HHS Public Access Author manuscript Author Manuscript
Int J Cancer. Author manuscript; available in PMC 2017 May 15. Published in final edited form as: Int J Cancer. 2016 May 15; 138(10): 2346–2356. doi:10.1002/ijc.29968.
Reproductive risk factors in relation to molecular subtypes of breast cancer: results from the Nurses' Health Studies Julia S. Sisti1,2,3, Laura C. Collins4, Andrew H. Beck4, Rulla M. Tamimi1,2, Bernard A. Rosner2, and A. Heather Eliassen1,2 1Department
of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA
Author Manuscript
2Channing
Division of Network Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA
3Department
of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New
York, NY 4Department
of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School
Abstract
Author Manuscript Author Manuscript
Several intrinsic breast cancer subtypes, possibly representing unique etiologic processes, have been identified by gene expression profiles. Evidence suggests that associations with reproductive risk factors may vary by breast cancer subtype. In the Nurses' Health Studies, we prospectively examined associations of reproductive factors with breast cancer subtypes defined using immunohistochemical staining of tissue microarrays. Multivariate-adjusted Cox proportional hazard models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). Over follow-up, we identified 2,063 luminal A, 1,008 luminal B, 209 HER2-enriched, 378 basallike and 110 unclassified tumors. Many factors appeared associated with luminal A tumors, including ages at menarche (Pheterogeneity=0.65) and menopause (Pheterogeneity=0.05), and current HT use (Pheterogeneity=0.33). Increasing parity was not associated with any subtype (Pheterogeneity=0.76), though age at first birth was associated with luminal A tumors only (per 1year increase HR=1.03 95%CI (1.02, 1.05), Pheterogeneity=0.04). Though heterogeneity was not observed, duration of lactation was inversely associated with risk of basal-like tumors only (7+ months vs. never HR=0.65 95%CI (0.49, 0.87), Ptrend=0.02), Pheterogeneity=0.27). Years between menarche and first birth was strongly positively associated with luminal A and non-luminal subtypes (e.g. 22-year interval vs. nulliparous HR=1.80, 95%CI (1.08, 3.00) for basal-like tumors; Pheterogeneity=0.003), and evidence of effect modification by breastfeeding was observed. In summary, many reproductive risk factors for breast cancer appeared most strongly associated with the luminal A subtype. Our results support previous reports that lactation is protective against basal-like tumors, representing a potential modifiable risk factor for this aggressive subtype.
Author to whom correspondence and/or reprint requests should be addressed: A. Heather Eliassen, Sc.D, Department of Epidemiology, Channing Laboratory, 181 Longwood Avenue, 3rd Floor, Boston, MA 02115, [email protected]. Competing interests: The authors declare that they have no competing interests.
Sisti et al.
Page 2
Author Manuscript
Keywords breast cancer; molecular subtypes; reproductive risk factors; pregnancy; lactation
INTRODUCTION
Author Manuscript
Genomic analysis suggests breast cancer heterogeneity is more complex than previous categorizations based on estrogen and progesterone receptor (ER/PR) status. Currently, at least 4 distinct molecular subtypes have been identified1–3 and can be reliably classified by immunohistochemistry of ER, PR and other markers.4–8 Tumors that express hormone receptors (HR+) are classified as either luminal A or luminal B, with the latter characterized by higher expression of the proliferative marker Ki-67. HER2-enriched tumors do not express HR, but overexpress human epidermal growth factor receptor 2 (HER2). Among tumors lacking expression of all three markers (triple-negative cancers), basal-like tumors express the basal markers cytokeratins 5/6 (CK 5/6) and/or endothelial growth factor (EGFR), while tumors that express none of these markers are termed unclassified. While these subtypes vary in prognosis and response to treatment9,10 they also may reflect distinct etiologic pathways given that subtype appears to be fixed from the time of tumor initiation.11 Because luminal A tumors comprise about 60% of breast tumors, many known risk factors are likely to be associated with this phenotype; however, less is known about risk factors for HR-negative (HR−) subtypes.
