Arshad, Muhammad Asad

Abstract Purpose Breast cancer risk is elevated in pathogenic germline BRCA 1/2 mutation carriers due to compromised DNA quality control and elevated mutagenic load. We hypothesized that if immunosurveillance promotes tumor suppression, then normal breast lobules from BRCA carriers may demonstrate higher immune cell densities. Methods We assessed immune cell composition in normal breast lobules from age-matched women with progressively increased breast cancer risk, including 1) 19 women who donated normal breast tissue to the Komen Tissue Bank (KTB) at Indiana University Simon Cancer Center, 2) 15 women with biopsy-identified benign breast disease (BBD), and 3) 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 Compared to BBD tissues, normal breast lobules from BRCA carriers had significantly higher densities of CD8 + cells (median 354.4 vs 200.0 cells/mm2, p = 0.01). Compared to KTB normal donor breast tissues, normal breast lobules from BRCA carriers also had significantly higher densities of CD4+ (median 116.3 vs 17.7 cells/mm2), CD8+ (354.4 vs 150.9 cells/mm2), CD11c+ (3.5% vs 0.4% pixels positive), and CD68+ (237.5 vs 57.8 cells/mm2) immune cells (each p < 0.001). Conclusions These preliminary analyses show that normal/benign breast lobules of BRCA mutation carriers contain increased immune cells compared with BBD and normal donated tissues. Future studies will be helpful to define relationships of mutations and immune responses.

Abstract Introduction: The immune microenvironment of the breast is critical in the development and progression of breast cancer. By analyzing the immune components of breast tissues from patients at varying degrees of breast cancer risk, we may gain enhanced understanding of breast cancer risk and mechanisms of carcinogenesis. Methods: We compared the immune cell composition in breast lobules of age-matched patients with normal and benign breast disease (BBD) at progressively lower breast cancer risk: 1) 18 women with germline mutations in (BRCA1/2); 2) women at intermediate risk (BBD), and 3) women at normal risk (Komen Tissue Bank- (KTB)). We performed immunohistochemical stains for T lymphocytes (CD4, CD8); dendritic cells (CD11c); B lymphocytes (CD20) and macrophages (CD68) and quantitated immune cell densities in up to 10 representative lobules per sample. Results: The median age of all groups was 43.8 years (range: 34-67). BRCA carriers and BBD patients had a higher percentage of fibrocystic lobules (50% fibrocystic) compared to KTB samples (15% fibrocystic), p&lt;0.001. BRCA carriers had significantly higher densities of CD4, CD8, CD11c, and CD68 positive immune cells compared to KTB donor breast tissues, see Table. Compared with BBD patients, mutation carriers had higher densities of CD8+ cells and suggestively higher CD4+ cells, with similar results for other markers assessed. Conclusions: Preliminarily, these data showed that benign prophylactic breast tissues from BRCA mutation carriers contain increased numbers of several immune cell types versus tissues from patients at lower breast cancer risk, including BBD patients and research tissue donors. Further investigation is needed to confirm and expand these findings in relation to breast cancer risk. Table: Pairwise Wilcoxon rank-sum p-values comparing immune cell densities in breast lobules from women with BRCA germline mutations, vs BBD and KTB groups. All Lobules BRCA BBD KTB BRCA vs BBD BRCA vs KTB Median Median Median p-value p-value (IQR) (IQR) (IQR) CD4+, cells/mm2 116.3 63.8 17.7 0.08 &lt;0.001 (46.4-183.6) (25.0-121.1) (13.3-40.6) CD8+, cells/mm2 354.4 200 150.9 0.01 &lt;0.001 (285.5-473.2) (118.9-385) (110.1-225.0) CD11c+, % pp 3.5 3.4 0.4 0.66 &lt;0.001 (1.9-6.8) (1.6-4.7) (0-0.6) CD20+, cells/mm2 6.2 16.7 0 0.16 0.65 (0-25.4) (0-58.3) (0-20.8) CD68, cells/mm2 237.5 219 57.8 0.99 &lt;0.001 (122.9-281.8) (75.0-459.4) (29.2-100.0) IQR - Interquartile Range pp - pixel positive Citation Format: Joshua W. Ogony, Tanya L. Hoskin, Melody L. Stallings Mann, Stacey J. Winham, Rushin Brahmbhatt, Muhammad Asad Arshad, Alvaro Pena Jimenez, Teresa M. Allers, Mark E. Sherrman, Daniel W. Visscher, Derek C. Radisky, Amy C. Degnim. Immune biomarkers in breast tissues of patients with BRCA1/2 mutations, benign breast disease, and normal tissue donors [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 5947.

