.8). 4. Discussion In the present study we investigated the relationship between ER, PR and HER2 status in the primary lesion and the corresponding ipsilateral event in a cohort with primary DCIS and a known relapse.
Our study demonstrates receptor conversion for ER, PR and HER2 being 15.1%, 29.2% and 10.5%, respectively. The receptor conversion was both from negative to positive and from positive to negative and we could not find any general tendency for either loss or gain of receptors for the different biomarkers, not even when stratified for either in situ relapse or invasive relapse. Previous studies have demonstrated discordances of receptor status between the primary invasive breast cancer and the corresponding relapse for ER, PR and HER2, in the range of 10–37%, 24–48% and 3–30%, respectively [7–14]. In addition, two prospective studies have indicated that the treating clinician changed the patient management in approximately 15% of cases as a result of biomarker change [12,13]. Moreover, impact on survival due to biomarker change between the primary invasive breast cancer and the corresponding relapse has been shown in a few studies including earlier data from our group [7–9]. In this study we did not look at survival as the prognosis for women with a DCIS was so good with a low number of breast cancer deaths. The majority of biomarker change were due to the loss of receptors between the primary invasive breast cancer to relapse, although a small proportion of those patients also gained receptors [8]. As can be seen, the primary DCIS both gains and loses a large amount of receptors (Table 3). There are several possible explanations for our findings of tumour markers changing during tumour progression. Both methodological issues and pure
522
E. Karlsson et al. / European Journal of Cancer 50 (2014) 517–524
biological explanations have been discussed [17,18]. The use of various tissues processing in laboratories and sampling techniques (fine needle aspirates, core biopsies or surgical excisions) can potentially lead to false discordance in tumour marker [17]. In the present study, we used TMA, and it is worth noting that the documentation of reliability for the evaluating of TMA material in the assessment of ER, PR and HER2 in tumour samples has been documented [19–21]. The described concordance, between TMA and corresponding whole sections slides for ER, PR and HER2 was approximately 85–95%, 81–88% and 90–100%, respectively. As can be seen, a lower correlation for PR was seen and a possible explanation for this might be a tendency towards a more heterogeneous expression of PR within the tumour compared to ER [20,22]. Also, we cannot exclude the possibility that an assumed local relapse of breast cancer actually represents a new primary breast cancer, thus influencing our results. Biological explanations that have been discussed concerning receptor change are tumour heterogeneity and clonal selection [23,24]. Moreover, several reports have shown that contributors of the stroma and the white adipose tissue can harbour important breast cancer functions. Bone marrow-derived mesenchymal stem cells and endothelial progenitor cells may have the capacity to influence tumour progression [25–28]. In addition, gene expression profiles have shown different quantity of certain genes, in particular of those genes relating to the extracellular matrix which were seen overexpressed in the invasive breast cancer compared to DCIS as well as in their adjacent stroma. These findings possibly indicate a step by step movement from in situ cancer to invasive cancer but also instability of tumour biology throughout tumour progression [29]. We used the 10% cut off for ER and PR positivity as this is the clinically used cut off in Sweden. Recently, the 1% cut off has been proposed for invasive breast cancer at the St Gallen International Expert Consensus [30,31]. We used the 1% cut off in a separate analysis and of course, some lesions that were classified as negative with the 10% cut off were reclassified as positive with the 1% cut off (six primaries and five recurrences for ER and eight and five for PR, respectively). But also, we found it very difficult to use this 1% cut off in DCIS biopsies in the TMAs. Actually, we do not know what true ER or PR positivity is. Today, we use a cut off based on IHC and a clinical assumption that this cut off is related to response to endocrine therapy. Instead, maybe in the future we should use the gene expression based molecular subtypes when looking for conversion between the primary tumour and the corresponding relapse. DCIS can be classified into the same molecular subtypes as invasive breast cancer [32] but still, we have little knowledge of its importance in DCIS.
