Molecular and clinical characterization of postpartum-associated breast cancer in the Carolina Breast Cancer Study Phase I-III, 1993–2013

Abstract

Background:

Breast cancers in recently postpartum women may have worse outcomes, but studies examining tumor molecular features by pregnancy recency have shown conflicting results.

Methods:

This analysis used Carolina Breast Cancer Study data to examine clinical and molecular tumor features among women <50 years of age who were recently (≤10 years prior), or remotely (>10 years prior) postpartum, or nulliparous. Prevalence odds ratios (PORs) and 95% confidence intervals (CIs) were estimated using multivariable models.

Results:

Recently postpartum women (N=618) were more frequently lymph node positive [POR (95% CI): 1.66 (1.26, 2.19)], ER negative [1.37 (1.02, 1.83)], and IHC-based triple negative [1.57 (1.00, 2.47)] compared to nulliparous (N=360) women. Some differences were identified between recent vs. remotely postpartum; smaller tumor size [0.67 (0.52, 0.86)], p53 wildtype [0.53 (0.36, 0.77)], and non-basal-like phenotype [0.53 (0.33, 0.84)] were more common among recently postpartum. Recently postpartum (vs. nulliparous) had significant enrichment for adaptive immunity, T cells, B cells, CD8 T cells, activated CD8 T cells/NK cells, Tfh cells and higher overall immune cell scores. These differences were attenuated in remotely (compared to recently) postpartum women.

Conclusions:

These results suggest a dominant effect of parity (vs. nulliparity) and a lesser effect of pregnancy recency on tumor molecular features, although tumor immune microenvironments were altered in association with pregnancy recency.

Impact:

Our study is unique in examining tumor immune microenvironment and RNA-based markers according to time since last childbirth. Future studies should examine the interplay between tumor features, post-diagnostic treatment and outcomes among recently postpartum women.

Introduction

Breast cancer is the most commonly diagnosed cancer among women aged 20–49 years [1] and the cancer most frequently temporally-related to pregnancy [2]. Several studies have found that breast cancer diagnosed during pregnancy or the postpartum years is associated with worse overall and disease-free survival, and plausible mechanisms have been proposed [3–9]. Elevated concentrations of estrogen, progesterone and prolactin during pregnancy may promote the growth and progression of breast tumors [10–12]. Postpartum involution, a breast remodeling process that generates a wound-healing/inflammatory microenvironment [13–16], promotes tumor growth in animal studies [17, 18]. However, despite the strength of the preclinical and mechanistic data, epidemiologic and clinical studies of breast tumors diagnosed during pregnancy or the postpartum years have reported incongruent results with respect to molecular characteristics [3–8, 19–24].

Inconsistencies in the molecular phenotypes of postpartum-associated breast cancer (PPBC) may be in part due to small sample size, varying definitions that combine breast cancers diagnosed during pregnancy (PrBC) with PPBC [25], and differences in the menopausal status of the study participants. Use of different reference groups has also complicated comparisons across studies. For instance, previous studies have either used only nulliparous women as the reference group [4, 21–24] or combined nulliparous women and parous women diagnosed with breast cancer remotely (after a given number of postpartum years) in one reference group [5–8, 19, 20]. To identify the effects of recent pregnancy, an ideal referent group would restrict to premenopausal women and account for age differences. Furthermore, adequate sample size, clear definitions of recently postpartum women (i.e., ≤10 years prior to diagnosis) and distinct comparison groups, including remotely postpartum (>10 years prior to diagnosis) and nulliparous women, would improve inferences and comparisons across studies.

We examined the distribution of a wide array of biological features including ER, PR, HER2, p53, IHC- and RNA-based subtypes, risk of recurrence multigene (ROR-P) scores, and multi-gene immune scores among premenopausal women <50 years of age according to parity and recency of pregnancy using data from a large population-based study. Based on prior literature, we used 10 years as the cutoff for our recently postpartum group [3, 25, 26], and we conducted sensitivity analyses and explored smaller windows of time since last childbirth.

