Kachergus, Jennifer M.

Abstract Brain white matter tracts undergo structural and functional changes linked to late-life cognitive decline, but the cellular and molecular contributions to their selective vulnerability are not well defined. In naturally aged mice, we demonstrate that senescent and disease-associated microglia (DAM) phenotypes converge in hippocampus-adjacent white matter. Through gold-standard gene expression and immunolabeling combined with high-dimensional spatial mapping, we identified microglial cell fates in aged white matter characterized by aberrant morphology, microenvironment reorganization, and expression of senescence and DAM markers, including galectin 3 (GAL3/Lgals3), B-cell lymphoma 2 (Bcl2), and cyclin dependent kinase inhibitors, including Cdkn2a/p16ink4a. Pharmacogenetic or pharmacological targeting of p16ink4a or BCL2 reduced white matter GAL3+ DAM abundance and rejuvenated microglial fimbria organization. Our results demonstrate dynamic changes in microglial identity in aged white matter that can be reverted by senotherapeutic intervention to promote homeostatic maintenance in the aged brain.

Abstract Cerebral malaria is the deadliest complication that can arise from Plasmodium infection. CD8 T-cell engagement of brain vasculature is a putative mechanism of neuropathology in cerebral malaria. To define contributions of brain endothelial cell major histocompatibility complex (MHC) class I antigen-presentation to CD8 T cells in establishing cerebral malaria pathology, we developed novel H-2Kb LoxP and H-2Db LoxP mice crossed with Cdh5-Cre mice to achieve targeted deletion of discrete class I molecules, specifically from brain endothelium. This strategy allowed us to avoid off-target effects on iron homeostasis and class I-like molecules, which are known to perturb Plasmodium infection. This is the first endothelial-specific ablation of individual class-I molecules enabling us to interrogate these molecular interactions. In these studies, we interrogated human and mouse transcriptomics data to compare antigen presentation capacity during cerebral malaria. Using the Plasmodium berghei ANKA model of experimental cerebral malaria (ECM), we observed that H-2Kb and H-2Db class I molecules regulate distinct patterns of disease onset, CD8 T-cell infiltration, targeted cell death and regional blood–brain barrier disruption. Strikingly, ablation of either molecule from brain endothelial cells resulted in reduced CD8 T-cell activation, attenuated T-cell interaction with brain vasculature, lessened targeted cell death, preserved blood–brain barrier integrity and prevention of ECM and the death of the animal. We were able to show that these events were brain-specific through the use of parabiosis and created the novel technique of dual small animal MRI to simultaneously scan conjoined parabionts during infection. These data demonstrate that interactions of CD8 T cells with discrete MHC class I molecules on brain endothelium differentially regulate development of ECM neuropathology. Therefore, targeting MHC class I interactions therapeutically may hold potential for treatment of cases of severe malaria.

Abstract Purpose: Young women’s breast cancers (YBCs), defined herein as breast cancer (BC) diagnosed at age ≤ 40 years, are etiologically and biologically distinct from postmenopausal BCs, demonstrating a higher proportion of estrogen receptor (ER) negative subtypes and a worse prognosis. Studies have not assessed whether risk factors such as recent parity and body mass index (BMI) are linked to protein expression profiles in YBCs. Accordingly, we examined protein expression assessed with GeoMx® Digital Spatial Profiling (DSP) in 640 YBCs prepared as tissue microarrays (TMAs) in the Young Women’s BC Study (YWBCS). Methods: The YWBCS is a multi-institutional cohort of 1,297 women with incident BC diagnosed at age <40 years identified through medical record review, with risk factor data collected via questionnaires. TMAs including 1,505 cores of 640 YBCs that were collected prior to therapy and were suitable for molecular analysis were evaluated for 72 markers with NanoString GeoMx DSP. Associations of log-transformed biomarker expression with BMI and time from last live birth to BC diagnosis were carried out using linear mixed modeling approaches, accounting for multiple tumor tissue cores per individual. Results: Mean age at BC was 35.5 years (range 17-40). Tumor subtypes based on clinical immunostaining were the following: luminal B 46%; luminal A 36%; triple negative 12% and HER2+ 6%. Of 72 biomarkers tested, 53 (74%) were evaluable after QC and normalization. GeoMx ER and PR protein levels were higher in luminal A and luminal B YBCs than in HER2+ and TNBC (p=2x10-94 and 2x10-52, respectively), supporting the accuracy of the GeoMx assay. Compared to nulliparas or those with most recent birth > 3 years before BC, those who gave birth ≤ 3 years prior to BC had tumors with higher expression of PR (8x10-6) and ER (p=0.006), and lower expression of BCL-2 (p=0.006). In contrast, women with last birth ≥ 6 years prior to BC had higher expression of CD8 (p=0.006) and PD-1 (p=0.001) than nulliparas and those with more recent births. Compared to women with BMI between 18.5 and 29.9, those with higher or lower BMIs had higher expression of CD34 (p=0.0003), CD4 (p=0.0009) and CD14 (p=0.001). BCL-2 expression was higher among women with BMI < 18.5 compared to those with higher values (p=0.005). Women with BMI of 30+ had higher β2-microglobulin levels than those with lower BMIs (p=0.003). Inverse dose-response associations (with higher BMI related to lower levels) were observed for expression of ER (p=0.01), pan cytokeratin (p=0.004) and four proteins in the p13K/AKT signaling pathway (p ≤ 0.01 for each). Conclusions: Preliminarily, we identify differences in hormonal and immune markers in YBCs in relation to timing of births and BMI, suggesting that these factors may contribute to etiologic heterogeneity. Multivariate modeling is ongoing to incorporate molecular subtype and other risk factors. Citation Format: Robert A. Vierkant, Laura M. Pacheco-Spann, Stacey J. Winham, Jennifer M. Kachergus, Ji Shi, Craig Snow, Fergus J. Couch, Janet E. Olson, Chen Wang, Derek C. Radisky, Amy C. Degnim, E. Aubrey Thompson, Laura C. Collins, Kathryn J. Ruddy, Ann H. Partridge, Mark E. Sherman. Associations of parity and body mass index with NanoString digital spatial protein profiling in early onset breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1000.

