Terminal Duct Lobular Units Research Articles

Deep learning assessment of senescence-associated nuclear morphologies in mammary tissue from healthy female donors to predict future risk of breast cancer: a retrospective cohort study

Cellular senescence has been associated with cancer as either a barrier mechanism restricting autonomous cell proliferation or a tumour-promoting microenvironmental mechanism that secretes proinflammatory paracrine factors. With most work done in non-human models and the heterogeneous nature of senescence, the precise role of senescent cells in the development of cancer in humans is not well understood. Furthermore, more than 1 million non-malignant breast biopsies are taken every year that could be a major resource for risk stratification. We aimed to explore the clinical relevance for breast cancer development of markers of senescence in mammary tissue from healthy female donors. In this retrospective cohort study, we applied single-cell deep learning senescence predictors, based on nuclear morphology, to histological images of haematoxylin and eosin-stained breast biopsy samples from healthy female donors at the Komen Tissue Bank (KTB) at the Indiana University Simon Cancer Center (Indianapolis, IN, USA). All KTB participants (aged ≥18 years) who underwent core biopsies for research purposes between 2009 and 2019 were eligible for the study. Senescence was predicted in the epithelial (terminal duct lobular units [TDLUs] and non-TDLU epithelium), stromal, and adipose tissue compartments using validated models, previously trained on cells induced to senescence by ionising radiation (IR), replicative exhaustion (or replicative senescence; RS), or antimycin A, atazanavir-ritonavir, and doxorubicin (AAD) exposures. To benchmark our senescence-based cancer prediction results, we generated 5-year Gail scores-the current clinical gold standard for breast cancer risk prediction-for participants aged 35 years and older on the basis of characteristics at the time of tissue donation. The primary outcome was estimated odds of breast cancer via logistic modelling for each tissue compartment based on predicted senescence scores in cases (participants who had been diagnosed with breast cancer as of data cutoff, July 31, 2022) and controls (those who had not been diagnosed with breast cancer). 4382 female donors (median age at donation 45 years [IQR 34-57]) were eligible for the study. As of data cutoff (median follow-up of 10 years [7-11]), 86 (2·0%) had developed breast cancer a mean of 4·8 years (SD 2·84) after date of donation and 4296 (98·0%) had not received a breast cancer diagnosis. Among the 86 cases, we found significant differences in adipose-specific IR and AAD senescence prediction scores compared with controls. Risk analysis showed that individuals in the upper half (above the median) of scores for the adipose tissue IR model had higher odds of developing breast cancer (odds ratio [OR] 1·71 [95% CI 1·10-2·68]; p=0·019), whereas the adipose AAD model revealed a reduced odds of developing breast cancer (OR 0·57 [0·36-0·88]; p=0·013). For the other tissue compartments and the RS model, no significant associations were found (except for stromal tissue via the IR model, had higher odds of developing breast cancer [OR 1·59, 1·03-2·49]). Individuals with both of the adipose risk factors had an OR of 3·32 (1·68-7·03; p=0·0009). Participants with 5-year Gail scores above the median had an OR for development of cancer of 2·33 (1·46-3·82; p=0·0012) compared with those with scores below the median. When combining Gail scores with our adipose AAD risk model, we found that individuals with both of these predictors had an OR of 4·70 (2·29-10·90; p<0·0001). When combining the Gail score with our adipose IR model, we found that individuals with both predictors had an OR of 3·45 (1·77-7·24; p=0·0002). Assessment of senescence-associated nuclear morphologies with deep learning allows prediction of future cancer risk from normal breast biopsy samples. The combination of multiple models improved prediction of future breast cancer compared with the current clinical benchmark, the Gail model. Our results suggest an important role for microscope image-based deep learning models in predicting future cancer development. Such models could be incorporated into current breast cancer risk assessment and screening protocols. Novo Nordisk Foundation, Danish Cancer Society, and the US National Institutes of Health.

Abstract C143: Molecular drivers of terminal duct lobular unit involution: Associations with gene expression, genetic ancestry, and breast cancer disparties

