Luminal A breast cancer defined as hormone receptor positive and human epidermal growth factor receptor 2 (HER2) negative is known to be heterogeneous. Previous study showed that luminal A tumours with the expression of basal markers ((cytokeratin (CK) 5 or CK5/6) or epidermal growth factor receptor (EGFR)) were associated with poorer prognosis compared with those that stained negative for basal markers. Prompted by this study, we assessed whether tumour characteristics and risk factors differed by basal marker status within luminal A tumours.
We pooled 5040 luminal A cases defined by immunohistochemistry (4490 basal-negative ((CK5 (or CK5/6))− and EGFR−) and 550 basal-positive ((CK5 (or CK5/6+)) or EGFR+)) from eight studies participating in the Breast Cancer Association Consortium. Case–case comparison was performed using unconditional logistic regression.
Tumour characteristics and risk factors did not vary significantly by the expression of basal markers, although results suggested that basal-positive luminal tumours tended to be smaller and node negative, and were more common in women with a positive family history and lower body mass index.
Most established breast cancer risk factors were similar in basal-positive and basal-negative luminal A tumours. The non-significant but suggestive differences in tumour features and family history warrant further investigations.
Breast cancer can be classified into several molecular subtypes based on gene expression profiling analyses (Perou et al, 2000; Sorlie et al, 2001), which can be approximated with the use of key immunohistochemical (IHC) markers, including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor 2 (HER2), and basal markers such as epidermal growth factor receptor (EGFR), cytokeratin 5 (CK5) or cytokeratin 5/6 (CK5/6). In general, well-known breast cancer hormonal and lifestyle risk factors, such as early age at menarche, late age at first birth, nulliparity, prolonged interval between menarche and age at first birth, and postmenopausal obesity showed stronger associations with ER-positive (luminal) subtypes (Yang et al, 2011; Anderson et al, 2014). In contrast, these factors showed either a lack of association or associations in the opposite direction for ER-negative (non-luminal) tumours. For example, parity and premenopausal obesity were protective for luminal cancers but associated with increased risk for non-luminal tumours, particularly triple-negative breast cancer (TNBC: ER−/PR−/HER2−; Millikan et al, 2008; Phipps et al, 2011). We have previously shown that risk factor associations differed most strikingly between luminal A (ER+ or PR+/HER2−) and core-basal phenotype (CBP: TNBC expressing (CK5 or CK5/6) or EGFR), suggesting that these two subtypes may develop from etiologically different pathways (Yang et al, 2011).
Experimental and clinical studies suggest more complex layers of heterogeneity within major breast cancer subtypes (Perou et al, 2000; Sotiriou et al, 2003; Colleoni et al, 2012; Ali et al, 2014). In particular, luminal cancers demonstrated substantial variability in molecular characteristics (Cancer Genome Atlas Network, 2012) and clinical behaviour, including responsiveness to endocrine treatment (Ciriello et al, 2013; Howell, 2013; Ignatiadis and Sotiriou, 2013). In line with this, in a recent large pooled analysis including >10 000 invasive breast cancer cases, Blows et al, 2010 showed that luminal A tumours expressing basal markers ((CK5 or CK5/6) or EGFR, luminal A basal-positive) had worse prognosis than luminal A tumours that were negative for basal markers (luminal A basal-negative). However, to our knowledge, there have been no reports on etiological heterogeneity within luminal A tumours so far.
To assess whether luminal A basal-positive tumours (ER+ or PR+/HER2−/basal markers+) represent a distinct disease entity from an etiologic perspective, we pooled individual data for 5040 luminal A breast cancer cases contributed by eight studies participating in the Breast Cancer Association Consortium (BCAC), with risk factor information and expression status for ER, PR, HER2, and basal markers. The goal of this study was to examine whether tumour characteristics and risk factors of luminal A basal-positive tumours are different from those of luminal A basal-negative tumours (ER+ or PR+/HER2−/basal markers−).