Author Manuscript Author Manuscript
Several reproductive risk factors are associated with breast cancer risk, possibly mediated by changes in sex hormones. Evidence suggests these risk factors may be more strongly associated with HR+ subtypes.12–14 While some reproductive factors are associated with HR− subtypes, these relationships do not always mirror those observed with HR+ disease. As recently reviewed,15 parity has been associated with decreased risk of ER+/PR+ breast cancers, while null or positive associations between parity and ER−/PR− tumors have been reported in some studies. Conversely, while the association between breastfeeding and ER+/PR+ tumors has been inconsistent, inverse associations have been observed with ER−/PR− and triple-negative tumors, suggesting lactation may mitigate the increased risk of these subtypes conferred by parity.15–17 Few studies have examined reproductive risk factors in relation to the fully characterized intrinsic breast cancer subtypes, including our prior analysis in the Nurses' Health Study (NHS) with 2,022 cases.18 Results suggest parity may protect against luminal subtypes, while significant18,19 or suggestive20 positive associations were observed for basal-like tumors. Conversely, breastfeeding was inversely associated with basal-like tumors.18,19 Associations with other risk factors were less consistent across studies. With the exception of the AMBER study of breast cancer in African American women, which included 678 triple-negative cases, each analysis to date included relatively few non-luminal cases, and immunohistological criteria used to define subtypes differed somewhat across studies. We examined associations of reproductive risk factors with molecular subtypes of breast cancer defined by comprehensive immunohistochemical profiling in the NHS and NHSII.
Int J Cancer. Author manuscript; available in PMC 2017 May 15.
Sisti et al.
Page 3
Author Manuscript
We added to our previous NHS analysis with an additional 8 years of follow-up, and included NHSII, for a total of nearly 4,000 cases with defined molecular subtypes.
METHODS Study population
Author Manuscript
The Nurses' Health Studies are ongoing prospective cohorts of registered nurses in the United States. The NHS was established in 1976 among 121,700 women ages 30–55 years; the NHSII was established in 1989 among 116,430 women ages 25–42 years. Participants have reported reproductive history, lifestyle factors and disease diagnoses at baseline and biennial questionnaires. Including surveillance for deaths, follow-up rates in NHS and NHSII were over 95% through the 2006 and 2003 questionnaire cycles, respectively. This study was approved by the Institutional Review Board of Brigham and Women's Hospital in Boston, MA. Outcome assessment Incident breast cancer diagnoses were reported on biennial questionnaires or identified through death records. Permission to access medical records for breast cancer cases was sought from participants or next of kin; requests were made to pathology departments from treating hospitals to obtain formalin-fixed paraffin-embedded (FFPE) tissue samples. Detailed description of block collection has been published previously21,22. Tissue blocks were obtained from approximately 70% of confirmed NHS cases and approximately 60% of NHSII cases reported through 2006. Tissue microarrays and subtype classification
Author Manuscript
Tissue microarrays (TMAs) that included tumors from 4,308 NHS participants and 1,253 NHSII participants were constructed at the Dana Farber/Harvard Cancer Center Tissue Microarray Core Facility in Boston, MA, as previously detailed elsewhere.22 Briefly, three 0.6mm diameter cores were obtained from each participant tumor tissue sample, and inserted into TMA blocks that were subsequently cut into 5um paraffin sections prior to immunohistochemical staining. TMA sections were stained for a panel of immunohistochemical markers (ER, PR, HER2, CK 5/6, EGFR) to classify molecular subtypes.4–6,8,23,24 A subset of NHS cases (N=3,281, 76%) was additionally stained for the proliferative marker Ki-67; no NHSII cases had Ki-67 data. Only invasive tumors (N=4,501), as confirmed by study pathologists, were eligible for inclusion in this analysis. Tissue microarrays and subtype classification
Author Manuscript
Positivity for each immunohistochemical marker was determined visually by a pathologist (LC) who reviewed each core under a microscope and assigned a score. The exception was Ki-67, which was evaluated automatically using an automated computational image analysis system (Definiens Tissue Studio software, Munich, Germany). Positivity for ER and PR was defined as any nuclear staining of these markers (≥1%), while HER2 over-expression was defined as strong membrane staining in >10% of cells in a given core. Cores were considered positive for CK 5/6 and EGFR if any cytoplasmic and/or membranous staining was present in any of a tumor's three cores. Lastly, we estimated the mean percentage of Int J Cancer. Author manuscript; available in PMC 2017 May 15.
