Abstract Background: Hormone receptors and proliferation markers are critical parameters for treatment selection of breast cancer patients. The expression of different parameters are currently assessed separately in different tissue sections but it is unclear how different parameters are co-expressed in a single cell in breast cancer tissues. Samples and Methods: Breast cancer tissues from fifty-one patients with ER-positive HER2-negative breast cancer were analyzed. Expressions of ER and PgR were assessed in association with Ki67 using dual fluorescence immunohistochemistry with specific antibodies: SP-1 (abcam, Tokyo), 1E2 (Roche Diagnostics GmBH, Germany), and MIB1 (Dako Japan, Tokyo), respectively. More than 500 cancer cells were assessed in each tissue. Expression levels of each marker in a single cell were semi-quantitatively assessed by MetaMorph image analyzer (Molecular Devices Japan, Tokyo). All statistical analyses were performed using JMP ver.8.01 (SAS Institute Japan, Tokyo). Results: To validate the system, Ki67 LI in breast cancer tissues were compared between this system and the regular DAB system. The two systems showed a good concordance (p < 0.0001). All cancer tissues expressed ER. There were two distinct populations among Ki67-positive proliferating cells according to PgR expression status: PgR-positive proliferating cells and PgR-negative proliferating cells. Since cell proliferation is regulated in cancer cells by at least two different drivers including hormone receptor and growth factor, it is conceivable that these two populations depend on different driving systems. Indeed, tissues with dominantly PgR-positive proliferating cells showed mostly histological grade 1 (15/20, 75%), while most of tissues with PgR-negative proliferating cells showed grade 2 and 3 (22/31, 71%) (p = 0.0025 by chi square test). Moreover, the multivariate analysis using the ordered logistic regression analysis showed that PgR status in proliferating cells is an independent factor associated with histological grade (p = 0.003) while PgR expression rate and Ki67 LI were not (p = 0.3 and 0.25, respectively). Conclusion: PgR status in Ki67-positive proliferating cells is associated with histological grade in ER positive HER2-negative breast cancers. Our results suggest that different driving systems give different expression patterns of PgR and Ki67 at a single cell level, which may distinguish between luminal A and luminal B cancers. Citation Format: Takayuki Ueno, Hirotsugu Isaka, Hiroki Itoh, Kaisuke Miyamoto, Manami Kitamura, Shigeru Imoto. Analysis of in situ expression of hormone receptors and proliferation marker at a single cell level in breast cancer tissues. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3484.