HER2 is of particular interest since it is both an important negative prognostic marker and a predictive factor regarding the efficacy of trastuzumab in the management of primary invasive breast cancer patients. Nevertheless, the role of HER2 in DCIS is unclear. For instance, it has been proposed that HER2 overexpression in DCIS is of major importance for tumour progression towards invasive cancer [33–35]. Some studies have demonstrated a more frequent overexpression of HER2 in DCIS compared to invasive breast cancer and particularly in high grade DCIS [36,37]. The findings from our present study cannot confirm such an influence regarding progression. HER2 overexpression was more frequent in the group of women with a primary DCIS who later developed an in situ relapse compared to those who developed an invasive relapse, 48.3% versus 29.8% (p = 0.014). Also, in our cohort of DCIS with a known recurrence HER2 status in the primary was positive in 40.3% of the cases, compared to 42.9% and 34.5% amongst the in situ relapses and invasive relapses, respectively. In line with this, other studies have shown a higher risk of developing a new in situ relapse amongst those women with a HER2 positive and Ki67 positive primary DCIS, while these women did not show a higher risk of developing an invasive relapse [38,39]. In a previous study (Lindstro¨m/Karlsson et al. JCO 2012), we have shown that usage of adjuvant therapy seemed to be driving clonal selection regarding hormonal receptor status between the primary invasive breast cancers and corresponding recurrences (e.g. the proportion of patients losing ER positivity was biggest in the groups treated with endocrine therapy). Moreover, a study from Niikura et al. demonstrate that loss of HER2 overexpression between primary invasive breast cancer and corresponding relapses was associated with chemotherapy but not with trastuzumab therapy [9]. In the present study, no patient with a primary DCIS received adjuvant endocrine therapy or chemotherapy. However, about a third of all patients received postoperative radiotherapy to the remaining breast. A lower proportion of patients who did receive radiotherapy demonstrated a change in ER and HER2 status from primary DCIS to relapse compared with the patients who received no treatment, although no statistically significant difference was seen between the groups. The decision to give radiotherapy was based on clinical data including NG and this can influence our results to some extent. Interestingly, in this study the proportion of patients with discordant biomarkers was higher in the group with high NG compared with the groups with lower NG. NG has been shown to be a risk factor for local recurrence [40] and in this cohort with DCIS with a known recurrence the proportion of lesions with NG 3
E. Karlsson et al. / European Journal of Cancer 50 (2014) 517–524
was larger than 50%. In comparison, a previous study with a cohort of DCIS selected by size and NG to form a low risk group was used to the Oncotype DX with a special DCIS score [18]. In this study, no difference in risk of local recurrence was seen in relation to NG but the gene expression data did predict local recurrence independently from classical risk factors. It would be highly interesting to test the receptor conversion in our cohort in relation to gene expression data, especially taking radiotherapy into consideration. In conclusion, we addressed the issue of biomarker alteration after the identification/treatment of the primary DCIS and during tumour progression. This study demonstrates receptor conversion for ER, PR and HER2 status between primary DCIS and corresponding local relapse in 10–30% of instances. However, no general pattern for the conversion was seen. And no pattern was seen when stratifying for either in situ or invasive relapse. Finally, this study does not support the premise that HER2 overexpression in primary DCIS is of any major importance for tumour progression towards invasive cancer which has been proposed. However, more studies are needed to evaluate the impact of biomarker discordances after the identification/treatment of the primary DCIS, with the aim of explaining the crucial step: the progression of in situ cancer to invasive cancer. Conflict of interest statement Professor Jonas Bergh has received research grants for molecular marker studies, functional imaging and unrestricted research grants for clinical studies from Amgen, Astra Zeneca, Bayer, Merck, Roche and Sanofi Aventis. No payments to any individuals; payments to Karolinska University Hospital and/or Karolinska Institutet. All remaining authors have declared no conflicts of interest. Acknowledgements ¨ rebro Regional Research Council, FOU Uppsala-O Va¨rmlands La¨ns Landsting, The Swedish Breast Cancer Association (BRO), The Swedish Cancer Society, The Stockholm Cancer Society, The King Gustav V Jubilee Fund, The Swedish Research Council, The Stockholm City Council, Karolinska Institutet and Stockholm County Council Research Strategy Committee, the Karolinska Institutet Research Funds, AstraZeneca and Ma¨rit and Hans Rausing. We thank Anita Ringberg, Harald Anderson, Lars-Gunnar Arnesson, Stefan Emdin, Hans Garmo, Lars Holmberg, Per Karlsson, Hans Nordgren and Arne Wallgren in the Swedish Breast Cancer Group for conducting the SweDCIS study.