Abstract Terminal duct lobular units (TDLU) are epithelial sub-structures located within breast tissue that function in lactation during child-bearing age. TDLU involution is a mechanism whereby shrinking of these units occurs once child-bearing age has been surpassed, inducing a decrease in acini sub-structures and TDLU counts in healthy breast tissues. Decreased TDLU involution has one of the largest associations with breast cancer risk, and is linked to factors such as breastfeeding, parity, hormone levels, mammographic density, age, BMI, menopausal status, and SNP risk scores. Decreased TDLU involution is seen more commonly in women of African ancestry and has been implicated to increase the risk of breast cancer development in this population. The molecular and environmental factors that influence TDLU involution have not been fully characterized. Environmentally, previous studies have shown that increased air pollution exposure with PM2.5 components is related to higher TDLU counts and lower involution. Molecularly, our previous work has shown that high TDLU counts are associated with a more pro-inflammatory immune environment and may indicate that lower TDLU involution is a result of inflammation in the breast due to persistence of at-risk epithelial tissue. TDLU involution is measured by TDLU counts, acini counts per TDLU, and TDLU diameter. Bulk RNAseq data from voluntary research biopsies of normal breast tissue donated by a cohort of 94 women (47 African American, 47 European American) was analyzed for gene expression associations with TDLU counts and self-reported race. Analysis revealed thirteen cancer- and/or immune-related genes with significant changes in expression associated with high TDLU counts indicating lower TDLU involution. For two out of three genes most relevant to mammary biology, the relationship between gene expression and increasing TDLU count was more significant among African American women than European American women. Additionally, CIBERSORTx RNAseq analysis estimated the abundance of immune cell sub- populations, and revealed significant differences between women with high and low TDLU counts. There are also differences observed in gene expression across race for women with high TDLU count that may indicate a link between lower TDLU involution and race. These initial findings provide greater insight into the molecular mechanisms that drive lower TDLU involution overall and for certain populations. Future analyses will further link these molecular factors to environmental exposures, such as air pollution, to provide context to risk factors associated with decreased TDLU involution and increased breast cancer risk. Citation Format: Jeri D. Hughes, Angel Pajimola, Brittney Davis-Lynn, Renata Cora, Alexandra Harris, Gretchen Gierach, Brittney Jenkins-Lord. Molecular drivers of terminal duct lobular unit involution: Associations with gene expression, genetic ancestry, and breast cancer disparties [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C143.

BreasTDLUSeg: A coarse-to-fine framework for segmentation of breast terminal duct lobular units on histopathological whole-slide images

Automatic segmentation of breast terminal duct lobular units (TDLUs) on histopathological whole-slide images (WSIs) is crucial for the quantitative evaluation of TDLUs in the diagnostic and prognostic analysis of breast cancer. However, TDLU segmentation remains a great challenge due to its highly heterogeneous sizes, structures, and morphologies as well as the small areas on WSIs. In this study, we propose BreasTDLUSeg, an efficient coarse-to-fine two-stage framework based on multi-scale attention to achieve localization and precise segmentation of TDLUs on hematoxylin and eosin (H&E)-stained WSIs. BreasTDLUSeg consists of two networks: a superpatch-based patch-level classification network (SPPC-Net) and a patch-based pixel-level segmentation network (PPS-Net). SPPC-Net takes a superpatch as input and adopts a sub-region classification head to classify each patch within the superpatch as TDLU positive or negative. PPS-Net takes the TDLU positive patches derived from SPPC-Net as input. PPS-Net deploys a multi-scale CNN-Transformer as an encoder to learn enhanced multi-scale morphological representations and an upsampler to generate pixel-wise segmentation masks for the TDLU positive patches. We also constructed two breast cancer TDLU datasets containing a total of 530 superpatch images with patch-level annotations and 2,322 patch images with pixel-level annotations to enable the development of TDLU segmentation methods. Experiments on the two datasets demonstrate that BreasTDLUSeg outperforms other state-of-the-art methods with the highest Dice similarity coefficients of 79.97% and 92.93%, respectively. The proposed method shows great potential to assist pathologists in the pathological analysis of breast cancer. An open-source implementation of our approach can be found at https://github.com/Dian-kai/BreasTDLUSeg.

CYB561 is a potential therapeutic target for breast cancer and is associated with immune cell infiltration

BackgroundBreast cancer (BC), a common malignant tumor originating from the terminal ductal lobular unit of the breast, poses a substantial health risk to women. Previous studies have associated cytochrome b561 (CYB561) with a poor prognosis in BC; however, its underlying mechanism of this association remains unclear.MethodsWe investigated the expression of CYB561 mRNA in BC using databases such as The Cancer Genome Atlas, Gene Expression Omnibus, Tumor–Normal–Metastatic plot, and Kaplan–Meier plotter databases. The prognostic value of CYB561 protein in BC was assessed in relation to its expression levels in tumor tissue samples from 158 patients with BC. The effect of CYB561 on BC progression was confirmed using in vivo and in vitro experiments. The biological functions and related signaling pathways of CYB561 in BC were explored using gene microarray, Innovative Pathway, Gene Ontology enrichment, and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. The correlation between CYB561 and the BC tumor immune microenvironment was evaluated using the CIBERSORT algorithm and single-cell analysis and further validated through immunohistochemistry of serial sections.ResultsOur study demonstrated that upregulation of CYB561 expression predicted poor prognosis in patients with BC and that CYB561 knockdown inhibited the proliferation, migration, and invasive ability of BC cells in vitro. CYB561 knockdown inhibited BC tumor formation in vivo.CYB561 was observed to modulate downstream tropomyosin 1 expression. Furthermore, CYB561 expression was associated with macrophage M2 polarization in the BC immune microenvironment.ConclusionsElevated CYB561 expression suggests a poor prognosis for patients with BC and is associated with macrophage M2 polarization in the BC microenvironment. Therefore, CYB561 could potentially serve as a therapeutic target for BC treatment.