AbstractBackgroundNeurofibrillary tangles are dynamic entities with a lifespan encompassing three maturity levels: intracellular pretangles, intracellular mature tangles, and extracellular ghost tangles. Proteins are suggested to be differentially expressed throughout the tangle lifespan; however, past studies may be limited by spatial expression or number of proteins investigated. We sought to assess differences in protein expression across the tangle lifespan using GeoMxTM Digital Spatial Profiler (DSP) by NanoString, a new technology allowing for spatially derived multiplex investigation.MethodWe used DSP to measure protein expression in hippocampi from Alzheimer’s disease (n=6) and nondemented controls (n=2) using the “Human Neural Cell Profiling Core” and “Alzheimer’s Pathology” module. Regions of interest (ROIs) were classified by the major tangle maturity level recognized by a primary fluorescent conjugated tau antibody. Protein expression was normalized to the geomean of the three housekeeping genes. The tau‐positive segment was specifically analyzed for comparisons. ANOVA was used for group‐wise comparisons and t‐test using the Benjamini‐Yekutieli adjustment for multiple tests was used for pair‐wise comparisons.ResultAs expected, cytoskeletal (e.g., MAP2, Tau) and neuronal (e.g., NeuN) markers generally decreased through the neurofibrillary tangle lifespan (Figure). Markers profiled as neurodegenerative (e.g., amyloid‐β1‐42, APOE, APP) generally increased through tangle maturity levels, except for TDP‐43 which decreased. Interestingly, there was no significant difference in ubiquitin expression. Astrocytic GFAP and S100B expression was significantly increased through each maturity level. Expression of microglial‐specific markers P2RY12 and TMEM119 was significantly elevated in ghost tangles compared to mature tangles. Additional microglial/macrophage markers including Iba‐1 and CD68 were generally increased as neurofibrillary tangles matured. Interestingly, APOE expression remained low in pretangles and mature tangles before dramatically increasing in ghost tangles (Figure).ConclusionOur findings suggest that proteins are differentially expressed throughout the tangle lifespan and the majority followed a monotonically directed pattern. Non‐monotonically directed protein patterns may reflect the death of the neuron during the ghost tangle maturity level. Future studies will test the hypothesis that the microenvironment of the hippocampus is altered with increasing vulnerability to tangle pathology.