Materials and Methods

Study population

The CBCS is a population-based study of breast cancer in North Carolina (phase I: 1993–1996; phase II: 1996–2001; and phase III: 2008–2013) (N=4806); details of the study have been described previously [27, 28] Briefly, the primary study enrolled women age 20–74 years diagnosed with first primary invasive breast cancer using rapid case ascertainment, with randomized recruitment to oversample Black and younger (age < 50 years) women [29]. The current analysis examined tumor characteristics of premenopausal women under 50 years of age, who contributed tumor tissue (N=2081) (Supplemental Figure 1). Written informed consent was obtained from each participant prior to data collection, and the study was conducted in accordance with U.S. Common Rule. The study was approved by the Office of Human Research Ethics/Institutional Review Board at the University of North Carolina at Chapel Hill.

In-person interviews were conducted by trained nurses to collect medical history including detailed information on pregnancy history. Date of breast cancer diagnosis was collected using medical record abstraction. Time since last full-term birth (≥7 months of pregnancy or live birth) was calculated by subtracting date of last full-term birth from date of breast cancer diagnosis. Cases with missing date of last full-term birth were excluded (N=2). Breast cancers diagnosed during pregnancy were excluded (N=15). Women diagnosed within 10 years of their last childbirth were included in the “recent” group (N=618). Women diagnosed more than 10 years after their last childbirth were assigned to the “remote” group (N=1086). Women who never had a full-term birth prior to their diagnosis were assigned to the “nulliparous” group (N=360).

Clinical tumor markers and IHC-based subtyping

Tumor size, grade, node status, and stage were abstracted from medical records and pathology reports. The multiple imputation plus outcome approach was used to impute missing tumor grade values for CBCS2 participants (n=578) [30]. Estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) status were abstracted from medical records or determined from formalin-fixed paraffin-embedded (FFPE) tumor tissue blocks, which were previously sectioned and stained for a panel of immunohistochemical (IHC) markers at the Translational Pathology Laboratory at the University of North Carolina, Chapel Hill; details have been described previously [31]. Briefly, ER and PR were considered positive if the percentage of positive cells was ≥10%, based on previous work showing that 10% positivity was most closely aligned with RNA-based subtypes [31], and HER2 positivity was defined as IHC 3+ [32]. Details of IHC-based breast cancer subtyping have been published previously [27, 33]. Briefly, IHC-based subtype was defined as luminal A-like (ER+ or PR+ and HER2−), luminal B-like (ER+ or PR+ and HER2+), HER2-type (ER− and PR− and HER2+), or triple-negative (ER− and PR− and HER2−).

Tumor-infiltrating lymphocytes (TILs) scores were calculated by adapting a digital algorithm similar to that previously described by Sandhu et al. [34] and optimized based on standardized international guidelines on TIL-assessment in breast cancer by the International TILs Working Group [35]. Briefly, composition algorithms were trained on several annotated representative hematoxylin and eosin (H&E)-stained tumor slides for each tissue feature (immune, epithelial, stromal, adipose) with accuracy optimized to match visual annotation by a trained pathologist’s assistant. Percentage area and nuclei per unit area (nuclear density) were calculated for each region.

Genomic assessment

Details of RNA isolation and quantification have been described previously [36]. Briefly, RNA was isolated and quantified using Qiagen FFPE RNeasy kit (Germantown, MD, USA) and NanoString assay, respectively [37]. Data that passed the NanoString nSolver software (Seattle, WA, USA) quality control were normalized using methods previously described by Bhattacharya and Hamilton et al. [38], including reference gene normalization, upper-quartile normalization [39], and Remove Unwanted Variation (RUV) using the RUVg function from the RUVSeq Bioconductor package [40]. Only FFPE tumor blocks with adequate tumor cellularity by manual review were included in RNA analyses. Relative to participants without RNA data, those with RNA data had larger tumors, higher tumor stage, and higher grade and were more likely to be lymph node positive, but age at diagnosis or time since last childbirth were not significantly different.

P53 status was determined using a previously validated TP53-dependent signature [41]. Tumors were classified as p53 mutant-like vs wild-type-like based on a similarity-to-centroid approach (Pearson coefficient) using distance-weighted discrimination [42]. RNA-based intrinsic subtype was determined using the PAM50 gene signature to classify tumors as luminal A, luminal B, HER2-enriched, basal-like and normal-like, as previously described [36]. Normal-like samples were excluded because this subtype is believed to arise due to insufficient tumor cellularity. The PAM50 predictor was used to calculate the risk of recurrence (ROR) score, which incorporates subtype and additional weighting by the proliferation signature (ROR-P) [36]. To identify the most aggressive tumors, ROR-P was categorized as low/medium vs. high.