Associations between quantitative measures of mammographic density and terminal ductal lobular unit involution in Chinese breast cancer patients

BackgroundHigher mammographic density (MD), a radiological measure of the proportion of fibroglandular tissue in the breast, and lower terminal duct lobular unit (TDLU) involution, a histological measure of the amount of epithelial tissue in the breast, are independent breast cancer risk factors. Previous studies among predominantly white women have associated reduced TDLU involution with higher MD.MethodsIn this cohort of 611 invasive breast cancer patients (ages 23–91 years [58.4% ≥ 50 years]) from China, where breast cancer incidence rates are lower and the prevalence of dense breasts is higher compared with Western countries, we examined the associations between TDLU involution assessed in tumor-adjacent normal breast tissue and quantitative MD assessed in the contralateral breast obtained from the VolparaDensity software. Associations were estimated using generalized linear models with MD measures as the outcome variables (log-transformed), TDLU measures as explanatory variables (categorized into quartiles or tertiles), and adjusted for age, body mass index, parity, age at menarche and breast cancer subtype.ResultsWe found that, among all women, percent dense volume (PDV) was positively associated with TDLU count (highest tertile vs. zero: Expbeta = 1.28, 95% confidence interval [CI] 1.08–1.51, ptrend = < .0001), TDLU span (highest vs. lowest tertile: Expbeta = 1.23, 95% CI 1.11–1.37, ptrend = < .0001) and acini count/TDLU (highest vs. lowest tertile: Expbeta = 1.22, 95% CI 1.09–1.37, ptrend = 0.0005), while non-dense volume (NDV) was inversely associated with these measures. Similar trend was observed for absolute dense volume (ADV) after the adjustment of total breast volume, although the associations for ADV were in general weaker than those for PDV. The MD-TDLU associations were generally more pronounced among breast cancer patients ≥ 50 years and those with luminal A tumors compared with patients < 50 years and with luminal B tumors.ConclusionsOur findings based on quantitative MD and TDLU involution measures among Chinese breast cancer patients are largely consistent with those reported in Western populations and may provide additional insights into the complexity of the relationship, which varies by age, and possibly breast cancer subtype.

An ovine model for investigation of the microenvironment of the male mammary gland.

The specific biology of the male breast remains relatively unexplored in spite of the increasing global prevalence of male breast cancer. Delineation of the microenvironment of the male breast is restricted by the low availability of human samples and a lack of characterisation of appropriate animal models. Unlike the mouse, the male ovine gland persists postnatally. We suggest that the male ovine mammary gland constitutes a promising adjunctive model for the male breast. In this study, we evaluate the male ovine mammary gland microenvironment, comparing intact and neutered males. Assessment of the glandular histo-anatomy highlights the resemblance of the male gland to that of neonatal female sheep and confirms the presence of rudimentary terminal duct lobular units. Irrespective of neutered status, cell proliferation in epithelial and stromal compartments is similarly low in males, and cell proliferation in epithelial cells and in the intralobular stroma is significantly lower than in pubertal female sheep. Between 42% and 72% of the luminal mammary epithelial cells in the male gland express the androgen receptor and expression is significantly reduced by neutering. Luminal epithelial cells within the intact and neutered male gland also express oestrogen receptor alpha, but minimal progesterone receptor expression is observed. The distribution of leukocytes within the ducts and stroma is similar to the mammary gland of female sheep and females of other species. Both macrophages and T lymphocytes are intercalated in the epithelial bilayer and are more abundant in the intralobular stroma than the interlobular stroma, suggesting that they may have a protective immunological function within the vestigial glandular tissue of the male sheep. Mast cells are also observed within the stroma. These cells cluster near the glandular tissue and are frequently located adjacent to blood vessels. The abundance of mast cells is significantly higher in intact males compared to neutered males, suggesting that hormone signalling may impact mast cell recruitment. In this study, we demonstrate the utility of the male ovine mammary gland as a model for furthering our knowledge of postnatal male mammary biology.

Abstract PO3-28-09: Does Diffusely Infiltrating Lobular Carcinoma of the Breast Arise from Epithelial-Mesenchymal Hybrid Cells?

Abstract Classic diffusely infiltrating lobular carcinoma has imaging features divergent from the breast cancers originating from the terminal ductal lobular units and from the major lactiferous ducts. Although the term “invasive lobular carcinoma” implies a site of origin within the breast lobular epithelium, we were unable to find evidence supporting this assumption. Exceptional excess of fibrous connective tissue and the unique cell architecture combined with the aberrant features at breast imaging suggest that this breast malignancy has not originated from cells lining the breast ducts and lobules. The only remaining relevant component of the fibroglandular tissue is the mesenchyme. The cells freshly isolated and cultured from diffusely infiltrating lobular carcinoma cases contained epithelial–mesenchymal hybrid cells with both epithelial and mesenchymal properties. The radiologic and histopathologic features of the tumours and expression of the mesenchymal stem cell positive markers CD73, CD90, and CD105 all suggest development in the direction of mesenchymal transition. These hybrid cells have tumour-initiating potential and have been shown to have poor prognosis and resistance to therapy targeted for malignancies of breast epithelial origin. Our work emphasizes the need for new approaches to the diagnosis and therapy of this highly fatal breast cancer subtype. Citation Format: Andras Voros, Laszlo Tabar, Renata Bozo, Orsolya Olah, Katalin Ormandi, Zoltan Vereb, Istvan Nemeth, Peter B. Dean, Olga Puchkova, Ming-Fang Yen, Li-Sheng Chen. Does Diffusely Infiltrating Lobular Carcinoma of the Breast Arise from Epithelial-Mesenchymal Hybrid Cells? [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-28-09.