Materials and methods
Among studies participating in the BCAC (Yang et al, 2011), eight studies that had IHC data on ER (and/or PR), HER2, and basal markers (CK5 (or CK5/6) and/or EGFR) as well as breast cancer risk factor information were eligible for inclusion. Study details are summarised in Supplementary Table 1. These include four population-based studies (Kuopio Breast Cancer Project (KBCP), Melbourne Collaborative Cohort Study (MCCS), Nurses’ Health Study (NHS), and NCI’s Polish Breast Cancer Study (PBCS)) and four hospital-based case-control studies or studies of mixed design (Helsinki Breast Cancer Study (HEBCS), Mayo Clinic Breast Cancer Study (MCBCS), Sheffield Breast Cancer Study (SBCS), and Study of Epidemiology and Risk factors in Cancer Heredity (SEARCH)). As the goal of our analysis was to determine whether tumour characteristics and risk factors differed by basal marker status within luminal tumours, we restricted the analysis to 5040 luminal A cases (ER+ or PR+/HER2−) that were known to express or not to express basal markers (CK5 (or CK5/6) or EGFR) (Table 1). Study participants were recruited under protocols approved by the institutional review board at each institution and all subjects provided written informed consent.
Tumour marker assessment and subtype classification
ER, PR, and HER2 status were primarily extracted from medical records. Accordingly, the source of tumour marker data and definition of positivity for each marker varied across studies (Supplementary Table 2). Among 5040 luminal A cases defined based on medical records for ER and PR, centralised quantitative scores for ER or PR status obtained through automated imaging analysis of tissue microarrays were available for 3702 participants (Supplementary Table 3). More than 99% of luminal A cases (n=3670/3072) had tumours with 1% cells and 97% (n=3592/3072) with 10% tumour cells stained positive for either ER or PR, respectively. Given the high concordance of clinical data and centralised measurements for ER and PR, we used clinical data for these markers in the main analyses because they were available for more cases. Data for CK5 (or CK5/6) and EGFR status were obtained from centralised visual scoring of tissue microarray slides by pathologists. Expression was determined to be positive if >10% tumour cells were stained. When the proportion of positive cells was missing, positivity was defined based on the intensity score (2 as positive).
The number of cases in each study by marker status is presented in Supplementary Table 4. In the current study, we focused on two subtypes within luminal A tumours: basal-negative (ER+ or PR+/HER2−/(CK5 (or CK5/6))−/EGFR−) and basal-positive (ER+ or PR+/HER2−/(CK5 (or CK5/6))+ or EGFR+).
Breast cancer risk factors
The collection of information on tumour characteristics and risk factors for BCAC studies has been previously described (Yang et al, 2011). Briefly, each study collected information on one or more of the following factors: family history of breast cancer in first-degree relatives, age at menarche, age at menopause, age at first full-term pregnancy, parity (never/ever), number of children, breast feeding (never/ever), and body mass index (BMI) at baseline (MCCS and KBCP) or at the time of diagnosis (all others). NHS was not included in risk factor analysis owing to the lack of data.
We compared the distribution of tumour characteristics and risk factors between luminal A basal-negative and basal-positive subtypes using unconditional logistic regression with luminal A basal-negative subtype as the reference group. Tumour characteristics included histology (ductal, lobular, other), grade (well, moderately, poorly differentiated), size (2 cm, >2 cm), and axillary node status (negative, positive). Breast cancer risk factors included family history of breast cancer among first-degree relatives (present, absent), age at menarche (12, 13–14, >14 years), parity (parous, nulliparous), and BMI (<25, 25–30, 30 kg m−2 or per 5 unit of increase); and in analyses restricted to parous women included age at first full-term birth (<20, 20–24, 25–29, 30 years), number of full-term pregnancies (1, 2, 3), and breast feeding (ever, never). Multivariable models were used in all analyses to control for age (10-year frequency), study, other tumour characteristics and risk factors. Given that risk associated with BMI is known to vary by menopausal status, we stratified the BMI analysis by menopausal status. We used age groups (<50 and 50 years) as a surrogate for menopausal status to maximise power. A sensitivity analysis using known menopausal status yielded similar results. Between-study heterogeneity was assessed with I2 statistics using study-specific odds ratio (ORs) and 95% confidence intervals (CIs). Analyses were conducted using SAS (version 9.3; SAS Institute, Cary, NC, USA) or Stata/SE (version 11.2; StataCorp LP, College Station, TX, USA).