We curated a 48-gene panel of immune markers that is representative of at least 10 individual immune cell types based on work from Bindea [43] and Danaher et al. [44]. As described in Hamilton et al. (in preparation), cell type scores for 10 immune cell types, including B Cells, T cells, T helper cells, T regulatory cells (Treg), T follicular helper cells (Tfh), CD8 T cells, Neutrophils, Eosinophils, Natural Killer (NK) cells, Macrophages, and the immune checkpoint marker, PDL1, were calculated by computing the median of the log2-transformed expression from each cell’s marker genes. For CD8 T cells specifically, we elected to include genes that were both specific and sensitive. These less-specific markers, such as GZMM and PRF1, have been shown to be expressed on both NK cells and CD8 T cells; thus, we refer to these cytotoxic cells as CD8 T cells/NK cells in downstream analysis. In addition to cell type scores, overall adaptive and innate immunity scores were calculated, where the median log2-transformed expression for all B cell, T cell, CD8 T cell, T helper cell, Treg and Tfh markers were computed for the adaptive scores, and the median of all eosinophil, neutrophil, NK cell and macrophage markers were calculated for the innate scores.

Covariate assessment

Race was determined by self-report and categorized as non-Black or Black. Less than 2% of non-Black participants self-identified as multiracial, Hispanic, or other race/ethnicities. Age at diagnosis was obtained from the baseline survey and used as a continuous variable in models. Age at last full-term pregnancy was obtained from baseline survey and categorized as <30 years and ≥ 30years. Information on parity was obtained from baseline survey and categories as nulliparous, or 1, 2 and ≥ 3 full-term births.

Statistical analyses

Descriptive statistics for patient sociodemographic and tumor characteristics of recently postpartum women were compared with distally postpartum and nulliparous women using chi-square tests (Fisher’s exact test when cell count <5). Binary logistic regression models were used to compute prevalence odds ratios (PORs) and 95% confidence intervals (95% CIs) as the measure of association between tumor characteristics and recency of pregnancy. The following tumor characteristics were studied in association with recency of pregnancy: tumor grade (low-intermediate, high), stage of disease (I/II, III/IV), lymph node status (positive, negative), tumor size (≤ 2 cm, > 2 cm), ER (negative, positive), PR status (negative, positive), HER2 status (negative, positive), IHC-based subtype (Luminal A, Luminal B, HER2-type, triple negative), p53 status (wild-type, mutant), PAM50 intrinsic breast cancer subtype (Luminal A, Luminal B, HER2-enriched, basal-like), and ROR-P score (low, medium, high). Models were adjusted for study phase (I, II, III), age at diagnosis (continuous), and race (Black, non-Black). Sensitivity analyses included using smaller intervals of time since last full-term birth (≤ 2, >2–5, >5–10 years postpartum) and stratifying by age at last full-term pregnancy (< 30 years, ≥ 30 years). We performed multiple sensitivity analyses, including adjusting for Body Mass Index (BMI) at diagnosis (<25, 25-<30, ≥30), parity (1, 2, ≥3), lactation history (ever, never), and oral contraceptive use (never, current, former). All analyses were conducted in SAS version 9.4 (SAS Institute, Cary, NC). P-values were two-sided with an alpha of 0.05 for statistical significance.

Data Availability

For participant confidentiality and due to ethical restrictions, data are available upon request and are subject to data use agreements and other stipulations. Permission to access data from the Carolina Breast Cancer Study may be obtained online (https://unclineberger.org/cbcs/) or by contacting the authors.

Results

Sociodemographic characteristics

Among premenopausal breast cancer cases <50 years at diagnosis in the Carolina Breast Cancer Study (Phase I-III, 1993–2013), Table 1 presents the distribution of age at diagnosis, age at last full-term pregnancy, race, parity, and study phase for recently postpartum (diagnosed ≤10 years postpartum), remotely postpartum (diagnosed >10 years postpartum), and nulliparous women diagnosed with breast cancer. Recently postpartum women had a significantly younger age at diagnosis (median = 39 years) compared to remotely postpartum (median = 45 years) and nulliparous (median = 42 years). Compared to remotely postpartum, recently postpartum also had an older age at last full-term pregnancy (median = 33 years vs. 26 years) and a higher frequency of Black race. There was no significant difference in multiparity or study phase between groups.