Abstract PO1-26-01: Leveraging Clinicopathological Factors and Deep Learning-Based Morphometrics for PDX Engraftment Success Prediction in Breast Cancer

Abstract Background: Patient-derived xenografts (PDXs) are pivotal in cancer research. Despite histopathological insights into factors driving PDX success, the role of artificial intelligence (AI) in predicting PDX engraftment remains unexplored. We aimed to bridge this gap by integrating clinicopathological data and AI-based morphometric analysis to predict PDX success in breast cancer. Methods: PDXs were generated from tumor tissues derived from breast cancer patients who had undergone surgical intervention. Clinicopathological information including subtypes, pathological diagnosis, modified Bloom-Richardson system histologic grades, treatment with neoadjuvant chemotherapy (NAC), Miller Payne grade, residual caner burden score, invasive tumor size, lymphovascular invasion status, AJCC 8th T and N stages and the percentage of tumor infiltrating lymphocytes were collected and analyzed. For the image analysis component, whole-slide images (WSIs) of hematoxylin and eosin–stained tissue samples from 64 surgically resected breast cancer patients were used as a training set for an AI model under the supervision of 2 pathologists to extract morphometric features. The model transformed image tiles into patches of morphologically similar patterns, and categorized them into adipose tissue, background, necrosis, invasive carcinoma, carcinoma in situ, stroma, and terminal ductal lobular unit. This trained model was subsequently applied to the WSIs employed in the establishment of PDXs. The classified patches and their relative ratios within the invasive tumor boundary were compiled. The consolidated data from clinicopathological and image analyses were subjected to logistic regression to discern correlates of successful PDX engraftment. Results: Out of the 311 patient tumor samples used for generating PDXs, (131 post-chemotherapy, 180 chemo-naïve), 47 PDXs were successfully established (15.1%). Logistic regression revealed several factors for successful engraftment including NAC treatment with an odds ratio of 6.71 (28.2% vs 5.6%, 95% CI: 2.53 - 17.80, p &amp;lt; 0.001), higher histologic grades (25.3% vs 2.2%, OR = 5.80, 95% CI: 1.49 - 22.63, p = 0.01), triple negative breast cancer compared to hormone receptor-positive cancers (32.0% vs 3.3%, OR = 10.99, 95% CI: 1.26 - 95.89, p = 0.03), and tumors of larger size (OR = 1.34, 95% CI: 1.02 - 1.76, p = 0.03). Interestingly, the percentage of specific tissue patch types within tumor did not significantly impact the likelihood of successful PDX engraftment. However, in our logistic regression analysis based solely on morphometric features, presence of necrosis within the tumor notably enhanced PDX establishment. Specifically, each percent increase in necrosis within tumor boosted the odds of successful PDX creation by 0.01% (OR = 1.0001, 95% CI: 1.00003 - 1.00024, p = 0.01). Conclusions: PDXs are often successfully established from clinically aggressive breast cancers, particularly those with NAC treatment, higher histologic grades, TNBC subtype, and larger tumor size. While morphometric features contribute to the prediction of PDX engraftment success, their importance is surpassed by these clinicopathological factors. However, presence of necrosis emerged as a key morphometric predictor of successful PDX engraftment. Keywords: breast cancer; patient-derived xenograft (PDX); Breast Cancer Morphometrics; Cancer Predictive Modeling. Citation Format: Jongwon Lee, GeonHee Lee, Gyungyub Gong, Hee Jin Lee. Leveraging Clinicopathological Factors and Deep Learning-Based Morphometrics for PDX Engraftment Success Prediction in Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-26-01.

Abstract PO3-08-08: Basal-Luminal (BL) Cells in Histologically Normal and Malignant Breast Tissue from BRCA1 and BRCA2 Carriers