Abstract Introduction: The role of macrophages in the tumor microenvironment is of increasing interest for tumor progression and invasion. Here we investigated the difference in CD68+ cell density in tissue samples from women with Benign Breast Disease (BBD) and women with no clinical breast disease from the Komen Tissue Bank (KTB) at Indiana University. Method: Archived breast tissue samples from women with BBD and no clinical breast disease were obtained for this age-matched case-control study. BBD samples included cases [later got Breast Cancer (BC)] and controls (did not get BC). Samples were characterized via H&E stain for epithelial proliferation [normal (N), fibrocystic non-proliferative (NP), proliferative (P), or atypia (A)]. CD68+ cell density was obtained by digital image quantitation on a per lobule basis (Total CD68+ cells/mm2). Statistical analysis was performed with mixed effects regression. Results: Among 276 women (median age 53, range 35-79) comprising 92 age-matched triplets, 2508 lobules were evaluated [789 KTB, 839 BBD case, 880 BBD control]. 1407 (56%) lobules were normal and 1101 (44%) were fibrocystic {NP [613 (56%)], P [448 (41%)] and A [40 (4%)]}. Out of 2508 lobules analyzed, 2362 (94%) had CD68+ cells, while 146 (6%) showed zero CD68+ cells [19 (2%) BBD cases, 20 (2%) BBD controls, 107 (14%) KTB, p&lt;0.0001]. KTB samples showed significantly lower (p&lt;0.0001) CD68+ density (median 136 cells/mm2) as compared to BBD cases (median 313 cells/mm2) and BBD controls (median 299 cells/mm2). (See Table) For each lobule type, KTB samples had lower CD68+ density compared with the same lobule type in BBD samples (p&lt;0.0001). Conclusion: CD68+ cells are more frequent in breast lobules from women with BBD compared to normal donors. This finding in premalignant breast tissues is consistent with inflammation as a driver of tumorigenesis. BBD case (n = 92 patients)BBD control (n = 92 patients)KTB(n = 92 patients)n lobulesMedian (IQR)n lobulesMedian (IQR)n lobulesMedian (IQR)Overall839313 (165 - 538)880299 (164 - 527)789136 (55 - 272)By Lobule TypeNormal344310 (177 - 557)434331 (177 - 567)629128 (55 - 255)All non-normal/fibrocystic lobules combined495314 (157 - 527)446274 (152 - 478)160159 (58 - 393)Fibrocystic non-proliferative231319 (153 - 543)263274 (140 - 496)119163 (71 - 359)Fibrocystic proliferative246295 (153 - 496)169277 (153 - 478)33128 (30 - 385)Atypia18484 (313 - 739)14260 (223 - 375)8216 (0 - 493)IQR - Interquartile range (25th percentile - 75th percentile) Citation Format: Muhammad A. Arshad, Daniel W. Visscher, Tanya L. Hoskin, Rushin D. Brahmbhatt, Alvaro Pena Jimenez, Melody L. Stallings Mann, Erin E. Miller, Linda M. Murphy, Jodi M. Carter, Stacey J. Winham, Keith L. Knutson, Derek C. Radisky, Amy C. Degnim. CD68+ immune cells show different infiltration patterns in tissue samples from women with no clinical breast disease and those who have benign breast disease. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2364. doi:10.1158/1538-7445.AM2015-2364