Cellular and Molecular Characterization of Breast Cancer Chemotherapy-Related B Cell Lymphomas

Chemotherapy can prolong life expectancy in patients with hematologic malignancies. However, the long-lasting effects of chemotherapy on the function of hematopoietic and immune systems remain to be fully characterized. The hematopoietic and immune systems are vital for lifelong supply of blood cells and defense against pathogens and malignancies. Cyclophosphamide (CTX) and Doxorubicin (DOX) are frequently used together as neoadjuvant and adjuvant therapy for breast cancer. The main goal of our project is to characterize in depth long-term adverse effects of CTX/DOX treatment on the function of hematopoietic and immune systems.

A cohort of Ly5.2+ C57BL6 female mice were treated once a week for 5 consecutive weeks with 30 mg/kg CTX and 3 mg/kg DOX. Cohorts of age-matched control mice and CTX/DOX-treated mice were analyzed 15 months after the end of treatment. Comprehensive flow cytometry analysis of >50 hematopoietic and immune cell types (HSPCs, B cells, T cells, myeloid, erythroid and megakaryocytic lineages) from the bone marrow (BM) and spleen (SPL) of individual age-matched control and CTX/DOX-treated mice, in combination with BM and SPL cell transplant from each mouse has revealed development of CTX/DOX-related B cell lymphoma (BCL) in 17% of CTX/DOX-treated mice. Moreover, 13% of the CTX/DOX-treated mice showed early stages of BCL development, while the remaining 70% of CTX/DOX-treated and all age-matched control mice did not show any signs of BCL. Chemo-treated mice developing BCL exhibited expansion of lymphoid-biased short-term HSCs (ST-HSC), significant reduction of Long-term HSCs (LT-HSC) in BM and SPL and increased population of immature B cell in the SPL. Transplant of BM and SPL cells from CTX/DOX-treated lymphoma-bearing (CTX/DOX-BCL) mice into Ly5.1+ syngeneic recipient mice resulted in massive splenomegaly, expansion of Ly5.2+ lymphoid-biased ST-HSCs and CLPs in the SPL, reduced Ly5.2+ myeloid-biased HSCs in BM and SPL and reduced Ly5.2+ MPPs and CMPs cells in the BM. Additionally, all Ly5.2+ B cell developmental stages were increased in CTX/DOX-BCL recipients. B1 cells were also significantly increased in the lymphoma transplant recipient mice, mimicking B1 cell overproduction in their donors. These results indicate that CTX/DOX treatment increases the incidence of cancer therapy-related B cell lymphoma development.

Further immunophenotypic characterization of donor Ly5.2+ B cells in recipients identified coexistence of two BCL types: 1) CLL/SLL (CD19+ CD23+ CD20+ CD5+ B220-), and 2) Mantle Cell Lymphoma (MCL; CD19+ CD23- CD20+ CD5+ B220-), where MCL is predominant in BM and SPL. In contrast, mice transplanted with cells from CTX/DOX-treated "early stage lymphoma" mice did not have exorbitant amount of these two immunophenotypic populations in the SPL as compared to CTX/DOX-BCL recipient mice; but BM had a 2-fold increase of these cells compared to control. Likewise, transplant recipients of cells from "early stage lymphoma" and "normal" CTX/DOX donor mice had increased number of lymphoid-biased ST-HSCs in the SPL and reduced LT-HSCs in BM.

Adding to the adverse effects of CTX/DOX on the immune system, populations of Natural Killer (NK) and Natural Killer T (NKT) cells were significantly decreased in recipients of cells from all CTX/DOX-treated mice compared to control transplant recipients. More importantly, RNAseq analysis revealed: 1) reduced expression of NK receptors (NKG2D, NKp46, CD94, CD69), 2) downregulated expression of PI3K, Fyn and PLCγ genes involved in NK cell-mediated cytotoxicity, and 3) upregulation of BAG6 and ULBP1 genes,

known as suppressors of NK receptors, in CTX/DOX-BCL recipients compared to control recipients. Combined, these results suggest that attenuated numbers and impaired function of NK/NKT cells could be accountable for development of chemotherapy-related B cell lymphomas.

Dysregulation of the hematopoietic system, expansion of BCL populations and attenuated NK/NKT function/surveillance in CTX/DOX-treated mice and mice transplanted with CTX/DOX-BCL cells demonstrate some of the harmful effects of CTX/DOX on the hematopoietic and immune system, causing the co-development of CLL and MCL B cell lymphomas. Thus, it is important to investigate direct effects of CTX/DOX on NK/NKT cells and determine the molecular and cellular causes and cellular origin (HSC, Ly-HSC, CLPs, B1 cells) of cancer therapy-related B cell lymphomas.