Table 1:

Distribution of select characteristics by recency of pregnancy among premenopausal women <50 years of age in the Carolina Breast Cancer Study, 1999–2013 (N=2064) ^a

	Nulliparous N = 360	Remote N = 1086	Recent N = 618	p-value
Age at diagnosis (years)				<0.0001
23–30	23 (6.4)	2 (0.2)	38 (6.2)
30–39	104 (28.9)	135 (12.4)	315 (51.0)
40–49	233 (64.7)	949 (87.4)	265 (42.9)
Median (Range)	42 (23, 49)	45 (29, 49)	39 (23, 49)
Age at last full-term pregnancy (years)				<0.0001
13–30	-	755 (69.5)	153 (24.8)
30–39	-	331 (30.5)	429 (69.4)
40–46	-	0 (0.0)	36 (5.8)
Median (Range)	-	26 (13, 38)	33 (17, 46)
Race				0.003
Black	145 (40.3)	535 (49.3)	267 (43.2)
Non-Black	215 (59.7)	551 (50.7)	351 (56.8)
Parity				0.41
1	-	287 (26.4)	157 (25.4)
2	-	469 (43.2)	254 (41.1)
3–8	-	330 (30.4)	207 (33.5)
Study phase				0.40
1	75 (20.8)	234 (21.6)	115 (18.6)
2	66 (18.3)	221 (20.4)	115 (18.6)
3	219 (60.8)	631 (58.1)	388 (62.8)

Definitions: Nulliparous, no full-term birth prior to diagnosis; Remote, diagnosed >10 years postpartum; Recent, diagnosed ≤10 years postpartum. P-values generated by chi-square test, except when expected cell count <5, they were calculated by Fisher's exact test. Missing values were excluded from percentage calculations. Percentages may not add up to 100 due to rounding.

Clinical and IHC-based tumor features

Although differences for several factors were observed when comparing recently postpartum to nulliparous women, there were few differences between recently and distally postpartum women (Table 2). Compared to nulliparous women, tumors among recently postpartum were more likely to be lymph node positive, ER/PR negative and IHC-based triple negative subtype, after adjusting for age at diagnosis, race, and study phase. Although not statistically significant, recently postpartum women tended to be diagnosed at a later stage and were more frequently IHC-based HER2-type subtype compared to nulliparous women. Sensitivity analysis considering smaller windows of time since last childbirth showed strongest associations with respect to lymph node positivity and later stage for those 0–2 years and 2.1–5 years postpartum vs. nulliparous women (Supplemental Tables 1–2). No substantial difference in grade, size, and HER2 status were observed between the recent and nulliparous groups.

Table 2:

Association between recent pregnancy (<10 years) and clinical and IHC-based tumor characteristics among premenopausal women <50 years of age in the Carolina Breast Cancer Study, 1999–2013 (N=2064)

