Emergency Department Visits Before Cancer Diagnosis Among Women at Mayo Clinic.

Objective: To determine associations of incident cancer diagnoses in women with recent emergency department (ED) care. Patients and Methods: A retrospective cohort study analyzing biological females aged 18 years and older, who were diagnosed with an incident primary cancer (12 cancer types studied) from January 1, 2015, to December 31, 2021, from electronic health records. The primary outcome was a cancer diagnosis within 6 months of a preceding ED visit. Secondary outcomes included patient factors associated with a preceding ED visit. Results: Of 25,736 patients (median age of 62 years, range 18-101) diagnosed with an incident primary cancer, 1938 (7.5%) had an ED visit ≤6 months before a diagnosis. The ED-associated cancer cases were highest in lung cancer (n=514, 14.7%) followed by acute lymphoblastic leukemia (n=22, 13.3%). Patient factors increasing the likelihood of ED evaluation before diagnosis included 18-50 years of age (OR=1.32; 95% CI, 1.09-1.61), Elixhauser score (measure of comorbidities) >4 (OR=17.90; 95% CI, 14.21-22.76), use of Medicaid or other government insurance (OR=2.10; 95% CI, 1.63-2.69), residence within the institutional catchment areas (OR=3.18; 95% CI, 2.78-3.66), non-Hispanic Black race/ethnicity (OR=1.41; 95% CI, 1.04-1.88), and established primary care provider at Mayo Clinic (OR=1.45; 95% CI, 1.28-1.65). The ED visits were more likely in those who died within 6 months of diagnosis (n=327, 37.8%) than those who did not die (n=1611, 6.5%). Conclusion: Patient characteristics identified in this study offer opportunities to provide cancer risk assessment and health navigation, particularly among individuals with comorbidities and limited health care access.

Leveraging GWAS: Path to Prevention?

Developing novel cancer prevention medication strategies is important for reducing mortality. Identification of common genetic variants associated with cancer risk suggests the potential to leverage these discoveries to define causal targets for cancer interception. Although each risk variant confers small increases in risk, researchers propose that blocking those that produce causal carcinogenic effects might have large impacts on cancer prevention. While a promising concept, we describe potential hurdles that may need to be scaled to reach this goal, including: (i) understanding the complexity of risk; (ii) achieving statistical power in studies with binary outcomes (cancer development: yes or no); (iii) characterization of cancer precursors; (iv) heterogeneity of cancer subtypes and the populations in which these diseases occur; (v) impact of static genetic markers across complex events of the life course; (vi) defining gene-gene and gene-environment interactions and (vii) demonstrating functional effects of markers in human populations. We assess short-term prospects for this research against the backdrop of these challenges and the potential to prevent cancer through other means. See related commentary by Peters and Tomlinson, p. 7.

Benign Breast Disease and Breast Cancer Risk in the Percutaneous Biopsy Era.

Importance: Benign breast disease (BBD) comprises approximately 75% of breast biopsy diagnoses. Surgical biopsy specimens diagnosed as nonproliferative (NP), proliferative disease without atypia (PDWA), or atypical hyperplasia (AH) are associated with increasing breast cancer (BC) risk; however, knowledge is limited on risk associated with percutaneously diagnosed BBD. Objectives: To estimate BC risk associated with BBD in the percutaneous biopsy era irrespective of surgical biopsy. Design, Setting, and Participants: In this retrospective cohort study, BBD biopsy specimens collected from January 1, 2002, to December 31, 2013, from patients with BBD at Mayo Clinic in Rochester, Minnesota, were reviewed by 2 pathologists masked to outcomes. Women were followed up from 6 months after biopsy until censoring, BC diagnosis, or December 31, 2021. Exposure: Benign breast disease classification and multiplicity by pathology panel review. Main Outcomes: The main outcome was diagnosis of BC overall and stratified as ductal carcinoma in situ (DCIS) or invasive BC. Risk for presence vs absence of BBD lesions was assessed by Cox proportional hazards regression. Risk in patients with BBD compared with female breast cancer incidence rates from the Iowa Surveillance, Epidemiology, and End Results (SEER) program were estimated. Results: Among 4819 female participants, median age was 51 years (IQR, 43-62 years). Median follow-up was 10.9 years (IQR, 7.7-14.2 years) for control individuals without BC vs 6.6 years (IQR, 3.7-10.1 years) for patients with BC. Risk was higher in the cohort with BBD than in SEER data: BC overall (standard incidence ratio [SIR], 1.95; 95% CI, 1.76-2.17), invasive BC (SIR, 1.56; 95% CI, 1.37-1.78), and DCIS (SIR, 3.10; 95% CI, 2.54-3.77). The SIRs increased with increasing BBD severity (1.42 [95% CI, 1.19-1.71] for NP, 2.19 [95% CI, 1.88-2.54] for PDWA, and 3.91 [95% CI, 2.97-5.14] for AH), comparable to surgical cohorts with BBD. Risk also increased with increasing lesion multiplicity (SIR: 2.40 [95% CI, 2.06-2.79] for ≥3 foci of NP, 3.72 [95% CI, 2.31-5.99] for ≥3 foci of PDWA, and 5.29 [95% CI, 3.37-8.29] for ≥3 foci of AH). Ten-year BC cumulative incidence was 4.3% for NP, 6.6% for PDWA, and 14.6% for AH vs an expected population cumulative incidence of 2.9%. Conclusions and Relevance: In this contemporary cohort study of women diagnosed with BBD in the percutaneous biopsy era, overall risk of BC was increased vs the general population (DCIS and invasive cancer combined), similar to that in historical BBD cohorts. Development and validation of pathologic classifications including both BBD severity and multiplicity may enable improved BC risk stratification.

