P1007: SPLENOMEGALY DEVELOPMENT PRECEDES THE PROGRESS OF HEMATOPOIETIC STEM CELLS MIGRATION TO SPLEEN IN MURINE MYELOFIBROSIS MODELS

Topic: 15. Myeloproliferative neoplasms - Biology & Translational Research Background: Primary myelofibrosis has characteristics such as myelofibrosis (MF), splenomegaly, and extramedullary hematopoiesis. It is typically believed that in primary MF, hematopoietic stem cells (HSCs) migrate out of the bone marrow niche and are trapped in the spleen, causing extramedullary hematopoiesis and splenomegaly; however, their sequence is not elucidated completely. Aims: Our objective was to elucidate the developmental mechanisms of extramedullary hematopoiesis and splenomegaly. We also evaluated the timing of their emergence and that of MF and peripheral blood stem cells. To clarify pathological changes over time, we have developed a quantitative assessment method for MF using deep learning. Methods: We measured CD34+ cells/CD45+ cells in the peripheral blood (PB) of 19 healthy controls and 52 patients with myeloproliferative neoplasm (MPN). In addition, sequential analysis of MF, splenomegaly and HSCs migration was performed in two MF model mice. In mice, fibrosis of the entire femur can be evaluated, and we also developed a quantitative assessment method for MF using deep learning. The sequence of this method is shown in Figure. Results: The mean percentage of CD34+/CD45+ cells in healthy controls and patients with MPN with MF-0 to MF-3 was 0.11%, 0.19%, 0.25%, 4.11%, and 7.75%, respectively. Compared with healthy controls, CD34+/CD45+ cells in patients with MPN with MF-2 and those with MF-3 significantly increased. The mean percentage of CD34+/CD45+ cells in patients with MPN without splenomegaly (n = 41) and those with splenomegaly (n = 11) was 0.17% and 3.81%. Splenomegaly tended to be observed in higher MF-grade cases. Four romiplostim (Rom) induced MF models were sacrificed on days 1, 3, 5, 7, 9, 11, and 13 after Rom administration. The mean spleen weights were 72.2 mg, 83.8 mg, 122.3 mg, 160.2 mg, 191.1 mg, 265.9 mg, and 311.9 mg, respectively. The mean fibrosis area was 0.015%, 0.021%, 0.081%, 0.31%, 1.20%, 1.31%, and 0.71%, respectively. MF appeared on day 7 after the Rom administration and reached its peak on days 9 and 11. The mean spleen HSC numbers/1.0 × 104 nucleated cells were 0.01, 0.01, 0.02, 0.58, 1.39, 2.14, and 4.16, respectively. The mean PB HSC numbers/1.0 × 104 nucleated cells were 0, 0, 0, 0.06, 0.05, 0.14, and 0.27, respectively. HSCs in PB and spleen increased around day 7 and continued to increase over time. In this murine model, MF and HSC migration occur in parallel but are preceded by spleen enlargement. In Jak2V617F TG mice, the mean spleen weights of 8, 12, 16, 20, 24, and 34 weeks of age were 1.01 g, 0.90 g, 1.14 g, 0.98 g, 1.05 g, and 1.18 g, respectively. Splenomegaly had already occurred at 8 weeks of age and did not change much as the weeks progressed. The mean fibrosis areas of 8, 12, 16, 20, 24, and 34 weeks of age were 18.5%, 33.0%, 40.4%, 28.6%, 41.3%, and 42.9%, respectively. The mean spleen HSC numbers/1.0 × 104 nucleated cells were 0.36, 1.58, 0.80, 0.21, 0.23, and 0.19, respectively. The mean PB HSC numbers/1.0 × 104 nucleated cells were 0.53, 0.63, 2.08, 0.03, 0.36, and 0.14, respectively. Around 8 weeks of age, MF and splenomegaly appeared, but there was no rise in the number of spleen HSCs. Instead, this happened after 12 weeks of age, after a delayed increase. In addition, after 20 weeks, no increase in spleen and PB HSCs was found, indicating that the migration of HSCs to the spleen was completed around 12 to 20 weeks. In this murine model, MF and splenomegaly are complete before HSC migration reaches its peak. Summary/Conclusion: The new deep-learning-based method is effective for assessing successional changes in MF, and splenomegaly precedes HSC migration.Keywords: Hematopoietic stem cell, Cell migration, Myelofibrosis, Splenomegaly