Abstract Background: Biopsy diagnoses of benign breast disease (BBD) confer a 1.5- to 4-fold increased risk of developing breast cancer (BC) compared with women without BBD. Previously, we reported that decreased numbers of specific immune cell types in lobules of BBD biopsies predicted increased BC risk, suggesting the promise of future tissue biomarker studies to define BC risk markers among BBD patients. Thus, we applied protein-based GeoMx® Digital Spatial Profiling (DSP) to BBD biopsies preceding BC (cases) and to BBD biopsies from cancer-free patients (controls) to identify possible BC risk markers, which we then evaluated in subsequent BC tissues and in surrounding normal lobules of cases. Methods: Archived pathology slides of BBD biopsies were reviewed and used to guide preparation of TMAs containing 1.0-mm diameter FFPE cores of lobules from an age- and cohort-period-matched set of 91 cases and 88 controls from the Mayo Clinic BBD Cohort. For patients who later developed BC, we prepared TMAs of BC tissue and surrounding mapped normal lobules. We applied GeoMx® DSP (immune and canonical signaling proteins) to both sets of TMAs. Following QC and data normalization, associations of case status with log-transformed biomarker expression in lobules of BBD biopsies were carried out using linear mixed modeling approaches, accounting for multiple ROIs per individual. Biomarkers significantly associated with case status (p<0.05) were further examined in BCs and adjacent normal lobules, using similar approaches. Results: The mean age at BBD biopsy was 52 years, and at BC diagnosis of cases, 61.4 years (mean time from BBD to BC was 10.2 years). A family history of BC was more frequent among cases (70% versus 43%; chi-square p=0.002). Of 72 biomarkers tested, 46 (64.4%) were evaluable after QC and normalization and 5 were associated with BC risk after adjustment for family history of BC: BCL2 (p=0.005), STING (p=0.006), CD44 (p=0.02), S100 protein (p=0.03) and pan-AKT (p=0.05); each showed higher levels in lobules of BBD biopsies of controls than cases. Three unique patterns appeared when examining these biomarkers across tissue type within cases: for BCL2 (p=5 x 10-9) and STING (p=2 x 10-19), levels were high in both BC and lobules surrounding BC but low in preceding BBD; for GAPDH (p=4 x 10-53) and pan-AKT (p=2 x 10-33), levels were high in BC, low in preceding BBD, and moderate in lobules surrounding BC; and for CD44 (p=2 x 10-6) and S100B (p=2 x 10-49), levels were low in BC, high in lobules surrounding BC and moderate in preceding BBD. Conclusions: Using a novel TMA of lobules in combination with DSP, we preliminarily identified immune-based and PI3 kinase-related protein biomarkers in BBD biopsies associated with BC risk. In case-only analyses, these markers demonstrated complex differences between lobules in BBD biopsies, subsequent BCs and adjacent normal lobules. Citation Format: Robert Alan Vierkant, Jodi M. Carter, Stacey J. Winham, Chen Wang, Jennifer M. Kachergus, Ji Shi, Raymond M. Moore, Bryan M. McCauley, Laura M. Pacheco-Spann, E A. Thompson, Derek C. Radisky, Amy C. Degnim, Mark E. Sherman. Towards prediction of breast cancer risk in benign biopsies with high-plex GeoMx spatial protein profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2203.