Intraluminal tumor cells and prognostic accuracy of endometrial cancer stage criteria: A multi-institution study.

OBJECTIVE: Endometrial cancer stage is a strong prognostic factor; however, the current stage classification does not incorporate transtubal spread as determined by intraluminal tumor cells (ILTCs). We examined relationships between ILTCs and survival outcomes according to histological subtype and stage and examined whether identification of ILTCs improves prognostic accuracy of endometrial cancer staging. METHODS: We conducted a retrospective cohort study of women diagnosed with endometrial cancer at five academic hospitals between 2007 and 2012. Pathologists determined ILTC presence (no vs. yes) and location (free in lumen vs. attached to epithelial surface) based on pathology review of hematoxylin and eosin-stained sections of fallopian tubes. Associations between ILTCs with time to recurrence (TTR) and overall survival (OS) were examined with Cox proportional hazards models adjusted for other prognostic factors. Model discrimination metrics were used to assess the addition of ILTCs to stage for prediction of 5-year TTR and OS. RESULTS: In the overall study population (N = 1303), ILTCs were not independently associated with TTR (HR = 0.95, 95% CI = 0.69-1.32) or OS (HR = 0.97, 95% CI = 0.72-1.31). Among 805 women with stage I disease, ILTCs were independently associated with worse TTR (HR = 2.31, 95% CI = 1.06-5.05) and OS (HR = 2.16, 95% CI = 1.14-4.11). Upstaging early-stage cases with ILTCs present did not increase model discrimination. CONCLUSION: While our data do not suggest that endometrial cancer staging guidelines should be revised to include ILTCs, associations between ILTCs and reduced survival observed among stage I cases suggest this tumor feature holds clinical relevance for subgroups of endometrial cancer patients.

Methylated DNA Markers for Sporadic Colorectal and Endometrial Cancer Are Strongly Associated with Lynch Syndrome Cancers.

Lynch syndrome (LS) markedly increases risks of colorectal and endometrial cancers. Early detection biomarkers for LS cancers could reduce the needs for invasive screening and surgical prophylaxis.To validate a panel of methylated DNA markers (MDM) previously identified in sporadic colorectal cancer and endometrial cancer for discrimination of these cancers in LS.In a case-control design, previously identified MDMs for the detection of colorectal cancer and endometrial cancer were assayed by qMSP on tissue-extracted DNA. Results were normalized to ACTB values within each sample. Least absolute shrinkage and selection operator models to classify colorectal cancer and endometrial cancer were trained on sporadic cases and controls and then applied to classify colorectal cancer and endometrial cancer, in those with LS, and cross-validated.We identified colorectal cancer cases (23 with LS, 48 sporadic), colorectal controls (32 LS, 48 sporadic), endometrial cancer cases (30 LS, 48 sporadic), and endometrial controls (29 LS, 37 sporadic). A 3-MDM panel (LASS4, LRRC4, and PPP2R5C) classified LS-CRC from LS controls with an AUC of 0.92 (0.84-0.99); results were similar for sporadic colorectal cancer. A 6-MDM panel (SFMBT2, MPZ, CYTH2, DIDO1, chr10.4479, and EMX2OS) discriminated LS-EC from LS controls with an AUC of 0.92 (0.83-1.0); the AUC for sporadic endometrial cancer versus sporadic controls was nominally higher, 0.99 (0.96-1.0).MDMs previously identified in sporadic endometrial cancer and colorectal cancer discriminate between endometrial cancer and benign endometrium and colorectal cancer and benign colorectum in LS. This supports the inclusion of patients with LS within future prospective clinical trials evaluating endometrial cancer and colorectal cancer MDMs and may provide a new avenue for cancer screening or surveillance in this high-risk population. PREVENTION RELEVANCE: Lynch syndrome (LS) markedly increases risks of colorectal and endometrial cancers. Early detection biomarkers for LS cancers could reduce the needs for invasive screening and surgery. Methylated DNA markers previously identified in sporadic endometrial cancer and colorectal cancer discriminate between benign and cancer tissue in LS.