Among all 7857 invasive breast cancer cases in the 8 studies, 63.3% (n=4490) and 7.8% (n=550) were classified as luminal A basal-negative and luminal A basal-positive subtype, respectively (Table 1). Mean age at diagnosis was not significantly different between the two subtypes, although women with luminal A basal-positive tumours were diagnosed less frequently after 60 years compared with the women with luminal A basal-negative tumours (Table 2). Compared with the luminal A basal-negative tumours, basal-positive tumours were more likely to be smaller (OR>2 cm vs 2 cm=0.83; 95% CI=0.67–1.03; P=0.09; I2=0%) and negative for axillary nodes (OR=0.83; 95% CI=0.67–1.02; P=0.08; I2=0%), however, the differences were not statistically significant. The association with tumour grade did not follow a logical trend, with luminal A basal-positive tumours showing a lower frequency of moderately differentiated tumours (OR=0.75; 95% CI=0.60–0.94; P=0.01), but a higher frequency of poorly differentiated tumours (OR=1.13; 95% CI=0.85–1.50; P=0.42) compared with luminal A basal-negative tumours (Table 2). Study heterogeneity was not significant in the former (I2=10.2%; P=0.35) but was significant in the latter association (I2=63.2%; P=0.01).
Compared with basal-negative cases, cases with luminal A basal-positive tumours were more likely to have a positive family history (OR=1.27; 95% CI=0.99–1.63; P=0.06; I2=1.1%; Table 3) particularly among younger (<50 years) women (OR=1.81; 95% CI=1.16–2.82; P=0.009). In addition, basal-positive cases tended to have lower BMI (ORper 5 unit=0.90; 95% CI=0.81–1.01, P=0.07; I2=0.0%) especially among older (50 years) women (ORper 5 unit=0.89; 95% CI=0.79–1.01, P=0.07; I2=15.5%) compared with basal-negative cases, but the differences were weak and the test of interaction by age group did not reach nominal significance (P<0.05). Other risk factors did not differ significantly between the two subtypes.
To reduce the impact of potential subtype misclassification, we conducted a sensitivity analysis by restricting our analyses to cases showing ER expression in 10% and PR expression in 20% tumour cells. Among 3015 basal-negative and 366 basal-positive cases with ER and PR percentage data available, 2372 (79%) basal-negative and 299 (82%) basal-positive cases were included in the sensitivity analysis. The only difference we observed was that luminal A basal-positive tumours now had a similar, rather than a higher, proportion of poorly differentiated tumours to luminal A basal-negative tumours. ORs for other tumour characteristics and risk factors did not change substantially (Supplementary Table 5).
In a previous BCAC analysis (Blows et al, 2010), we showed that all-cause mortality among cases with luminal A basal-positive tumours was slightly but significantly higher than that of cases with luminal A basal-negative tumours, and the difference was persistent up to 15 years after diagnosis. Similar but non-significant difference in survival by basal marker expression (adjusted hazard ratio=1.20basal-positive vs basal-negative; 95% CI=0.69–2.08, P=0.51) was observed in our study when we analysed a subset of cases (1245 luminal A cases; 1124 basal-negative cases, and 121 basal-positive cases) with the follow-up data available. Interestingly, luminal A basal-positive tumours were not associated with more aggressive features, rather, they tended to be less aggressive (smaller, lower grade, and node negative) compared with basal-negative tumours.
The apparent discrepancy between less aggressive tumour features and poorer prognosis in basal-positive cases might be explained by different responses to endocrine therapy among cases with luminal tumours. Previous studies using luminal tumour xenografts identified a subpopulation of ER-PR-CK5+ cells that were resistant to endocrine therapies (Horwitz et al, 2008); when ER+ tumours with ER-PR-CK5+ cells were treated with 17β-estradiol plus anti-estrogens tamoxifen or fulvestrant, the number of CK5+ cells in post compared with pre-treatment tumours coupled with decreased expression of ER and increased expression of CK5 (Kabos et al, 2011). Studies with detailed pathology data incorporating cellular subpopulation, as well as treatment regimens with long-term follow-up are needed to definitively address this question.
Known breast cancer risk factors did not appear to vary significantly by basal marker expression within luminal A tumours, although we observed weak associations between basal-positive tumours and higher frequency of positive family history especially among younger women and lower prevalence of obesity. The higher frequency of slim women with luminal A basal-positive tumours might be also related to smaller tumour size of luminal A basal-positive tumours as we observed a significant correlation between tumour size and BMI among our study subjects. Indeed, when we adjusted for BMI, the association between luminal A basal-positive subtype and smaller tumour size was attenuated (OR=0.87; 95% CI=0.69–1.09; P=0.22). This finding is consistent with previous reports that obese breast cancer patients had larger tumours and higher rates of lymph node metastases (Ewertz et al, 2011; Haakinson et al, 2012).