	Nulliparous N = 360		Remote N = 1086		Recent N = 618		Adjusted POR (95% CI)a
	n	% b	n	% b	n	% b	Referent = Remote	Referent = Nulliparous
Grade
Low-Intermediate	193	53.6	561	51.7	290	46.9	Ref.	Ref.
High	167	46.4	525	48.3	328	53.1	0.94 (0.73, 1.20)	1.13 (0.86, 1.48)
Missing	0	-	0	-	0	-
Stage
I-II	297	85.3	859	81.4	490	81.3	Ref.	Ref.
III-IV	51	14.7	196	18.6	113	18.7	0.88 (0.64, 1.21)	1.28 (0.88, 1.85)
Missing	12	-	31	-	15	-
Node
Negative	228	64.4	613	57.1	311	50.9	Ref.	Ref.
Positive	126	35.6	460	42.9	300	49.1	1.13 (0.89, 1.45)	1.66 (1.26, 2.19)
Missing	6	-	13	-	7	-
Size
≤ 2cm	155	45.1	466	44.4	281	46.8	Ref.	Ref.
> 2cm	189	54.9	584	55.6	320	53.2	0.67 (0.52, 0.86)	0.81 (0.61, 1.07)
Missing	16	-	36	-	17	-
ER Status
Positive	231	67.0	620	59.3	352	58.6	Ref.	Ref.
Negative	114	33.0	426	40.7	249	41.4	0.95 (0.73, 1.23)	1.37 (1.02, 1.83)
Missing	15	-	40	-	17	-
PR Status
Positive	216	62.6	568	54.5	299	50.3	Ref.	Ref.
Negative	129	37.4	474	45.5	296	49.8	0.98 (0.76, 1.26)	1.48 (1.11, 1.97)
Missing	15	-	44	-	23	-
HER2 Status
Negative	253	76.9	820	83.1	449	79.2	Ref.	Ref.
Positive	76	23.1	167	16.9	118	20.8	1.20 (0.87, 1.66)	0.87 (0.62, 1.21)
Missing	31	-	99	-	51	-
IHC-based Subtype
Luminal A	114	45.6	319	41.4	160	36.2	Ref.	Ref.
Luminal B	80	32.0	177	23.0	122	27.6	1.06 (0.73, 1.54)	0.98 (0.66, 1.46)
HER2-type	14	5.6	58	7.5	41	9.3	1.16 (0.67, 2.01)	1.86 (0.95, 3.64)
Triple negative	42	16.8	216	28.1	119	26.9	0.75 (0.51, 1.08)	1.57 (1.00, 2.47)
Missing	110	-	316	-	176	-

Definitions: Nulliparous, no full-term birth prior to diagnosis; Remote, diagnosed >10 years postpartum; Recent, diagnosed ≤10 years postpartum.

Abbreviations: IHC, Immunohistochemistry; POR, prevalence odds ratio; CI, confidence interval

^aAdjusted for age at diagnosis, race and study phase

^bPercentages may not add up to 100 due to rounding

Compared to remotely postpartum women, recently postpartum women did not have more aggressive clinical and molecular tumor characteristics (Table 2). After adjusting for age at diagnosis, race, and study phase, no significant differences in tumor grade, stage, lymph node status, and ER/PR/HER2 status were observed between recent and remote groups and recently postpartum women had significantly smaller tumors compared to remotely postpartum women.

RNA-based tumor characteristics

No significant differences in RNA-based intrinsic subtypes, TP53 status, and ROR-P scores were observed between the recent and nulliparous groups (Table 3). However, compared to remotely postpartum, recently postpartum women had significantly more frequently wildtype TP53 status, and more frequent non-basal-like subtype. No substantial or significant differences in ROR-P scores were observed between the recent and remote groups. Sensitivity analyses conducted with time since pregnancy as a continuous variable showed similar results. Adjusting for BMI at diagnosis, parity, lactation history, and oral contraceptive use did not substantially alter POR estimates. Sensitivity analysis stratifying on age at last full-term pregnancy or including smaller time intervals of recency did not alter the relationships between parity, recency and RNA-based features (Supplemental Tables 3–4). Similarly, stratifying on prior parity status or restricting to only CBCS phase 3 participants did not change these results.

Table 3:

Association between recent pregnancy (<10 years) and RNA-based tumor characteristics among premenopausal women <50 years of age in the Carolina Breast Cancer Study, 1999–2013 (N=966)

	Nulliparous N = 154		Remote N = 520		Recent N = 289		Adjusted POR (95% CI) a
	n	% b	n	% b	n	% b	Referent = Remote	Referent = Nulliparous
RNA-based P53 Status
Wildtype	74	48.0	243	46.7	137	47.4	Ref.	Ref.
Mutant-like	80	52.0	277	53.3	152	52.6	0.53 (0.36, 0.77)	0.80 (0.52, 1.24)
RNA-based Subtype (PAM50)
Luminal A	69	45.7	203	40.6	108	38.7	Ref.	Ref.
Luminal B	25	16.6	76	15.2	46	16.5	0.67 (0.39, 1.16)	1.00 (0.54, 1.84)
HER2-enriched	16	10.6	49	9.8	35	12.5	0.64 (0.34, 1.23)	1.14 (0.56, 2.34)
Basal-like	41	27.2	172	34.4	90	32.3	0.53 (0.33, 0.84)	1.04 (0.61, 1.79)
Unknown	3	-	20	-	10	-
ROR-P
Low	33	21.4	135	26.0	64	22.2	Ref.	Ref.
Medium	71	46.1	231	44.4	124	42.9	0.85 (0.54, 1.34)	0.89 (0.51, 1.54)
High	50	32.5	154	29.6	101	35.0	0.67 (0.39, 1.14)	0.74 (0.40, 1.38)

Definitions: Nulliparous, no full-term birth prior to diagnosis; Remote, diagnosed >10 years postpartum; Recent, diagnosed ≤10 years postpartum.

