Background We investigated the impact of gender-affirming testosterone therapy (TT) on breast cancer (BC) risk and tumor progression. Materials and methods We leveraged a large human breast tissue dataset (n=417) to assess TT and terminal duct lobular unit (TDLU) involution, complemented with tissue markers (ER, PR, AR, and Ki67; n=24) and transcriptome profiling (n=8). Preclinical models assessed the effect of TT on BC incidence (MMTV-Cre Pik3caf/wt n=149 and K14-Cre Brcaf/fTp53f/f n=153), murine mammary gland architecture (n=60), and tumor transcriptome (n=10). Lastly, we discuss trans masculine invasive BC cases and summarize tumor characteristics in this population (n=24). Results TT promotes TDLU involution by reducing epithelial proliferation via altered estrogen signaling and increases ER+, PR+, and Ki67+ extralobular stromal cells. In mice, TT similarly reduced mammary gland ductal branching and terminal end buds. TT decreased Pik3ca-related ER+ BC incidence by 81% compared to female controls (adj RR 0.19, 95% CI 0.08-0.45), but did not affect Brca1-related triple negative BC incidence. TT did not influence tumor progression in either model but shaped the Pik3ca-related ER+ tumor microenvironment toward a pro-tumor phenotype. Most trans masculine BC cases were ER+ (83.3%), small and node-negative, but were also moderately to poorly differentiated (70.8%). Conclusion TT reduces ER+ BC risk but does not eliminate risk, and has a negligible impact on BRCA1-related triple-negative BC risk. TT does not affect tumor growth once tumors are established but modulates the tumor microenvironment. Our work supports the need for breast cancer screening in TT users.Competing Interest StatementThe authors have declared no competing interest.Funding StatementThis work was supported by the National Cancer Institute (NCI) R56CA284564 (YJH and GMW), R21CA267088 (YJH and GMW), R01CA226776 (GMW), P50CA168504 SPORE Career Enhancement Program (YJH), and Columbia Innovation Grant (LCH and KC). GMW is supported by the Breast Cancer Research Foundation 24-177. The University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core is supported by the NICHD R24HD102061.Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:This study was approved by the BIDMC Committee for Clinical Investigations (2018P000814), Columbia University IRB (AAAT0129), and BIDMC IACUC protocol (#052-2020-23). The Dana Farber/Harvard Cancer Center Office for Human Research Studies IRB determined that the medical records review of the four new invasive BC cases meets the criteria for exemption from IRB review (25-223).I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesRNASeqdata are available at the National Center for Biotechnology Information Gene Expression Omnibus (GSE306236). All other data that support the findings of this study are available from Dr. Jan Heng.
BACKGROUND: Early menarche is an established risk factor for breast cancer but its molecular contribution to tumor biology and prognosis remains unclear.
METHODS: We profiled transcriptome-wide gene expression in breast tumors (N = 846) and tumor-adjacent normal tissues (N = 666) from women in the Nurses' Health Studies (NHS) to investigate whether early menarche (age < 12) is associated with tumor molecular and prognostic features in women with breast cancer. Multivariable linear regression and pathway analyses using competitive gene set enrichment analysis were conducted in both tumor and adjacent-normal tissue and externally validated in TCGA (N = 116). Subgroup analyses stratified on ER-status based on the tumor were also performed. PAM50 signatures were used for tumor molecular subtyping and to generate proliferation and risk of recurrence scores. We created a gene expression score using LASSO regression to capture early menarche based on 28 genes from FDR-significant pathways in breast tumor tissue in NHS and tested its association with 10-year disease-free survival in both NHS (N = 836) and METABRIC (N = 952).
RESULTS: Early menarche was significantly associated with 369 individual genes in adjacent-normal tissues implicated in extracellular matrix, cell adhesion, and invasion (FDR ≤ 0.1). Early menarche was associated with upregulation of cancer hallmark pathways (18 significant pathways in tumor, 23 in tumor-adjacent normal, FDR ≤ 0.1) related to proliferation (e.g. Myc, PI3K/AKT/mTOR, cell cycle), oxidative stress (e.g. oxidative phosphorylation, unfolded protein response), and inflammation (e.g. pro-inflammatory cytokines IFN α and IFN γ ). Replication in TCGA confirmed these trends. Early menarche was associated with significantly higher PAM50 proliferation scores (β = 0.082 [0.02-0.14]), odds of aggressive molecular tumor subtypes (basal-like, OR = 1.84 [1.18-2.85] and HER2-enriched, OR = 2.32 [1.46-3.69]), and PAM50 risk of recurrence score (β = 4.81 [1.71-7.92]). Our NHS-derived early menarche gene expression signature was significantly associated with worse 10-year disease-free survival in METABRIC (N = 952, HR = 1.58 [1.10-2.25]).