Reproductive Events and Risk of Women's Cancers: From Parturition to Prevention.

Reproductive events beginning with pregnancy and ending with remodeling of the breast after cessation of breastfeeding alter breast structure and function and produce dramatic changes in systemic biology. In aggregate, these processes lower overall risk for breast, tubo-ovarian and endometrial cancers, albeit differentially by molecular subtypes of these tumors. Herein, we explore opportunities for research on protective mechanisms operative during this period of the life course, with the goal of encouraging studies to advance cancer prevention. See related article by Getz et al., p. 353.

Detection of endometrial cancer using tampon-based collection and methylated DNA markers.

OBJECTIVE: Alterations in DNA methylation are early events in endometrial cancer (EC) development and may have utility in EC detection via tampon-collected vaginal fluid. METHODS: For discovery, DNA from frozen EC, benign endometrium (BE), and benign cervicovaginal (BCV) tissues underwent reduced representation bisulfite sequencing (RRBS) to identify differentially methylated regions (DMRs). Candidate DMRs were selected based on receiver operating characteristic (ROC) discrimination, methylation level fold-change between cancers and controls, and absence of background CpG methylation. Methylated DNA marker (MDM) validation was performed using qMSP on DNA from independent EC and BE FFPE tissue sets. Women ≥45 years of age with abnormal uterine bleeding (AUB) or postmenopausal bleeding (PMB) or any age with biopsy-proven EC self-collected vaginal fluid using a tampon prior to clinically indicated endometrial sampling or hysterectomy. Vaginal fluid DNA was assayed by qMSP for EC-associated MDMs. Random forest modeling analysis was performed to generate predictive probability of underlying disease; results were 500-fold in-silico cross-validated. RESULTS: Thirty-three candidate MDMs met performance criteria in tissue. For the tampon pilot, 100 EC cases were frequency matched by menopausal status and tampon collection date to 92 BE controls. A 28-MDM panel highly discriminated between EC and BE (96% (95%CI 89-99%) specificity; 76% (66-84%) sensitivity (AUC 0.88). In PBS/EDTA tampon buffer, the panel yielded 96% (95% CI 87-99%) specificity and 82% (70-91%) sensitivity (AUC 0.91). CONCLUSION: Next generation methylome sequencing, stringent filtering criteria, and independent validation yielded excellent candidate MDMs for EC. EC-associated MDMs performed with promisingly high sensitivity and specificity in tampon-collected vaginal fluid; PBS-based tampon buffer with added EDTA improved sensitivity. Larger tampon-based EC MDM testing studies are warranted.

Endometrial safety of low-dose vaginal estrogens.

ABSTRACT: It is estimated that up to 50% to 90% of postmenopausal women may experience genitourinary syndrome of menopause (GSM), which may have a detrimental impact on quality of life. One of the most effective modes of treatment of GSM is low-dose vaginal estrogens. Numerous studies have addressed the safety of these estrogens using endometrial biopsy and/or endometrial thickness on ultrasound. Based on these studies, the consensus is that low-dose vaginal estrogens do not substantially increase the risk of endometrial hyperplasia or cancer; however, the data are severely limited by short duration of follow-up. Although long-term trials are warranted, they are difficult to carry out, costly, and will not yield data for years. More immediate information regarding endometrial safety may be obtained from studies measuring endometrial tissue and serum concentrations of estradiol, estrone, and relevant equine estrogens after administration of different estrogen formulations and doses. This would allow us to understand better the metabolism of estrogens by the vagina and endometrium, and how much estrogen is reaching the endometrium. Here, we discuss metabolism, receptor binding, and signaling of estrogens in vaginal and endometrial tissue, and summarize the existing studies on the endometrial impact of low-dose vaginal estrogen treatment in postmenopausal women.