Our study has limitations. Although it is one of the largest consortium studies with breast tumour subtype information and risk factor data collected, statistical power was limited to assess risk factors in uncommon subtypes especially when controlling for potential confounding factors such as breastfeeding, menopausal hormone therapy usage, and tumour size. As expected for any analysis pooling data from multiple studies, there were variations in study populations, study designs, data collection methods, and marker measurement, which may cause study heterogeneity and subtype misclassification. However, we found no significant heterogeneity across studies at least for the associations in risk factors we analysed. In addition, the proportions of CBP (8.5%) and 5-NP (6.3%) subtypes were also comparable to those reported previously (Cheang et al, 2008; Blows et al, 2010; Yang et al, 2011; Liu et al, 2012). Of note, although we used centralised measurement for CK5/6 and EGFR expression, we used ER, PR, and HER2 status retrieved from clinical records in each study instead of centralised data to maximise the power of our study. Accordingly, IHC methods and cut-point for positivity varied substantially among studies. However, we observed high concordance for ER and PR status between clinical records and centralised quantitative measurements among a subset of study subjects with both data available. Further, the overall proportions of positivity for these five markers (ER, 78.0%; PR, 64.2%; HER2, 14.5%; CK5/6, 13.5%; EGFR, 12.8%) were generally consistent with what was reported in previous studies (El-Rehim et al, 2004; Carey et al, 2006; Rakha et al, 2006; Cheang et al, 2008; Liu et al, 2012). Finally, information on proliferation marker (such as Ki-67) was not available for most studies, which made the accurate classification of real luminal A tumours a challenge. However, results from the sensitivity analysis restricting to cases with high ER and PR expression levels using centralised data did not change results significantly, suggesting that the potential subtype misclassification caused by study heterogeneity or marker measurement and scoring did not significantly influence our conclusion.
In conclusion, we found that tumour characteristics and known risk factors were generally similar in basal-positive and basal-negative luminal A tumours. The small differences in tumour features and family history between the two luminal A subtypes warrant further investigations in future studies with larger number of subjects and detailed annotation of subtype and risk factor information.
The Helsinki Breast Cancer Study (HEBCS) thanks Kristiina Aittomäki, Kirsimari Aaltonen, Taru A Muranen, Karl von Smitten, and Irja Erkkilä for their kind help with the patient samples and data. The Study of Epidemiology and Risk factors in Cancer Heredity (SEARCH) thanks Elena Provenzano and Marie Mack. The HEBCS was supported by The Helsinki University Central Hospital Research Fund, Academy of Finland (266528), the Finnish Cancer Society, and The Nordic Cancer Union and the Sigrid Juselius Foundation. The Mayo Clinic Breast Cancer Study (MCBCS) was supported by The Breast Cancer Research Foundation, the National Institutes of Health Specialized Program of Research Excellence (SPORE) in Breast Cancer (CA116201), R01 grants (CA128978 and CA176785), and the Grohne Family. The Melbourne Collaborative Cohort Study (MCCS) was supported by The Cancer Council Victoria, VicHealth, NHMRC (209057, 251533, 396414, and 504711), Victorian Cancer Registry (VCR) and the Australian Institute of Health and Welfare (AIHW), including the National Death Index (NDI). The Nurses’ Health Study (NHS) was supported by National Institutes of Health/National Cancer Institute (UM1 CA186107 and P01 CA 87969). The NHS thanks the following state cancer registries for their help: AL, AZ, AR, CA, CO, CT, DE, FL, GA, ID, IL, IN, IA, KY, LA, ME, MD, MA, MI, NE, NH, NJ, NY, NC, ND, OH, OK, OR, PA, RI, SC, TN, TX, VA, WA, WY. The Polish Breast Cancer Study (PBCS) was supported by Intramural Research Program of US NIH, NCI, Division of Cancer Epidemiology and Genetics (DCEG). The Sheffield Breast Cancer Study (SBCS) was supported by Yorkshire Cancer Research (S295, S299, and S305PA) and Sheffield Experimental Cancer Medicine Centre. The SEARCH was supported by Cancer Research UK (C490/A16561), the Biomedical Research Centre at the University of Cambridge. The Kuopio Breast Cancer Project (KBCP) was supported by The special Government Funding of Kuopio University Hospital grants, Cancer Fund of North Savo, the Finnish Cancer Organizations, the strategic funding of the University of Eastern Finland.
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Supplementary Information accompanies this paper on British Journal of Cancer website (http://www.nature.com/bjc)
Breast Cancer Research and Treatment (2017)