523
References [1] Wai ES, Lesperance ML, Alexander CS, Truong PT, Moccia P, Culp M, et al. Predictors of local recurrence in a population-based cohort of women with ductal carcinoma in situ treated with breast conserving surgery alone. Ann Surg Oncol 2011;18(1):119–24 [Epub 2010/07/21]. [2] Bijker N, Meijnen P, Peterse JL, Bogaerts J, Van Hoorebeeck I, Julien JP, et al. Breast-conserving treatment with or without radiotherapy in ductal carcinoma-in-situ: ten-year results of European Organisation for Research and Treatment of Cancer randomized phase III trial 10853 – a study by the EORTC Breast Cancer Cooperative Group and EORTC Radiotherapy Group. J Clin Oncol 2006;24(21):3381–7 [Epub 2006/06/28]. [3] Emdin SO, Granstrand B, Ringberg A, Sandelin K, Arnesson LG, Nordgren H, et al. SweDCIS: Radiotherapy after sector resection for ductal carcinoma in situ of the breast. Results of a randomised trial in a population offered mammography screening. Acta Oncol 2006;45(5):536–43 [Epub 2006/07/26]. [4] Julien JP, Bijker N, Fentiman IS, Peterse JL, Delledonne V, Rouanet P, et al. Radiotherapy in breast-conserving treatment for ductal carcinoma in situ: first results of the EORTC randomised phase III trial 10853. EORTC Breast Cancer Cooperative Group and EORTC Radiotherapy Group. Lancet 2000;355(9203): 528–33 [Epub 2000/02/22]. [5] Provenzano E, Hopper JL, Giles GG, Marr G, Venter DJ, Armes JE. Biological markers that predict clinical recurrence in ductal carcinoma in situ of the breast. Eur J Cancer 2003;39(5):622–30 [Epub 2003/03/12]. [6] Ringberg A, Anagnostaki L, Anderson H, Idvall I, Ferno M. Cell biological factors in ductal carcinoma in situ (DCIS) of the breast-relationship to ipsilateral local recurrence and histopathological characteristics. Eur J Cancer 2001;37(12):1514–22 [Epub 2001/08/17]. [7] Liedtke C, Broglio K, Moulder S, Hsu L, Kau SW, Symmans WF, et al. Prognostic impact of discordance between triplereceptor measurements in primary and recurrent breast cancer. Ann Oncol 2009;20(12):1953–8 [Epub 2009/07/15]. [8] Lindstrom LS, Karlsson E, Wilking UM, Johansson U, Hartman J, Lidbrink EK, et al. Clinically used breast cancer markers such as estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 are unstable throughout tumor progression. J Clin Oncol 2012;30(21):2601–8 [Epub 2012/06/20]. [9] Niikura N, Liu J, Hayashi N, Mittendorf EA, Gong Y, Palla SL, et al. Loss of human epidermal growth factor receptor 2 (HER2) expression in metastatic sites of HER2-overexpressing primary breast tumors. J Clin Oncol 2012;30(6):593–9 [Epub 2011/11/30]. [10] Kamby C, Rasmussen BB, Kristensen B. Oestrogen receptor status of primary breast carcinomas and their metastases. Relation to pattern of spread and survival after recurrence. Br J Cancer 1989;60(2):252–7 [Epub 1989/08/01]. [11] Curigliano G, Bagnardi V, Viale G, Fumagalli L, Rotmensz N, Aurilio G, et al. Should liver metastases of breast cancer be biopsied to improve treatment choice? Ann Oncol 2011;22(10):2227–33 [Epub 2011/02/24]. [12] Thompson AM, Jordan LB, Quinlan P, Anderson E, Skene A, Dewar JA, et al. Prospective comparison of switches in biomarker status between primary and recurrent breast cancer: the Breast Recurrence In Tissues Study (BRITS). Breast Cancer Res 2010;12(6):R92 [Epub 2010/11/10]. [13] Amir E, Miller N, Geddie W, Freedman O, Kassam F, Simmons C, et al. Prospective study evaluating the impact of tissue confirmation of metastatic disease in patients with breast cancer. J Clin Oncol 2012;30(6):587–92 [Epub 2011/11/30]. [14] Wilking U, Karlsson E, Skoog L, Hatschek T, Lidbrink E, Elmberger G, et al. HER2 status in a population-derived breast