Short-term changes in ultrasound tomography measures of breast density and treatment-associated endocrine symptoms after tamoxifen therapy.

Although breast density decline with tamoxifen therapy is associated with greater therapeutic benefit, limited data suggest that endocrine symptoms may also be associated with improved breast cancer outcomes. However, it is unknown whether endocrine symptoms are associated with reductions in breast density after tamoxifen initiation. We evaluated treatment-associated endocrine symptoms and breast density change among 74 women prescribed tamoxifen in a 12-month longitudinal study. Treatment-associated endocrine symptoms and sound speed measures of breast density, assessed via novel whole breast ultrasound tomography (m/s), were ascertained before tamoxifen (T0) and at 1-3 (T1), 4-6 (T2), and 12 months (T3) after initiation. CYP2D6 status was genotyped, and tamoxifen metabolites were measured at T3. Using multivariable linear regression, we estimated mean change in breast density by treatment-associated endocrine symptoms adjusting for age, race, menopausal status, body mass index, and baseline density. Significant breast density declines were observed in women with treatment-associated endocrine symptoms (mean change (95% confidence interval) at T1:-0.26 m/s (-2.17,1.65); T2:-2.12 m/s (-4.02,-0.22); T3:-3.73 m/s (-5.82,-1.63); p-trend = 0.004), but not among women without symptoms (p-trend = 0.18) (p-interaction = 0.02). Similar declines were observed with increasing symptom frequency (p-trends for no symptoms = 0.91; low/moderate symptoms = 0.03; high symptoms = 0.004). Density declines remained among women with detectable tamoxifen metabolites or intermediate/efficient CYP2D6 metabolizer status. Emergent/worsening endocrine symptoms are associated with significant, early declines in breast density after tamoxifen initiation. Further studies are needed to assess whether these observations predict clinical outcomes. If confirmed, endocrine symptoms may be a proxy for tamoxifen response and useful for patients and providers to encourage adherence.

Reproducibility and intratumoral heterogeneity of the PAM50 breast cancer assay.

BACKGROUND: The PAM50 assay is used routinely in clinical practice to determine breast cancer prognosis and management; however, research assessing how technical variation and intratumoral heterogeneity contribute to misclassification and reproducibility of these tests is limited. METHODS: We evaluated the impact of intratumoral heterogeneity on the reproducibility of results for the PAM50 assay by testing RNA extracted from formalin-fixed paraffin embedded breast cancer blocks sampled at distinct spatial locations. Samples were classified according to intrinsic subtype (Luminal A, Luminal B, HER2-enriched, Basal-like, or Normal-like) and risk of recurrence with proliferation score (ROR-P, high, medium, or low). Intratumoral heterogeneity and technical reproducibility (replicate assays on the same RNA) were assessed as percent categorical agreement between paired intratumoral and replicate samples. Euclidean distances between samples, calculated across the PAM50 genes and the ROR-P score, were compared for concordant vs. discordant samples. RESULTS: Technical replicates (N = 144) achieved 93% agreement for ROR-P group and 90% agreement on PAM50 subtype. For spatially distinct biological replicates (N = 40 intratumoral replicates), agreement was lower (81% for ROR-P and 76% for PAM50 subtype). The Euclidean distances between discordant technical replicates were bimodal, with discordant samples showing higher Euclidian distance and biologic heterogeneity. CONCLUSION: The PAM50 assay achieved very high technical reproducibility for breast cancer subtyping and ROR-P, but intratumoral heterogeneity is revealed by the assay in a small proportion of cases.

Methylation Signature Implicated in Immuno-Suppressive Activities in Tubo-Ovarian High-Grade Serous Carcinoma.