Sisti et al.
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Author Manuscript
cells displaying nuclear staining Ki-67 by weighting each core by its total cell count; we considered cases positive if >14% of cells were positive for Ki-67.5
Author Manuscript
We classified tumors by molecular subtype using definitions that correlate with gene expression profile classifications.4–8,23 For tumors missing Ki-67 expression data, we used histologic grade as a surrogate; NHS results for luminal tumors were similar when restricted to tumors with Ki-67 data. Luminal A tumors were ER-positive and/or PR-positive, HER2negative, and Ki-67-negative (or histologic grade 1); luminal B tumors were either i) ERpositive and/or PR-positive and HER2-positive or ii) ER-positive and/or PR-positive, HER2negative, and Ki-67-positive (or histologic grade 2/3); HER2-enriched tumors were ERnegative, PR-negative and HER2-positive; basal-like tumors were ER-negative, PR-negative, HER2-negative and CK 5/6-positive and/or EGFR-positive; unclassified tumors were ERnegative, PR-negative, HER2-negative, CK 5/6-negative and EGFR-negative. Tumors missing complete data on markers necessary to classify them into one of the five subtypes (N=368) were excluded from analysis. Statistical analyses
Author Manuscript
Information on reproductive factors and other potential covariates was obtained from baseline and subsequent biennial questionnaires. No strong evidence of between-cohort heterogeneity in associations with reproductive factors was observed; therefore data from NHS and NHSII was pooled for all analyses. Multivariate-adjusted Cox proportional hazards models were used to calculate hazard ratios (HR) and 95% confidence intervals (CI) for each subtype and across all subtypes combined. Women contributed person-time from baseline questionnaire return until the first date of diagnosis of breast or other cancer (excluding nonmelanoma skin cancer), death, or June 1, 2006. Models were stratified by cohort, age and calendar time, and updated through follow-up as available. Models included mutual adjustment for reproductive variables of interest, and the following known breast cancer risk factors: body mass index (BMI) at age 18, weight change since age 18, height, personal history of benign breast disease, family history of breast cancer, total physical activity (MET-hr/wk) and alcohol intake. We excluded women who were missing age at menarche, and person-time where parity and age at first birth were unknown. These criteria resulted in the exclusion of an additional 365 cases from the analyses. We included women with missing data on breastfeeding, age at menopause, or menopausal status by coding these exposures as missing; results were similar when restricted to women with complete data on these variables.
Author Manuscript
In secondary models, we examined the timing of reproductive events, and their interactions. These Cox models used calendar time as the metameter, and included terms for duration of pre- and postmenopause. We included a term for years between menarche and first birth to represent the one-time increase in breast cancer risk following a first pregnancy;25(add Rosner et al., 1994) nulliparous women were assigned a value of zero. To capture the effect of timing and spacing of births on risk, we derived a birth index term, defined as:
Int J Cancer. Author manuscript; available in PMC 2017 May 15.
Sisti et al.