524
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
E. Karlsson et al. / European Journal of Cancer 50 (2014) 517–524 cancer cohort: discordances during tumor progression. Breast Cancer Res Treat 2011;125(2):553–61 [Epub 2010/07/16]. Holmberg L, Garmo H, Granstrand B, Ringberg A, Arnesson LG, Sandelin K, et al. Absolute risk reductions for local recurrence after postoperative radiotherapy after sector resection for ductal carcinoma in situ of the breast. J Clin Oncol 2008;26(8):1247–52 [Epub 2008/02/06]. Zhou W, Jirstrom K, Johansson C, Amini RM, Blomqvist C, Agbaje O, et al. Long-term survival of women with basal-like ductal carcinoma in situ of the breast: a population-based cohort study. BMC Cancer 2010;10:653 [Epub 2010/12/02]. Foukakis T, Astrom G, Lindstrom L, Hatschek T, Bergh J. When to order a biopsy to characterise a metastatic relapse in breast cancer. Ann Oncol 2012;23(Suppl. 10):x349–53 [Epub 2012/09/ 26]. Pusztai L, Viale G, Kelly CM, Hudis CA. Estrogen and HER-2 receptor discordance between primary breast cancer and metastasis. Oncologist 2010;15(11):1164–8 [Epub 2010/11/03]. Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S, et al. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med 1998;4(7):844–7 [Epub 1998/07/14]. Torhorst J, Bucher C, Kononen J, Haas P, Zuber M, Kochli OR, et al. Tissue microarrays for rapid linking of molecular changes to clinical endpoints. Am J Pathol 2001;159(6):2249–56 [Epub 2001/ 12/06]. Warnberg F, Amini RM, Goldman M, Jirstrom K. Quality aspects of the tissue microarray technique in a population-based cohort with ductal carcinoma in situ of the breast. Histopathology 2008;53(6):642–9 [Epub 2008/12/17]. Zidan A, Christie Brown JS, Peston D, Shousha S. Oestrogen and progesterone receptor assessment in core biopsy specimens of breast carcinoma. J Clin Pathol 1997;50(1):27–9 [Epub 1997/01/ 01]. Fidler IJ, Kripke ML. Metastasis results from preexisting variant cells within a malignant tumor. Science 1977;197(4306):893–5 [Epub 1977/08/26]. Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 2012;366(10):883–92 [Epub 2012/03/09]. Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, Bell GW, et al. Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature 2007;449(7162):557–63 [Epub 2007/10/05]. Farmer P, Bonnefoi H, Anderle P, Cameron D, Wirapati P, Becette V, et al. A stroma-related gene signature predicts resistance to neoadjuvant chemotherapy in breast cancer. Nat Med 2009;15(1):68–74 [Epub 2009/01/06]. Finak G, Bertos N, Pepin F, Sadekova S, Souleimanova M, Zhao H, et al. Stromal gene expression predicts clinical outcome in breast cancer. Nat Med 2008;14(5):518–27 [Epub 2008/04/29]. Martin-Padura I, Gregato G, Marighetti P, Mancuso P, Calleri A, Corsini C, et al. The white adipose tissue used in lipotransfer procedures is a rich reservoir of CD34+ progenitors able to promote cancer progression. Cancer Res 2012;72(1):325–34 [Epub 2011/11/05].