Abstract Background Single-cell studies of normal breast tissue in BRCA mutation carriers have led to the identification of a candidate cancer precursor cell, the basal-luminal (BL) cell. The localization of BL cells within normal, pre-malignant and malignant breast tissue architecture is not well characterized. Here, using cyclic immunofluorescence (CyCIF), we quantify and localize BL cells within normal and malignant breast tissues from BRCA1 and BRCA2 carriers. Methods Formalin-fixed paraffin-embedded breast tissue sections from 9 BRCA1 and 9 BRCA2 consented patients treated at Dana Farber Cancer Institute (2001-2019) were retrieved from the clinical archives and subjected to CyCIF for luminal and basal markers including CK5, CK14 and CK19. Using one section/case, cells co-expressing basal and luminal cytokeratins (CK14/CK19 and/or CK5/CK19) were visually identified in invasive carcinoma, ductal carcinoma in situ (DCIS) and in adjacent normal breast tissue. In histologically normal tissue, regions of interest (ROIs) with co-expressing cells were classified as terminal duct lobular units (TDLUs) or isolated ducts. Within each ROI, the quantity of BL cells was scored as low (1-5 cells), moderate (6-10 cells), or high ( &amp;gt;10 cells). Results The 9 BRCA1 tumors included 8 invasive ductal carcinomas/no special type and 1 tubular carcinoma. The 9 BRCA2 tumors included 4 invasive ductal carcinomas/no special type and 5 invasive carcinomas with ductal and lobular features. 496 ROIs were evaluated in histologically normal tissue: 297 from BRCA1 cases and 199 from BRCA2 cases. BL cells co-expressing CK14/CK19 and/or CK5/CK19 were identified in normal TDLUs and isolated ducts in both BRCA1 and BRCA2 cases. BL cells were more frequently identified in BRCA2 cases, with at least one BL cell present in 97/297 ROIs (33%) in BRCA1 cases and in 133/199 ROIs (67%) in BRCA2 cases (p&amp;lt; 0.01). 55% of BRCA2 ROIs had &amp;gt;10 BL cells each versus 42% of BRCA1 ROIs; however, this difference was not significant. Four BRCA1 cases showed a diffuse BL cell phenotype within the invasive carcinoma with either a diffuse or scattered BL cell phenotype within the DCIS. The BL cell phenotype was not seen diffusely in invasive carcinoma or DCIS in the other 14 cases; however individual tumor cells or small cell clusters with the BL cell phenotype were occasionally found. Conclusions BL cells are found in histologically normal TDLUs and isolated ducts in breast tissue from BRCA1 and BRCA2 carriers, with greater prevalence in BRCA2 carriers. The BL cell phenotype is present diffusely within tumor cells in almost half of BRCA1-associated cancers and none of the BRCA2-associated cancers in this small cohort. Further study of this interesting cell population is warranted in efforts to understand early changes in epithelium at high risk for the development of cancer. Citation Format: Rodrigo Fonseca Abreu, Tabata Alves Domingos, Roberto Bonfim Pimenta Peixoto, Ashka Patel, Krishan Taneja, McKenna Moore, Alicia Pollaci, Judy Garber, Nomeda Girnius, Deborah Dillon. Basal-Luminal (BL) Cells in Histologically Normal and Malignant Breast Tissue from BRCA1 and BRCA2 Carriers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-08-08.

Integrating X-ray phase-contrast imaging and histology for comparative evaluation of breast tissue malignancies in virtual histology analysis

Detecting breast tissue alterations is essential for cancer diagnosis. However, inherent bidimensionality limits histological procedures’ effectiveness in identifying these changes. Our study applies a 3D virtual histology method based on X-ray phase-contrast microtomography (PhC μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mu$$\\end{document}CT), performed at a synchrotron facility, to investigate breast tissue samples including different types of lesions, namely intraductal papilloma, micropapillary intracystic carcinoma, and invasive lobular carcinoma. One-to-one comparisons of X-ray and histological images explore the clinical potential of 3D X-ray virtual histology. Results show that PhC μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mu$$\\end{document}CT technique provides high spatial resolution and soft tissue sensitivity, while being non-destructive, not requiring a dedicated sample processing and being compatible with conventional histology. PhC μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mu$$\\end{document}CT can enhance the visualization of morphological characteristics such as stromal tissue, fibrovascular core, terminal duct lobular unit, stromal/epithelium interface, basement membrane, and adipocytes. Despite not reaching the (sub) cellular level, the three-dimensionality of PhC μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mu$$\\end{document}CT images allows to depict in-depth alterations of the breast tissues, potentially revealing pathologically relevant details missed by a single histological section. Compared to serial sectioning, PhC μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mu$$\\end{document}CT allows the virtual investigation of the sample volume along any orientation, possibly guiding the pathologist in the choice of the most suitable cutting plane. Overall, PhC μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mu$$\\end{document}CT virtual histology holds great promise as a tool adding to conventional histology for improving efficiency, accessibility, and diagnostic accuracy of pathological evaluation.

Abstract A087: Morphometric analysis of the terminal ductal lobular unit architecture in human breast

Abstract The major lactiferous ducts of the human breast branch out and ultimately end at terminal ductal lobular units (TDLUs). These glandular structures are the source of milk upon lactation, and also the most common origin of breast cancer. Despite their functional and clinical importance, the three dimensional (3D) architecture of TDLUs has remained undetermined, largely due to their absence in rodent animal models. By utilizing recent technological advances in optical tissue clearing and microscopy, we imaged the 3D structure of healthy human breast tissue to determine whether general branching patterns or cell type specific niches exist in the TDLU. Our data demonstrate that TDLUs have a consistent shape and their branch parameters are largely comparable between different TDLUs and individuals irrespective of age or parity. Simulation of TDLU branching morphogenesis in 3D by mathematical modelling suggests that evolutionarily conserved mechanisms regulate mammary gland branching in humans and mice, despite their anatomical differences. The data also demonstrate a new level of organization within the TDLU structure identifying a main branch that dominates in bifurcation events and exhibits a more duct-like keratin expression pattern. In all, our data provide the first structural insights into 3D human breast anatomy and branching, and exemplify the power of volumetric imaging in deeper understanding of human breast biology. Citation Format: Markus Peurla, Oona Paavolainen, Ella Tammelin, Suvi-Riitta Sulander, Larissa Mourao, Pia Boström, Nina Brück, Colinda LGJ Scheele, Pauliina Hartiala, Emilia Peuhu. Morphometric analysis of the terminal ductal lobular unit architecture in human breast [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A087.