CONCLUSIONS: Early menarche is associated with more aggressive molecular tumor characteristics and its gene expression signature within tumors is associated with worse 10-year disease-free survival among women with breast cancer. As the age of onset of menarche continues to decline, understanding its relationship to breast tumor characteristics and prognosis may lead to novel secondary prevention strategies.
BACKGROUND: We examined associations of CD44, CD24 and ALDH1A1 breast stem cell markers with mammographic breast density (MBD), a well-established breast cancer (BCa) risk factor.
METHODS: We included 218 cancer-free women with biopsy-confirmed benign breast disease within the Nurses' Health Study (NHS) and NHSII. The data on BCa risk factors were obtained from biennial questionnaires. Immunohistochemistry (IHC) was done on tissue microarrays. For each core, the IHC expression was assessed using a semi-automated platform and expressed as percent of positively stained cells for each marker out of the total cell count. MBD was assessed with computer-assisted techniques. Generalised linear regression was used to examine the associations of each marker with square root-transformed percent density (PD), absolute dense and non-dense areas (NDA), adjusted for BCa risk factors.
RESULTS: Stromal CD44 and ALDH1A1 expression was positively associated with PD (≥ 10% vs. <10% β = 0.56, 95% confidence interval [CI] [0.06; 1.07] and β = 0.81 [0.27; 1.34], respectively) and inversely associated with NDA (β per 10% increase = -0.17 [-0.34; -0.01] and β for ≥10% vs. <10% = -1.17 [-2.07; -0.28], respectively). Epithelial CD24 expression was inversely associated with PD (β per 10% increase = -0.14 [-0.28; -0.01]. Stromal and epithelial CD24 expression was positively associated with NDA (β per 10% increase = 0.35 [0.2 × 10-2; 0.70] and β per 10% increase = 0.34 [0.11; 0.57], respectively).
CONCLUSION: Expression of stem cell markers is associated with MBD.
BACKGROUND: Breast tumor immune infiltration is clearly associated with improved treatment response and outcomes in breast cancer. However, modifiable patient factors associated with breast cancer immune infiltrates are poorly understood. The Nurses' Health Study (NHS) offers a unique cohort to study immune gene expression in tumor and adjacent normal breast tissue, immune cell-specific immunohistochemistry (IHC), and patient exposures. We evaluated the association of body mass index (BMI) change since age 18, physical activity, and the empirical dietary inflammatory pattern (EDIP) score, all implicated in systemic inflammation, with immune cell-specific expression scores.
METHODS: This population-based, prospective observational study evaluated 882 NHS and NHSII participants diagnosed with invasive breast cancer with detailed exposure and gene expression data. Of these, 262 women (training cohort) had breast tumor IHC for four classic immune cell markers (CD8, CD4, CD20, and CD163). Four immune cell-specific scores were derived via lasso regression using 105 published immune expression signatures' association with IHC. In the remaining 620 patient evaluation cohort, we evaluated association of each immune cell-specific score as outcomes, with BMI change since age 18, physical activity, and EDIP score as predictors, using multivariable-adjusted linear regression.
RESULTS: Among women with paired expression/IHC data from breast tumor tissue, we identified robust correlation between novel immune cell-specific expression scores and IHC. BMI change since age 18 was positively associated with CD4+ (β = 0.16; p = 0.009), and CD163 novel immune scores (β = 0.14; p = 0.04) in multivariable analyses. In other words, for each 10 unit (kg/m2) increase in BMI, the percentage of cells positive for CD4 and CD163 increased 1.6% and 1.4%, respectively. Neither physical activity nor EDIP was significantly associated with any immune cell-specific expression score in multivariable analyses.
CONCLUSIONS: BMI change since age 18 was positively associated with novel CD4+ and CD163+ cell scores in breast cancer, supporting further study of the effect of modifiable factors like weight gain on the immune microenvironment.