Page 5
Author Manuscript
where t* = min(current age, age at menopause); bit= 1 if parity ≥ i at age t, and 0 if nulliparous; and ti = age at ith birth,26 such that a higher birth index represents a higher number of births occurring at earlier ages. For example, a 50-year old woman with four births occurring at ages 20, 23, 26 and 29 would be assigned a value of 102, while a 50-year old woman with a single birth at age 35 would be assigned a value of 15. These models additionally excluded women with unknown menopausal status and incomplete reproductive histories. We examined whether the associations of menarche to first birth and birth index with risk varied by lactation status. To evaluate whether associations differed by molecular subtype, we conducted competing risks analyses using the approach described by Lunn and McNeill.27 P-values were twosided and tests of significance were performed at the α=0.05 level. Analyses were conducted using SAS v. 9.3 (SAS Institute, Cary, NC).
Author Manuscript
RESULTS
Author Manuscript
Over follow-up, we identified 2,063 luminal A, 1,008 luminal B, 209 HER2-enriched, 378 basal-like and 110 unclassified tumors (Table 1). The frequency of subtypes appeared to differ somewhat across the 2,935 NHS and 832 NHSII tumors included in our analysis. Compared to tumors in the NHSII, we observed that NHS tumors included a higher proportion of luminal A (55.5% v. 52.3%), HER2-type (6.0% v. 4.1%), and unclassified subtypes (3.4% vs. 1.1%) and a lower proportion of luminal B (25.8% v. 29.9%) and basallike subtypes (9.3% v. 12.6%). As in our previous study,18 basal-like tumors were diagnosed at the youngest mean age (54.1y) and were most frequently poorly differentiated (72.3%), while unclassified tumors were most likely to be metastatic at diagnosis (15.2%) (Table 1). Luminal A tumors were most likely to be small (0.1–1.0 cm: 26.9%) and have no nodal involvement at diagnosis (65.9%), while HER2-enriched tumors were most frequently stage III or IV (28.7%).
Author Manuscript
Associations with some reproductive factors varied by breast cancer subtype (Table 2). For example, age at menopause was positively associated with risk of luminal A, luminal B and unclassified tumors, and suggestively associated with HER2-enriched tumors (per 1-year increase HRs=1.04–1.12), though no association with basal-like was observed (HR=1.01, Pheterogeneity=0.05). The inverse association with age at menarche was not heterogeneous across subtypes (>14 vs.
Int J Cancer. Author manuscript; available in PMC 2017 May 15. Published in final edited form as: Int J Cancer. 2016 May 15; 138(10): 2346–2356. doi:10.1002/ijc.29968.
Reproductive risk factors in relation to molecular subtypes of breast cancer: results from the Nurses' Health Studies Julia S. Sisti1,2,3, Laura C. Collins4, Andrew H. Beck4, Rulla M. Tamimi1,2, Bernard A. Rosner2, and A. Heather Eliassen1,2 1Department
of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA
Author Manuscript
2Channing
Division of Network Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA
3Department
of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New
York, NY 4Department
of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School
Abstract
Author Manuscript Author Manuscript
Several intrinsic breast cancer subtypes, possibly representing unique etiologic processes, have been identified by gene expression profiles. Evidence suggests that associations with reproductive risk factors may vary by breast cancer subtype. In the Nurses' Health Studies, we prospectively examined associations of reproductive factors with breast cancer subtypes defined using immunohistochemical staining of tissue microarrays. Multivariate-adjusted Cox proportional hazard models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). Over follow-up, we identified 2,063 luminal A, 1,008 luminal B, 209 HER2-enriched, 378 basallike and 110 unclassified tumors. Many factors appeared associated with luminal A tumors, including ages at menarche (Pheterogeneity=0.65) and menopause (Pheterogeneity=0.05), and current HT use (Pheterogeneity=0.33). Increasing parity was not associated with any subtype (Pheterogeneity=0.76), though age at first birth was associated with luminal A tumors only (per 1year increase HR=1.03 95%CI (1.02, 1.05), Pheterogeneity=0.04). Though heterogeneity was not observed, duration of lactation was inversely associated with risk of basal-like tumors only (7+ months vs. never HR=0.65 95%CI (0.49, 0.87), Ptrend=0.02), Pheterogeneity=0.27). Years between menarche and first birth was strongly positively associated with luminal A and non-luminal subtypes (e.g. 22-year interval vs. nulliparous HR=1.80, 95%CI (1.08, 3.00) for basal-like tumors; Pheterogeneity=0.003), and evidence of effect modification by breastfeeding was observed. In summary, many reproductive risk factors for breast cancer appeared most strongly associated with the luminal A subtype. Our results support previous reports that lactation is protective against basal-like tumors, representing a potential modifiable risk factor for this aggressive subtype.