Evidence of steady-state fibroblast subtypes in the normal human breast as cells-of-origin for perturbed-state fibroblasts in breast cancer

BackgroundHuman breast cancer most frequently originates within a well-defined anatomical structure referred to as the terminal duct lobular unit (TDLU). This structure is endowed with its very own lobular fibroblasts representing one out of two steady-state fibroblast subtypes—the other being interlobular fibroblasts. While cancer-associated fibroblasts (CAFs) are increasingly appreciated as covering a spectrum of perturbed states, we lack a coherent understanding of their relationship—if any—with the steady-state fibroblast subtypes. To address this, we here established two autologous CAF lines representing inflammatory CAFs (iCAFs) and myofibroblast CAFs (myCAFs) and compared them with already established interlobular- and lobular fibroblasts with respect to their origin and impact on tumor formation.MethodsPrimary breast tumor-derived CAFs were transduced to express human telomerase reverse transcriptase (hTERT) and sorted into CD105low and CD105high populations using fluorescence-activated cell sorting (FACS). The two populations were tested for differentiation similarities to iCAF and myCAF states through transcriptome-wide RNA-Sequencing (RNA-Seq) including comparison to an available iCAF-myCAF cell state atlas. Inference of origin in interlobular and lobular fibroblasts relied on RNA-Seq profiles, immunocytochemistry and growth characteristics. Osteogenic differentiation and bone formation assays in culture and in vivo were employed to gauge for origin in bone marrow-derived mesenchymal stem cells (bMSCs). Functional characteristics were assessed with respect to contractility in culture and interaction with tumor cells in mouse xenografts. The cells’ gene expression signatures were tested for association with clinical outcome of breast cancer patients using survival data from The Cancer Genome Atlas database.ResultsWe demonstrate that iCAFs have properties in common with interlobular fibroblasts while myCAFs and lobular fibroblasts are related. None of the CAFs qualify as bMSCs as revealed by lack of critical performance in bone formation assays. Functionally, myCAFs and lobular fibroblasts are almost equally tumor promoting as opposed to iCAFs and interlobular fibroblasts. A myCAF gene signature is found to associate with poor breast cancer-specific survival.ConclusionsWe propose that iCAFs and myCAFs originate in interlobular and lobular fibroblasts, respectively, and more importantly, that the tumor-promoting properties of lobular fibroblasts render the TDLU an epicenter for breast cancer evolution.

Huddled and Preternatural- Atypical Lobular Hyperplasia

Atypical lobular hyperplasia manifests as a clonal proliferation of dis-cohesive epithelial cells emerging from terminal duct lobular units. Neoplasm appears reminiscent of lobular carcinoma in situ (LCIS) although cellular quantification is reduced. Characteristically, the incidentally discovered lesion is associated with dysfunction or decimation of E-cadherin. Additionally designated as lobular intraepithelial neoplastic 1 (LIN1), tumefaction exemplifies a female preponderance. Typically, adult subjects are implicated. Tumefaction is commonly encountered within the fifth decade although no age of disease emergence is exempt. Atypical lobular hyperplasia is detected within&lt;1% of core needle biopsies adopted for evaluating BI-RADS 4 lesions [1,2]. Atypical lobular hyperplasia may incriminate bilateral breasts wherein specific lesion localization is absent. Tumefaction exhibits loss of chromosome 16qand deletion of CDH1 genes with loss of function of E-cadherin, a Tran’s membrane cell adhesion molecule. Alternatively, altered function of E-cadherin may occur. Exceptionally, various adhesion proteins are implicated wherein E-cadherin molecule appears stable [1,2]. Characteristically, atypical lobular hyperplasia is a diploid neoplasm. Tumefaction exhibits loss of heterozygosis (LOH) at chromosome 16q. A subset of neoplasms demonstrates methylation of CDH1 gene. Several tumefaction express loss of chromosome 22q and 16p, gain of chromosome 2p11.2, 5q32- 33.1 (CSF1R), 6q, 11q13 or 14q32.33.