[29] Vargas AC, Reed AE, Waddell N, Lane A, Reid LE, Smart CE, et al. Gene expression profiling of tumour epithelial and stromal compartments during breast cancer progression. Breast Cancer Res Treat 2012;135(1):153–65 [Epub 2012/06/22]. [30] Goldhirsch A, Wood WC, Coates AS, Gelber RD, Thurlimann B, Senn HJ. Strategies for subtypes – dealing with the diversity of breast cancer: highlights of the St. Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2011. Ann Oncol 2011;22(8):1736–47 [Epub 2011/06/29]. [31] Goldhirsch A, Winer EP, Coates AS, Gelber RD, PiccartGebhart M, Thurlimann B, et al. Personalizing the treatment of women with early breast cancer: highlights of the St Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2013. Ann Oncol 2013;24(9):2206–23 [Epub 2013/ 08/07]. [32] Muggerud AA, Hallett M, Johnsen H, Kleivi K, Zhou W, Tahmasebpoor S, et al. Molecular diversity in ductal carcinoma in situ (DCIS) and early invasive breast cancer. Mol Oncol 2010;4(4):357–68 [Epub 2010/07/29]. [33] Roses RE, Paulson EC, Sharma A, Schueller JE, Nisenbaum H, Weinstein S, et al. HER-2/neu overexpression as a predictor for the transition from in situ to invasive breast cancer. Cancer Epidemiol Biomarkers Prev 2009;18(5):1386–9 [Epub 2009/04/ 23]. [34] Harada S, Mick R, Roses RE, Graves H, Niu H, Sharma A, et al. The significance of HER-2/neu receptor positivity and immunophenotype in ductal carcinoma in situ with early invasive disease. J Surg Oncol 2011;104(5):458–65 [Epub 2011/05/11]. [35] Liao N, Zhang GC, Liu YH, Li XR, Yao M, Xu FP, et al. HER2positive status is an independent predictor for coexisting invasion of ductal carcinoma in situ of the breast presenting extensive DCIS component. Pathol Res Pract 2011;207(1):1–7 [Epub 2010/ 11/26]. [36] Latta EK, Tjan S, Parkes RK, O’Malley FP. The role of HER2/ neu overexpression/amplification in the progression of ductal carcinoma in situ to invasive carcinoma of the breast. Mod Pathol 2002;15(12):1318–25 [Epub 2002/12/14]. [37] Allred DC, Clark GM, Molina R, Tandon AK, Schnitt SJ, Gilchrist KW, et al. Overexpression of HER-2/neu and its relationship with other prognostic factors change during the progression of in situ to invasive breast cancer. Hum Pathol 1992;23(9):974–9 [Epub 1992/09/01]. [38] Kerlikowske K, Molinaro AM, Gauthier ML, Berman HK, Waldman F, Bennington J, et al. Biomarker expression and risk of subsequent tumors after initial ductal carcinoma in situ diagnosis. J Natl Cancer Inst 2010;102(9):627–37 [Epub 2010/ 04/30]. [39] Rakovitch E, Nofech-Mozes S, Hanna W, Narod S, Thiruchelvam D, Saskin R, et al. HER2/neu and Ki-67 expression predict non-invasive recurrence following breast-conserving therapy for ductal carcinoma in situ. Br J Cancer 2012;106(6):1160–5 [Epub 2012/03/01]. [40] Ringberg A, Nordgren H, Thorstensson S, Idvall I, Garmo H, Granstrand B, et al. Histopathological risk factors for ipsilateral breast events after breast conserving treatment for ductal carcinoma in situ of the breast – results from the Swedish randomised trial. Eur J Cancer 2007;43(2):291–8 [Epub 2006/11/23].