Author to whom correspondence and/or reprint requests should be addressed: A. Heather Eliassen, Sc.D, Department of Epidemiology, Channing Laboratory, 181 Longwood Avenue, 3rd Floor, Boston, MA 02115, [email protected]. Competing interests: The authors declare that they have no competing interests.
Sisti et al.
Page 2
Author Manuscript
Keywords breast cancer; molecular subtypes; reproductive risk factors; pregnancy; lactation
INTRODUCTION
Author Manuscript
Genomic analysis suggests breast cancer heterogeneity is more complex than previous categorizations based on estrogen and progesterone receptor (ER/PR) status. Currently, at least 4 distinct molecular subtypes have been identified1–3 and can be reliably classified by immunohistochemistry of ER, PR and other markers.4–8 Tumors that express hormone receptors (HR+) are classified as either luminal A or luminal B, with the latter characterized by higher expression of the proliferative marker Ki-67. HER2-enriched tumors do not express HR, but overexpress human epidermal growth factor receptor 2 (HER2). Among tumors lacking expression of all three markers (triple-negative cancers), basal-like tumors express the basal markers cytokeratins 5/6 (CK 5/6) and/or endothelial growth factor (EGFR), while tumors that express none of these markers are termed unclassified. While these subtypes vary in prognosis and response to treatment9,10 they also may reflect distinct etiologic pathways given that subtype appears to be fixed from the time of tumor initiation.11 Because luminal A tumors comprise about 60% of breast tumors, many known risk factors are likely to be associated with this phenotype; however, less is known about risk factors for HR-negative (HR−) subtypes.
Author Manuscript Author Manuscript
Several reproductive risk factors are associated with breast cancer risk, possibly mediated by changes in sex hormones. Evidence suggests these risk factors may be more strongly associated with HR+ subtypes.12–14 While some reproductive factors are associated with HR− subtypes, these relationships do not always mirror those observed with HR+ disease. As recently reviewed,15 parity has been associated with decreased risk of ER+/PR+ breast cancers, while null or positive associations between parity and ER−/PR− tumors have been reported in some studies. Conversely, while the association between breastfeeding and ER+/PR+ tumors has been inconsistent, inverse associations have been observed with ER−/PR− and triple-negative tumors, suggesting lactation may mitigate the increased risk of these subtypes conferred by parity.15–17 Few studies have examined reproductive risk factors in relation to the fully characterized intrinsic breast cancer subtypes, including our prior analysis in the Nurses' Health Study (NHS) with 2,022 cases.18 Results suggest parity may protect against luminal subtypes, while significant18,19 or suggestive20 positive associations were observed for basal-like tumors. Conversely, breastfeeding was inversely associated with basal-like tumors.18,19 Associations with other risk factors were less consistent across studies. With the exception of the AMBER study of breast cancer in African American women, which included 678 triple-negative cases, each analysis to date included relatively few non-luminal cases, and immunohistological criteria used to define subtypes differed somewhat across studies. We examined associations of reproductive risk factors with molecular subtypes of breast cancer defined by comprehensive immunohistochemical profiling in the NHS and NHSII.
Int J Cancer. Author manuscript; available in PMC 2017 May 15.
Sisti et al.
Page 3
Author Manuscript
We added to our previous NHS analysis with an additional 8 years of follow-up, and included NHSII, for a total of nearly 4,000 cases with defined molecular subtypes.