Immune infiltration, aggressive pathology, and poor survival outcomes in RECQL helicase deficient breast cancers

RECQL is essential for genomic stability. Here, we evaluated RECQL in 449 pure ductal carcinomas in situ (DCIS), 152 DCIS components of mixed DCIS/invasive breast cancer (IBC) tumors, 157 IBC components of mixed DCIS/IBC and 50 normal epithelial terminal ductal lobular units (TDLUs). In 726 IBCs, CD8+, FOXP3+, IL17+, PDL1+, PD1+ T-cell infiltration (TILs) were investigated in RECQL deficient and proficient cancers. Tumor mutation burden (TMB) was evaluated in five RECQL germ-line mutation carriers with IBC by genome sequencing. Compared with normal epithelial cells, a striking reduction in nuclear RECQL in DCIS was evident with aggressive pathology and poor survival. In RECQL deficient IBCs, CD8+, FOXP3+, IL17+ or PDL1+ TILs were linked with aggressive pathology and shorter survival. In germline RECQL mutation carriers, increased TMB was observed in 4/5 tumors. We conclude that RECQL loss is an early event in breast cancer and promote immune cell infiltration.

Tubular Adenoma of the Breast: Radiologic-Pathologic Correlation.

Breast tubular adenomas (TAs) are rare, benign glandular epithelial tumors that arise from a proliferation of acini in the terminal duct lobular units. In the literature, 40 TA cases have previously been reported, and we describe 5 additional cases in this article. In the small number of reported cases, TAs present most often in women of reproductive age but may also occur in postmenopausal women. Mammographically and sonographically, TAs are almost indistinguishable from fibroadenomas (FAs), and they typically present on US as hypoechoic, oval, circumscribed, parallel masses with variable internal vascularity. TAs can also be seen on mammography as oval masses with microlobulated margins, or as grouped coarse, heterogeneous microcalcifications with or without associated mass or asymmetry. On MRI, TAs present as heterogeneously enhancing, T2-hyperintense oval masses with persistent kinetics. Histopathologically, TAs consist of closely packed round tubules with minimal stroma, in distinction to FAs, which have a prominent stromal component that surrounds and can distort the associated tubules. Because of their benign classification and excellent prognosis, patients with biopsy-confirmed TAs may resume routine screening. Complete surgical excision may be considered for cosmetic purposes or for TAs exhibiting associated suspicious calcifications or rapid growth.

The term “classic invasive lobular carcinoma” of the breast defines breast malignancies of vastly different nature

PurposeTo describe in detail the special features of a previously unappreciated “classic invasive lobular carcinoma” which is confined to the terminal ductal lobular units (TDLUs) and differs considerably from the extensive classic invasive lobular carcinoma, and to suggest specific terminology. MethodAll invasive breast cancer cases without associated microcalcifications diagnosed in our Institution with the histopathologic diagnosis of classic invasive lobular carcinoma during the years 1996–2019 (n = 560) formed the basis of this study. The cases were prospectively classified according to their imaging biomarkers (mammographic features) and followed up to Dec 31, 2021, to determine long-term patient outcome. An additional 2600 invasive breast cancer cases (diagnosed other than invasive lobular carcinoma) without associated microcalcifications served as a reference group. Detailed histopathologic analysis used large format (10x8 cm) thin section technique and staining methods including hematoxylin-eosin (H&E), E-cadherin, cytokeratin CK 5/6, a transmembrane glycoprotein (CD44) and anti-actin or anti-smooth muscle myosin heavy chain. ResultsThe imaging biomarkers differentiated two separate disease subgroups, having the same histopathologic diagnosis, classic invasive lobular carcinoma. One of these has the imaging biomarker of extensive architectural distortion with no central tumour mass, occupies the extralobular mesenchyme and has a long-term survival of 56%. The other subgroup forms stellate or circular non-calcified tumour masses usually smaller than 20 mm, which appear to arise in the intralobular mesenchyme, and has a significantly better long-term survival of 84%. ConclusionsThere is a striking difference between the subgross histopathology and the mammographic appearance (imaging biomarkers) of two breast malignancies having the same histopathologic diagnosis, “classic invasive lobular carcinoma”. The large difference in the long-term outcome of these two tumour types is even more striking. Using the same specific term, “classic invasive lobular carcinoma”, to describe these two separate entities can adversely affect management decisions.

Does Diffusely Infiltrating Lobular Carcinoma of the Breast Arise from Epithelial-Mesenchymal Hybrid Cells?

Classic diffusely infiltrating lobular carcinoma has imaging features divergent from the breast cancers originating from the terminal ductal lobular units and from the major lactiferous ducts. Although the term "invasive lobular carcinoma" implies a site of origin within the breast lobular epithelium, we were unable to find evidence supporting this assumption. Exceptional excess of fibrous connective tissue and the unique cell architecture combined with the aberrant features at breast imaging suggest that this breast malignancy has not originated from cells lining the breast ducts and lobules. The only remaining relevant component of the fibroglandular tissue is the mesenchyme. The cells freshly isolated and cultured from diffusely infiltrating lobular carcinoma cases contained epithelial-mesenchymal hybrid cells with both epithelial and mesenchymal properties. The radiologic and histopathologic features of the tumours and expression of the mesenchymal stem cell positive markers CD73, CD90, and CD105 all suggest development in the direction of mesenchymal transition. These hybrid cells have tumour-initiating potential and have been shown to have poor prognosis and resistance to therapy targeted for malignancies of breast epithelial origin. Our work emphasizes the need for new approaches to the diagnosis and therapy of this highly fatal breast cancer subtype.