BACKGROUND: Better understanding of prognostic factors in tubo-ovarian high-grade serous carcinoma (HGSC) is critical, as diagnosis confers an aggressive disease course. Variation in tumor DNA methylation shows promise predicting outcome, yet prior studies were largely platform-specific and unable to evaluate multiple molecular features. METHODS: We analyzed genome-wide DNA methylation in 1,040 frozen HGSC, including 325 previously reported upon, seeking a multi-platform quantitative methylation signature that we evaluated in relation to clinical features, tumor characteristics, time to recurrence/death, extent of CD8+ tumor-infiltrating lymphocytes (TIL), gene expression molecular subtypes, and gene expression of the ATP-binding cassette transporter TAP1. RESULTS: Methylation signature was associated with shorter time to recurrence, independent of clinical factors (N = 715 new set, hazard ratio (HR), 1.65; 95% confidence interval (CI), 1.10-2.46; P = 0.015; N = 325 published set HR, 2.87; 95% CI, 2.17-3.81; P = 2.2 × 10-13) and remained prognostic after adjustment for gene expression molecular subtype and TAP1 expression (N = 599; HR, 2.22; 95% CI, 1.66-2.95; P = 4.1 × 10-8). Methylation signature was inversely related to CD8+ TIL levels (P = 2.4 × 10-7) and TAP1 expression (P = 0.0011) and was associated with gene expression molecular subtype (P = 5.9 × 10-4) in covariate-adjusted analysis. CONCLUSIONS: Multi-center analysis identified a novel quantitative tumor methylation signature of HGSC applicable to numerous commercially available platforms indicative of shorter time to recurrence/death, adjusting for other factors. Along with immune cell composition analysis, these results suggest a role for DNA methylation in the immunosuppressive microenvironment. IMPACT: This work aids in identification of targetable epigenome processes and stratification of patients for whom tailored treatment may be most beneficial.

Benign Breast Disease, NSAIDs, and Postmenopausal Breast Cancer Risk in the CPS-II Cohort.

ABSTRACT: Nonsteroidal anti-inflammatory agents (NSAID) are associated with modest inconsistent reductions in breast cancer risk in population-based cohorts, whereas two focused studies of patients with benign breast disease (BBD) have found lower risk with NSAID use. Given that BBD includes fibroinflammatory lesions linked to elevated breast cancer risk, we assessed whether NSAID use was associated with lower breast cancer risk among patients with BBD.Participants were postmenopausal women in the Cancer Prevention Study-II (CPS-II), a prospective study of cancer incidence and mortality, who completed follow-up surveys in 1997 with follow-up through June 30, 2015. History of BBD, NSAID use, and covariate data were updated biennially. This analysis included 23,615 patients with BBD and 36,751 patients with non-BBD, including 3,896 incident breast cancers over an average of 12.72 years of follow-up among participants. NSAID use, overall and by formulation, recency, duration, and pills per month was analyzed versus breast cancer risk overall and by BBD status using multivariable-adjusted Cox models; BBD status and NSAID use were modeled as time-dependent exposures.Patients with BBD who reported using NSAIDs experienced lower breast cancer risk (HR, 0.87; 95% CI, 0.78-0.97), with similar effects for estrogen receptor (ER)-positive breast cancers [HR, 0.85; 95% confidence interval (CI), 0.74-0.97] and ER-negative breast cancers (HR, 0.87; 95% CI, 0.59-1.29); among women without BBD, NSAID use was unrelated to risk (HR, 1.02; 95% CI, 0.92-1.13; Pinteraction = 0.04). Associations stratified by age, obesity, menopausal hormone use, and cardiovascular disease were similar.Among patients with BBD, NSAID use appears linked to lower breast cancer risk. Further studies to assess the value of NSAID use among patients with BBD are warranted. PREVENTION RELEVANCE: We examined whether NSAID use, a modifiable exposure, is associated with breast cancer risk in postmenopausal women from the Cancer Prevention Study-II with self-reported benign breast disease, an often inflammatory condition associated with higher rates of breast cancer.

Immune cells are increased in normal breast tissues of BRCA1/2 mutation carriers.