METHODS Study population
Author Manuscript
The Nurses' Health Studies are ongoing prospective cohorts of registered nurses in the United States. The NHS was established in 1976 among 121,700 women ages 30–55 years; the NHSII was established in 1989 among 116,430 women ages 25–42 years. Participants have reported reproductive history, lifestyle factors and disease diagnoses at baseline and biennial questionnaires. Including surveillance for deaths, follow-up rates in NHS and NHSII were over 95% through the 2006 and 2003 questionnaire cycles, respectively. This study was approved by the Institutional Review Board of Brigham and Women's Hospital in Boston, MA. Outcome assessment Incident breast cancer diagnoses were reported on biennial questionnaires or identified through death records. Permission to access medical records for breast cancer cases was sought from participants or next of kin; requests were made to pathology departments from treating hospitals to obtain formalin-fixed paraffin-embedded (FFPE) tissue samples. Detailed description of block collection has been published previously21,22. Tissue blocks were obtained from approximately 70% of confirmed NHS cases and approximately 60% of NHSII cases reported through 2006. Tissue microarrays and subtype classification
Author Manuscript
Tissue microarrays (TMAs) that included tumors from 4,308 NHS participants and 1,253 NHSII participants were constructed at the Dana Farber/Harvard Cancer Center Tissue Microarray Core Facility in Boston, MA, as previously detailed elsewhere.22 Briefly, three 0.6mm diameter cores were obtained from each participant tumor tissue sample, and inserted into TMA blocks that were subsequently cut into 5um paraffin sections prior to immunohistochemical staining. TMA sections were stained for a panel of immunohistochemical markers (ER, PR, HER2, CK 5/6, EGFR) to classify molecular subtypes.4–6,8,23,24 A subset of NHS cases (N=3,281, 76%) was additionally stained for the proliferative marker Ki-67; no NHSII cases had Ki-67 data. Only invasive tumors (N=4,501), as confirmed by study pathologists, were eligible for inclusion in this analysis. Tissue microarrays and subtype classification
Author Manuscript
Positivity for each immunohistochemical marker was determined visually by a pathologist (LC) who reviewed each core under a microscope and assigned a score. The exception was Ki-67, which was evaluated automatically using an automated computational image analysis system (Definiens Tissue Studio software, Munich, Germany). Positivity for ER and PR was defined as any nuclear staining of these markers (≥1%), while HER2 over-expression was defined as strong membrane staining in >10% of cells in a given core. Cores were considered positive for CK 5/6 and EGFR if any cytoplasmic and/or membranous staining was present in any of a tumor's three cores. Lastly, we estimated the mean percentage of Int J Cancer. Author manuscript; available in PMC 2017 May 15.
Sisti et al.
Page 4
Author Manuscript
cells displaying nuclear staining Ki-67 by weighting each core by its total cell count; we considered cases positive if >14% of cells were positive for Ki-67.5
Author Manuscript
We classified tumors by molecular subtype using definitions that correlate with gene expression profile classifications.4–8,23 For tumors missing Ki-67 expression data, we used histologic grade as a surrogate; NHS results for luminal tumors were similar when restricted to tumors with Ki-67 data. Luminal A tumors were ER-positive and/or PR-positive, HER2negative, and Ki-67-negative (or histologic grade 1); luminal B tumors were either i) ERpositive and/or PR-positive and HER2-positive or ii) ER-positive and/or PR-positive, HER2negative, and Ki-67-positive (or histologic grade 2/3); HER2-enriched tumors were ERnegative, PR-negative and HER2-positive; basal-like tumors were ER-negative, PR-negative, HER2-negative and CK 5/6-positive and/or EGFR-positive; unclassified tumors were ERnegative, PR-negative, HER2-negative, CK 5/6-negative and EGFR-negative. Tumors missing complete data on markers necessary to classify them into one of the five subtypes (N=368) were excluded from analysis. Statistical analyses
Author Manuscript
Information on reproductive factors and other potential covariates was obtained from baseline and subsequent biennial questionnaires. No strong evidence of between-cohort heterogeneity in associations with reproductive factors was observed; therefore data from NHS and NHSII was pooled for all analyses. Multivariate-adjusted Cox proportional hazards models were used to calculate hazard ratios (HR) and 95% confidence intervals (CI) for each subtype and across all subtypes combined. Women contributed person-time from baseline questionnaire return until the first date of diagnosis of breast or other cancer (excluding nonmelanoma skin cancer), death, or June 1, 2006. Models were stratified by cohort, age and calendar time, and updated through follow-up as available. Models included mutual adjustment for reproductive variables of interest, and the following known breast cancer risk factors: body mass index (BMI) at age 18, weight change since age 18, height, personal history of benign breast disease, family history of breast cancer, total physical activity (MET-hr/wk) and alcohol intake. We excluded women who were missing age at menarche, and person-time where parity and age at first birth were unknown. These criteria resulted in the exclusion of an additional 365 cases from the analyses. We included women with missing data on breastfeeding, age at menopause, or menopausal status by coding these exposures as missing; results were similar when restricted to women with complete data on these variables.