Abbreviations: RNA, ribonucleic acid; POR, prevalence odds ratio; CI, confidence interval

^aAdjusted for age at diagnosis, race and study phase

^bPercentages may not add up to 100 due to rounding

Tumor microenvironment and immune markers

Despite few changes in epithelial tumor features, we observed an enrichment for adaptive immune cells and higher immune cell composition among recently postpartum women compared to nulliparous women (Figure 1A). After adjusting for intrinsic subtype, race, age, and study phase, we saw an enrichment for overall adaptive immunity and adaptive immune cells, such as T cells, B cells, CD8 T cells, activated CD8 T cells/ Natural Killer (NK) cells, and T follicular helper cells (Tfh) in recent vs. nulliparous women (Figure 1A). Consistent with these findings, tumors from recently and remotely postpartum participants had higher immune cell composition than tumors from nulliparous participants (Figure 1B). When comparing remote vs. nulliparous women, CD8T cells were also significantly enriched in remote vs. nulliparous women (Figure 1C); however, many fewer immune cell scores were significant. Nonetheless, no significant differences in immune cell scores were observed between recently vs. remotely postpartum women (Figure 1D). Box plots for signatures that were significant in Figure 1A showed that the level of each immune cell scores was elevated in both recently and remotely postpartum compared to nulliparous, but were modestly attenuated in remotely postpartum (Figure 1E).

Figure 1: Tumor immune microenvironment and adaptive immune cell types by recency of pregnancy.

(A) Volcano plot of immune cell score, adjusted for intrinsic subtype, race, age, and study phase; showing comparison by Recent vs. Nulliparous. (B) Boxplot of log2 transformed immune cell composition scores, or TIL scores, from H&E analysis by Recent, Remote and Nulliparous groups. (C and D) Volcano plots of immune cell score, adjusted for intrinsic subtype, race, age, and study phase; showing comparisons by Remote vs. Nulliparous, and Recent vs. Remote, respectively. (E) Boxplot of immune cell composition scores by Recent, Remote and Nulliparous groups (P=Kruskal-Wallis p-value).

Discussion

Among premenopausal women, we observed few differences between the tumors of recently and remotely postpartum women in the Carolina Breast Cancer Study. While parity status seems to be associated with breast cancer clinical and molecular tumor characteristics, recent pregnancy among parous women had weak or null associations with clinical and molecular characteristics, except when considering the most recent postpartum (≤ 5 years) and lymph node status and stage. However, consistent with the post-pregnancy wound healing microenvironment hypothesis [26], we observed an enrichment for adaptive immune cells and higher immune cell composition among recently postpartum women compared to nulliparous women. CD8 immune changes appeared to persist long term, but other immune response changes were attenuated in remotely postpartum vs. nulliparous women.

Three previous studies have reported significant differences in some key tumor characteristics between recently postpartum compared to nulliparous women [3, 21, 22]. Consistent with our findings, Goddard et al. observed an increased lymph node involvement among PPBC women compared to nulliparous women [3]. Pilewskie et al. found breast cancers diagnosed within 2 years of pregnancy were more likely to be lymph node positive, hormone receptor negative and triple negative compared to nulliparous women, after conditioning multivariate models on categorical age at diagnosis [21]. Similarly, Nagatsuma et al. reported ER/PR negative and triple negative tumors to be more common among recently postpartum (≤2 years before diagnosis) women compared to nulliparous women, in a Japanese hospital-based study [22]. While these latter studies used a narrower postpartum window than we did to define recency, we observed that several of the differences between recently postpartum and nulliparous women may continue to persist for many years postpartum similar to Goddard et al. [3].