PURPOSE: Breast cancer risk is elevated in pathogenic germline BRCA 1/2 mutation carriers due to compromised DNA quality control. We hypothesized that if immunosurveillance promotes tumor suppression, then normal/benign breast lobules from BRCA carriers may demonstrate higher immune cell densities. METHODS: We assessed immune cell composition in normal/benign breast lobules from age-matched women with progressively increased breast cancer risk, including (1) low risk: 19 women who donated normal breast tissue to the Komen Tissue Bank (KTB) at Indiana University Simon Cancer Center, (2) intermediate risk: 15 women with biopsy-identified benign breast disease (BBD), and (3) high risk: 19 prophylactic mastectomies from women with germline mutations in BRCA1/2 genes. We performed immunohistochemical stains and analysis to quantitate immune cell densities from digital images in up to 10 representative lobules per sample. Median cell counts per mm2 were compared between groups using Wilcoxon rank-sum tests. RESULTS: Normal/benign breast lobules from BRCA carriers had significantly higher densities of immune cells/mm2 compared to KTB normal donors (all p < 0.001): CD8 + 354.4 vs 150.9; CD4 + 116.3 vs 17.7; CD68 + 237.5 vs 57.8; and CD11c + (3.5% vs 0.4% pixels positive). BBD tissues differed from BRCA carriers only in CD8 + cells but had higher densities of CD4 + , CD11c + , and CD68 + immune cells compared to KTB donors. CONCLUSIONS: These preliminary analyses show that normal/benign breast lobules of BRCA mutation carriers contain increased immune cells compared with normal donor breast tissues, and BBD tissues appear overall more similar to BRCA carriers.

Reproductive risk factors associated with breast cancer in young women by molecular subtype.

BACKGROUND: Few studies have examined detailed features of pregnancy and the postpartum period as potential risk factors for early onset breast cancer (BC) by molecular subtype. These data may have value for improving risk assessment and prevention. METHODS: We surveyed parous enrollees in the prospective Mayo Clinic Breast Disease Registry (MCBDR) who had been diagnosed with BC at age <55 years between 2015 and 2020. Summary statistics were used to describe survey responses and reproductive risk factors by BC subtype (defined by estrogen/progesterone receptors and human epidermal growth factor receptor expression, nurse-abstracted from the medical record). Associations were assessed with Kruskal-Wallis and Chi-Square tests, followed by age-adjusted linear and logistic regression models. We compared results from this parous cohort to those from a separate cohort of nulliparous MCBDR participants with BC diagnosed at age <55 years. RESULTS: In 436 parous respondents with subtype data abstracted, we identified a higher frequency of BRCA1 mutation, earlier age at diagnosis, and lower BI in patients with triple negative BC. Comparing parous to nulliparous young women with breast cancer, the proportion with TNBC was larger in the latter (12.2% vs. 15.1%, p = 0.03). CONCLUSIONS: Early age at diagnosis and deleterious BRCA1 mutation were more frequent among TNBC patients. In addition, parous young women with TNBC had a lower BI than those with other BC subtypes, a hypothesis-generating finding that supports the need for additional research on the cycle of pregnancy-lactation-postpartum involution and BC etiology.

DNA barcoded competitive clone-initiating cell analysis reveals novel features of metastatic growth in a cancer xenograft model.

A problematic feature of many human cancers is a lack of understanding of mechanisms controlling organ-specific patterns of metastasis, despite recent progress in identifying many mutations and transcriptional programs shown to confer this potential. To address this gap, we developed a methodology that enables different aspects of the metastatic process to be comprehensively characterized at a clonal resolution. Our approach exploits the application of a computational pipeline to analyze and visualize clonal data obtained from transplant experiments in which a cellular DNA barcoding strategy is used to distinguish the separate clonal contributions of two or more competing cell populations. To illustrate the power of this methodology, we demonstrate its ability to discriminate the metastatic behavior in immunodeficient mice of a well-established human metastatic cancer cell line and its co-transplanted LRRC15 knockdown derivative. We also show how the use of machine learning to quantify clone-initiating cell (CIC) numbers and their subsequent metastatic progeny generated in different sites can reveal previously unknown relationships between different cellular genotypes and their initial sites of implantation with their subsequent respective dissemination patterns. These findings underscore the potential of such combined genomic and computational methodologies to identify new clonally-relevant drivers of site-specific patterns of metastasis.

Towards defining morphologic parameters of normal parous and nulliparous breast tissues by artificial intelligence.