Abstract Background: The importance of the antitumor immune response in TNBC is well established. TNBC with higher TILs are less likely to recur and more responsive to systemic therapy. Likewise, PD-L1+ TNBC are more likely to benefit from chemoimmunotherapy. However, TNBC is highly heterogeneous. Of the TNBC molecular subtypes, LAR TNBC is less sensitive to systemic therapy, has lower TILs and lower rates of PD-L1 positivity. The role of other immune related proteins in LAR TNBC is not well established. Here, we evaluated differentially expressed (DE) immune related proteins in the stromal and intratumoral compartments of LAR vs non-LAR TNBC tumors. Methods: We used the Nanostring GeoMX DSP platform to quantitate 58 proteins within spatially distinct intraepithelial, cytokeratin (CK)-positive tumor segments and adjacent CK-negative/nuclei-positive stromal segments in 248 TNBC tumors included in a tissue microarray generated from a cohort of pts with centrally confirmed TNBC who underwent breast surgery without prior neoadjuvant therapy. A subset (n=111) underwent bulk tumor RNA sequencing and were classified as LAR or non-LAR TNBC. DE proteins were identified using a negative binomial generalized linear model (SNR>2, p<0.05). A targeted set of DE proteins was dichotomized at the 80th percentile. Results: Of 111 TNBC tumors, 17 (15%) were LAR and 94 (85%) non-LAR. Compared to non-LAR TNBC, pts with LAR TNBC were older (age ≥50: 82% vs 52%, p<0.01), with tumors that were more often of apocrine histology (35% vs 0%, p <0.01), grade 1-2 (24% vs 1%, p<0.01), and had lower Ki67 (Ki67 ≤15: 24% vs 11%, p=0.06). Most tumors were T1-2 (94% vs 93%, p=0.82) and N0 (53% vs 62%, p=0.09), respectively. As expected, expression of most immune-related proteins was higher in the stromal vs the intratumoral compartment for both LAR and non-LAR TNBC. When focusing on the stromal compartment, expression of multiple immune related proteins was significantly lower in LAR compared to non-LAR TNBC, including the pan-leukocyte marker CD45 (log-2 fold change [log2FC]: 0.552, p=0.05), the macrophage marker CD14 (log2FC: 0.834, p=0.06), CD44 (lof2FC: 0.637, p=0.07), and the immune checkpoint proteins IDO1 (log2FC: 0.914, p=0.04), VISTA (log2FC: 0.471, p=0.07), ICOS (log2FC: 0.444, p=0.08), and STING (log2FC: 0.544, p=0.09). Proteins with expression levels too low for comparisons included PD-L1, LAG3, FOXP3 and BCL-2. When focusing on the intratumoral compartment, expression of most immune-related proteins was very low in both LAR and non-LAR TNBC. Like in the stromal compartment, CD45 expression was lower in LAR TNBC (log2FC: 0.78, p=0.02). Expression of the immune checkpoint B7-H3 was lower in LAR TNBC (log2FC: 0.737, p=0.02), while expression of the T cell marker CD127 was higher (log2FC: -0.528, p=0.34). With regards to relevant non-immune markers, expression of Ki67 was lower in LAR TNBC (log2FC: 0.5498, p=0.05), consistent with the clinical assay. Conclusion: In this ultra high-plex spatial analysis, we provide first insights into the differential expression at the protein level of several targetable immune checkpoint molecules in LAR vs non-LAR TNBC. The lower expression of several immune related proteins in LAR TNBC is consistent with the hypothesis that LAR TNBC exhibits a “cold” immune microenvironment compared to other TNBC subtypes, potentially rendering itself less susceptible to immunotherapy-based strategies. These data support the need to consider TNBC molecular subtypes in future evaluations of immune-based therapeutic approaches. Funding: This work was supported by NIH grant P50CA116201 to RLF, JMC, KRK, FJC, DZ, JNI, and MPG; BCRF grant 19-161 to EAT and NCATS grant CTSA KL2 TR002379 to RLF. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH Citation Format: Roberto A Leon-Ferre, Jodi M. Carter, David M. Zahrieh, David W. Hillman, Saranya Chumsri, Yaohua Ma, Jennifer M. Kachergus, Xue Wang, Judy C. Boughey, Minetta C. Liu, James N. Ingle, Krishna R. Kalari, Jose C. Villasboas Bisneto, Fergus J. Couch, E. Aubrey Thompson, Matthew P. Goetz. Digital spatial profiling of immune-related proteins in luminal androgen receptor (LAR) vs non-LAR triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-01.

Abstract 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.

Abstract Purpose: Programmed death ligand 1 [PD-(L)1]-targeted therapies have shown modest survival benefit in triple-negative breast cancer (TNBC). PD-L1+ microenvironments in TNBC are not well characterized and may inform combinatorial immune therapies. Herein, we characterized clinicopathologic features, RNA-based immune signatures, and spatially defined protein-based tumor–immune microenvironments (TIME) in early-stage PD-L1+ and PD-L1− TNBC. Experimental Design: From a large cohort of chemotherapy-naïve TNBC, clinicopathologic features, deconvoluted RNA immune signatures, and intraepithelial and stromal TIME (Nanostring GeoMX) were identified in subsets of PD-L1+ and PD-L1− TNBC, as defined by FDA-approved PD-L1 companion assays. Results: 228 of 499 (46%) TNBC were PD-L1+ (SP142: ≥1% immune cells-positive). Using PD-L1 22C3, 46% had combined positive score (CPS) ≥ 1 and 16% had CPS ≥10. PD-L1+ TNBC were higher grade with higher tumor-infiltrating lymphocytes (TIL; P &lt; 0.05). PD-L1 was not associated with improved survival following adjustment for TILs and other variables. RNA profiles of PD-L1+ TNBC had increased dendritic cell, macrophage, and T/B cell subset features; and decreased myeloid-derived suppressor cells. PD-L1+ stromal and intraepithelial TIMEs were highly enriched in IDO-1, HLA-DR, CD40, and CD163 compared with PD-L1-TIME, with spatially specific alterations in CTLA-4, Stimulator of Interferon Genes (STING), and fibronectin. Macrophage- and antigen presentation–related proteins correlated most strongly with PD-L1 protein. Conclusions: In this early-stage TNBC cohort, nearly 50% were PD-L1+ (SP142 companion assay) while 16% were PD-L1+ with the 22C3 companion assay. PD-L1+ TNBC had specific myeloid-derived and lymphoid features. Spatially defined PD-L1+ TIME were enriched in several clinically actionable immune proteins. These data may inform future studies on combinatorial immunotherapies for patients with PD-L1+ TNBC. See related commentary by Symmans, p. 5446