Changes in the mammary gland during aging and its links with breast diseases.

The functional capacity of organisms declines in the process of aging. In the case of breast tissue, abnormal mammary gland development can lead to dysfunction in milk secretion, a primary function, as well as the onset of various diseases, such as breast cancer. In the process of aging, the terminal duct lobular units (TDLUs) within the breast undergo gradual degeneration, while the proportion of adipose tissue in the breast continues to increase and hormonal levels in the breast change accordingly. Here, we review changes in morphology, internal structure, and cellular composition that occur in the mammary gland during aging. We also explore the emerging mechanisms of breast aging and the relationship between changes during aging and breast-related diseases, as well as potential interventions for delaying mammary gland aging and preventing breast disease.

Correlation Among Tumor Size, Histopathological Grade and the Expression of RAC1 Protein with Breast Cancer Metastasis to Ipsilateral Axillary Lymph Nodes

Breast cancer (BC) is the most common cancer affecting women worldwide. Mastectomy is the standard treatment for breast cancer. The purpose of axillary lymph nodes (ALN) dissection in mastectomy is to examine the status and the presence of metastasis in ALN. This study is an analytic observational cross-sectional study coupled with a categorical comparative study in 60 BC patient’s with- and without-ALN metastasis. The data that used in this study were histopathological data from patients medical record and immunohistochemistry (IHC) of RAC1 expression data. The immunohistochemistry was performed with a cutoff point based on the result of a receiver operating characteristics (ROC). This study combined BC tumor size, histopathological grade, and expression of RAC1 protein in correlation with known risk factors on metastasis to ALN. From 60 subjects, there were 63.3 % (38) ALN (+) and 36.7 % (22), ALN (−). BC of T2 tumor size, grade 2 and RAC1(−), the ALN metastasis chance was 0.6 %. T2 tumor size, grade 2 and RAC1(+), the ALN metastasis chance was 4.5 %. T2 tumor size, grade 3 and RAC1(−), the ALN metastasis chance was 3.0 %. T2 tumor, grade 3 and RAC1(+), the ALN metastasis chance was 18.8 %; T3 tumor size, grade 3 and RAC1(−), the ALN metastasis chance was 12.7 %. T3 tumor size, grade 3 and RAC1(+), the ALN metastasis chance was 52.2 %; T4b tumor size, grade 3, RAC1(−) and RAC1(+), the ALN metastasis chance was 40.8 and 83.8 %, respectively. We conclude that smaller tumor size and lower histopathological grading with RAC1(−) has lesser possibility to ALN metastasize. HIGHLIGHTS Breast cancer (BC) is a malignant neoplasm originating from epithelial cells of the ducts and breast glands in the area of the lobules and terminal ducts known as the terminal duct lobular unit (TDLU) and the surgery performed is breast-conserving treatment or mastectomy that examine the status and the presence of metastasis in ALN RAC1 is a member of the RHO-GTPase family that plays an important role in the migration process, loss of adhesions between cells, and tumor metastasis also found in BC so it was used as parameter in this study Tumor size, histopathological grading, and the expression of RAC1 protein has correlation with with BC metastasis to the axillary lymph nodes that the smaller the tumor size, the lower the histopathological grading with RAC1(-) GRAPHICAL ABSTRACT

The cell polarity protein Scribble is downregulated by the water channel aquaporin-5 in breast cancer cells.

Breast carcinomas originate from cells in the terminal duct-lobular unit. Carcinomas are associated with increased cell proliferation and migration, altered cellular adhesion, as well as loss of epithelial polarity. In breast cancer, aberrant and high levels of AQP5 are associated with increased metastasis, poor prognosis and cancer recurrence. AQP5 increases proliferation and migration of cancer cells, and ectopic expression of AQP5 in normal epithelial cells reduces cell-cell adhesion and increases cell detachment and dissemination from migrating cell sheets, the latter via AQP5 mediated activation of the Ras pathway. Here, we investigated if AQP5 also affects cellular polarity by examining the relationship between the essential polarity protein Scribble and AQP5. In tissue samples from invasive lobular and ductal carcinomas, the majority of cells with high AQP5 expression displayed low Scribble levels, indicating an inverse relationship. Probing for interactions via a GST pull-down experiment revealed that AQP5 and Scribble interacted. Moreover, overexpression of AQP5 in the breast cancer cell line MCF7 reduced both size and circularity of 3D spheroids and induced cell detachment and dissemination from migrating cell sheets. In addition, Scribble levels were reduced. An AQP5 mutant cell line, that cannot activate Ras (AQP5S156A), displayed unchanged spheroid size and circularity and an intermediate level of Scribble, indicating that the effect of AQP5 on Scribble is, at least in part, dependent of AQP5 mediated activation of Ras. Thus, our results suggest that high AQP5 expression negatively regulates the essential polarity protein Scribble and thus, can affect cellular polarity in breast cancer.