Some previous literature seems to contradict the findings of our research. Specifically, we found ER/PR negative and IHC-based triple negative subtype tumors to be more common among recently postpartum (≤10 years postpartum) compared to nulliparous women, but two other groups found no difference in ER/PR status or IHC-based subtype between recently postpartum (<5 years postpartum) compared to nulliparous young women [4, 24]. However, compared to Callihan et al. we had several underlying methodologic differences. For example, we adjusted for age, while Callihan et al. did not [4]. The population studied in Callihan et al. has been expanded and the analysis has been updated by Goddard et al. in a recent publication corroborating our findings [3]. The study by Collins et al. also found no significant differences in ER/PR status and IHC-based subtype distribution among women diagnosed within 5 years of their last pregnancy compared to nulliparous women. However, patient ER status definition varied within their study population as their enrollment period began in 2006 and lasted beyond 2010, when ER positivity classification changed from ≥10% to ≥1% of tumor cells; thus, women who would have been classified as ER negative before 2010 were classified as ER positive if enrolled after 2010, leading to a slight reduction in ER negative and triple negative cases [24]. Additionally, the Collins et al. study population largely consisted of white women and the distribution of ER status has been shown to vary by race [24]. An advantage of the current analysis is that it included a significant representation of Black women, a group that has been underrepresented in previous analyses. Some findings of Collins et al. were consistent with ours, notably, neither study observed significant difference in tumor grade or stage comparing recently postpartum women to nulliparous women [24].

Only two previous studies have reported on the distribution of tumor characteristics among women >10 years postpartum (remotely postpartum) [3, 4]. Similar to our results, both of these studies have reported no significant difference in breast cancer grade, stage, nodal status, ER/PR status, HER2 status, and IHC-based subtypes between the recently and remotely postpartum groups.

To our knowledge, no other studies have examined RNA-based p53 signature, RNA-based intrinsic subtype, or ROR-P scores with respect to recently postpartum and nulliparous premenopausal women at breast cancer diagnosis. However, one previous study did evaluate p53 status by IHC. Our p53 findings corroborate those reported by Pilewskie et al., who found no significant difference in IHC-based p53 status between recently postpartum and nulliparous women at breast cancer diagnosis [21].

Our study is unique among the aforementioned studies in that we examined the immune microenvironment of tumors diagnosed according to time since last childbirth (recently vs. remotely postpartum women). Our results showed an enrichment in adaptive immune cells and a higher immune cell composition among recently postpartum women (≤10 years postpartum), which is attenuated among remotely postpartum women, compared to nulliparous women. Our findings are consistent with previous literature, which has noted an upregulation of immune-related genes in tumor-associated stroma of animal models [45, 46] or pregnant women [47]. Within normal breast tissue, this enrichment of inflammatory and immune response genes may persist many years after the completion of a pregnancy [48, 49]. In animal models, tumors during pregnancy or within the postpartum had an enrichment of immune responsiveness genes and immune cell infiltration.[45, 46]. Harvell et al. noted that compared to nulliparous or women diagnosed >5 years postpartum, tumors diagnosed during pregnancy overexpressed immune response genes [47]. Lastly, in normal breast tissue, genes in the immune response and inflammation pathways have been observed to be upregulated among parous compared to nulliparous women [48, 49].

Previously in the CBCS population, we observed that breast cancer-specific and overall survival were worse in recently postpartum women compared to both remotely postpartum and nulliparous women [50]. Several previous studies have also reported worse overall and disease-free survival among recently postpartum women [3–9]. Therefore, we hypothesized that tumor characteristics might explain the poor survival observed within this population. However, defining the ideal comparison group for recently postpartum women is complex because age, menopausal status and parity are strongly associated with tumor characteristics. Addressing this complexity was a strength of our analyses as we showed comparisons to two different populations (nulliparous women and remotely postpartum parous women), restricted our analysis to premenopausal women less <50 years of age, and controlled for the variation in age between comparison groups. To fully understand how recency of pregnancy affects outcomes, analyses should examine breast cancer recurrence or disease-specific mortality and incorporate both molecular data and treatment information, but our study population (CBCS3) has too few recurrences among recently postpartum women (n=56) to address these subgroups.

Results suggesting a distinct immune microenvironment in recently postpartum women is an intriguing finding. It is increasingly recognized that immune microenvironment plays a key role in response to treatment, and as the science of immune response and chemotherapy response is better defined, there may be implications for understanding PPBC outcomes. Future studies should examine the interplay between tumor microenvironment, post-diagnostic treatment and breast cancer recurrence/progression among recently postpartum women.