BACKGROUND: Breast terminal duct lobular units (TDLUs), the source of most breast cancer (BC) precursors, are shaped by age-related involution, a gradual process, and postpartum involution (PPI), a dramatic inflammatory process that restores baseline microanatomy after weaning. Dysregulated PPI is implicated in the pathogenesis of postpartum BCs. We propose that assessment of TDLUs in the postpartum period may have value in risk estimation, but characteristics of these tissues in relation to epidemiological factors are incompletely described. METHODS: Using validated Artificial Intelligence and morphometric methods, we analyzed digitized images of tissue sections of normal breast tissues stained with hematoxylin and eosin from donors ≤ 45 years from the Komen Tissue Bank (180 parous and 545 nulliparous). Metrics assessed by AI, included: TDLU count; adipose tissue fraction; mean acini count/TDLU; mean dilated acini; mean average acini area; mean "capillary" area; mean epithelial area; mean ratio of epithelial area versus intralobular stroma; mean mononuclear cell count (surrogate of immune cells); mean fat area proximate to TDLUs and TDLU area. We compared epidemiologic characteristics collected via questionnaire by parity status and race, using a Wilcoxon rank sum test or Fisher's exact test. Histologic features were compared between nulliparous and parous women (overall and by time between last birth and donation [recent birth: ≤ 5 years versus remote birth: > 5 years]) using multivariable regression models. RESULTS: Normal breast tissues of parous women contained significantly higher TDLU counts and acini counts, more frequent dilated acini, higher mononuclear cell counts in TDLUs and smaller acini area per TDLU than nulliparas (all multivariable analyses p < 0.001). Differences in TDLU counts and average acini size persisted for > 5 years postpartum, whereas increases in immune cells were most marked ≤ 5 years of a birth. Relationships were suggestively modified by several other factors, including demographic and reproductive characteristics, ethanol consumption and breastfeeding duration. CONCLUSIONS: Our study identified sustained expansion of TDLU numbers and reduced average acini area among parous versus nulliparous women and notable increases in immune responses within five years following childbirth. Further, we show that quantitative characteristics of normal breast samples vary with demographic features and BC risk factors.

Protocol for the Detection of Organoid-Initiating Cell Activity in Patient-Derived Single Fallopian Tube Epithelial Cells.

Identification of serous tubal intraepithelial carcinomas (STIC) in the fallopian tubes of women who are carriers of germ line pathogenic variants in tubo-ovarian cancer predisposition genes (i.e., BRCA1 and BRCA2) has led to the hypothesis that many high-grade serous carcinomas (HGSC) arise from the fimbria of the fallopian tube. However, the primitive (stem and progenitor) tubal epithelial cells that give rise to STIC and HGSC have not been defined. Further, as putative HGSC precursors are discovered at salpingectomy, the natural history of such lesions is truncated at diagnosis. Thus, living cultures of human fallopian tubes suitable for experimental studies are needed to define and characterize the cellular origin of HGSCs and thereby advance the discovery of improved methods to assess risk, develop effective early detection tests and identify novel prevention approaches. Accordingly, patient-derived tissue-organoids and isolated epithelial stem cell derived-organoids generated from average and high-risk patients are vital resources to understand the developmental biology of aging fallopian tubes and pathogenesis of HGSCs. With a vision to boost HGSC prevention research, we have established state-of-the-art protocols for the collection, processing, storage, distribution, and management of fallopian tube tissues. Here we describe the protocol for preparing these organoids, with emphasis on the key steps that require meticulous attention to achieve success.

Serum hormone levels and normal breast histology among premenopausal women.

PURPOSE: Breast terminal duct lobular units (TDLUs) are the main source of breast cancer (BC) precursors. Higher serum concentrations of hormones and growth factors have been linked to increased TDLU numbers and to elevated BC risk, with variable effects by menopausal status. We assessed associations of circulating factors with breast histology among premenopausal women using artificial intelligence (AI) and preliminarily tested whether parity modifies associations. METHODS: Pathology AI analysis was performed on 316 digital images of H&E-stained sections of normal breast tissues from Komen Tissue Bank donors ages ≤ 45 years to assess 11 quantitative metrics. Associations of circulating factors with AI metrics were assessed using regression analyses, with inclusion of interaction terms to assess effect modification. RESULTS: Higher prolactin levels were related to larger TDLU area (p < 0.001) and increased presence of adipose tissue proximate to TDLUs (p < 0.001), with less significant positive associations for acini counts (p = 0.012), dilated acini (p = 0.043), capillary area (p = 0.014), epithelial area (p = 0.007), and mononuclear cell counts (p = 0.017). Testosterone levels were associated with increased TDLU counts (p < 0.001), irrespective of parity, but associations differed by adipose tissue content. AI data for TDLU counts generally agreed with prior visual assessments. CONCLUSION: Among premenopausal women, serum hormone levels linked to BC risk were also associated with quantitative features of normal breast tissue. These relationships were suggestively modified by parity status and tissue composition. We conclude that the microanatomic features of normal breast tissue may represent a marker of BC risk.