Author Manuscript
In secondary models, we examined the timing of reproductive events, and their interactions. These Cox models used calendar time as the metameter, and included terms for duration of pre- and postmenopause. We included a term for years between menarche and first birth to represent the one-time increase in breast cancer risk following a first pregnancy;25(add Rosner et al., 1994) nulliparous women were assigned a value of zero. To capture the effect of timing and spacing of births on risk, we derived a birth index term, defined as:
Int J Cancer. Author manuscript; available in PMC 2017 May 15.
Sisti et al.
Page 5
Author Manuscript
where t* = min(current age, age at menopause); bit= 1 if parity ≥ i at age t, and 0 if nulliparous; and ti = age at ith birth,26 such that a higher birth index represents a higher number of births occurring at earlier ages. For example, a 50-year old woman with four births occurring at ages 20, 23, 26 and 29 would be assigned a value of 102, while a 50-year old woman with a single birth at age 35 would be assigned a value of 15. These models additionally excluded women with unknown menopausal status and incomplete reproductive histories. We examined whether the associations of menarche to first birth and birth index with risk varied by lactation status. To evaluate whether associations differed by molecular subtype, we conducted competing risks analyses using the approach described by Lunn and McNeill.27 P-values were twosided and tests of significance were performed at the α=0.05 level. Analyses were conducted using SAS v. 9.3 (SAS Institute, Cary, NC).
Author Manuscript
RESULTS
Author Manuscript
Over follow-up, we identified 2,063 luminal A, 1,008 luminal B, 209 HER2-enriched, 378 basal-like and 110 unclassified tumors (Table 1). The frequency of subtypes appeared to differ somewhat across the 2,935 NHS and 832 NHSII tumors included in our analysis. Compared to tumors in the NHSII, we observed that NHS tumors included a higher proportion of luminal A (55.5% v. 52.3%), HER2-type (6.0% v. 4.1%), and unclassified subtypes (3.4% vs. 1.1%) and a lower proportion of luminal B (25.8% v. 29.9%) and basallike subtypes (9.3% v. 12.6%). As in our previous study,18 basal-like tumors were diagnosed at the youngest mean age (54.1y) and were most frequently poorly differentiated (72.3%), while unclassified tumors were most likely to be metastatic at diagnosis (15.2%) (Table 1). Luminal A tumors were most likely to be small (0.1–1.0 cm: 26.9%) and have no nodal involvement at diagnosis (65.9%), while HER2-enriched tumors were most frequently stage III or IV (28.7%).
Author Manuscript
Associations with some reproductive factors varied by breast cancer subtype (Table 2). For example, age at menopause was positively associated with risk of luminal A, luminal B and unclassified tumors, and suggestively associated with HER2-enriched tumors (per 1-year increase HRs=1.04–1.12), though no association with basal-like was observed (HR=1.01, Pheterogeneity=0.05). The inverse association with age at menarche was not heterogeneous across subtypes (>14 vs.
