Immune Reconstitution after Allogenic Stem Cell Transplantation in Patients with Myelofibrosis
Authors: Farina Mirko , Chiarini Marco , Terlizzi Sofia , Galvagni Anna , Bernardi Simona , Leoni Alessandro , Morello Enrico , Radici Vera , Magliano Gabriele , Masina Lorenzo , Galli Marco , Re Federica , Quaresima Virginia , Moratto Daniele , Brugnoni Duilio , Malagola Michele , Polverelli Nicola , Russo Domenico 
Published: December 2024

Background
Allogeneic hematopoietic stem cell transplantation (alloHSCT) remains the only curative treatment for patients (pts) diagnosed with myelofibrosis (MF). Despite several improvements over time, post-transplant complications can still be severe and significantly affect pts' survival. Inflammation and bone marrow fibrosis may hinder immune reconstitution (IR) in MF pts, but there is limited data available. This study aimed to evaluate IR in MF pts undergoing alloHSCT and its impact on their outcomes.
Methods
We retrospectively evaluated IR in MF pts who underwent alloHSCT at the Bone Marrow Transplantation Unit of Spedali Civili di Brescia between April 2017 and April 2024. Absolute lymphocyte counts (ALC) and lymphocyte subtypes were assessed at +3, +6, +9, +12, +18, and +24 months (M) post-transplant.
IR was evaluated by flow cytometry using fresh whole blood samples. Monoclonal antibodies were employed to evaluate main lymphocyte populations, CD4/CD8 ratios, and T-cell subset (CD45RA; CD45RO).
Lymphocyte recovery was defined as ALC &gt;500/uL at M+3, and ALC &gt;750/uL from M+6 onward. The recovery of subtypes CD3+CD4+, CD3+CD8+ and CD19+ was classified as absolute counts &gt;200/uL.
Results
Since April 2017, 33 MF pts were transplanted at our Institution. The median age was 60.7 years (range 44-71), with 76% (25) being male. Seventeen out of 29 evaluable pts (59%) had an unfavorable karyotype. Twenty-four pts were JAK2 mutated, while 8 were CALR+ and 1 MPL+. Overall, 13 on 28 evaluable pts harbored a high molecular risk mutation. Five pts had DIPSS intermediate-1, 20 pts had intermediate-2, and 8 had high risk. Twenty-eight pts received JAK2 inhibitor before alloHSCT. Regarding transplant features, 16 pts received a myeloablative conditioning, and 27 received double-alkylators regimens (Thiothepa-Busulfan-Fludarabine). Donors were sibling, unrelated or haploidentical in 18.2%, 48.5% and 33.3% of cases, respectively. HCT-CI was high in 15 pts. Almost all pts (85%) received peripheral blood stem cell grafts. In haploidentical alloHSCT, pts received post-transplant cyclophosphamide, while the other pts received GvHD prophylaxis with cyclosporine and methotrexate (or mycophenolate); 21 pts received rabbit antithymocyte globulin (ATG).
After a median follow-up of 15.9 months (IQ 8-43), 12 pts had died (5 relapse, 4 infections, 3 acute GVHD) for a 5-year CIR, NRM and OS of 27.2% (95%CI 10.8-46.7), 19% (95%CI 7.5-34.6) and 54.3% (95%CI 28.5-74.4), respectively.
Serial ALC assessments showed a gradual increase from M+3 to M+18. Median counts reached 426/uL (115-1758) at M+3, 587/uL (254-2794) at M+6, 891/uL (148-3181) at M+9, and 1643/uL (438-4978) at M+18, representing 41%, 42%, 53% and 83% of recovered pts, respectively. Similarly, CD4+ recovery improved over time, with median CD4+ counts surpassing 200/uL only at M+9. CD4+ reconstitution at M+3 varied significatively according to donor type: haploidentical alloHSCT had a median of 104/uL, whereas sibling and MUD transplants were 171/uL and 142/uL, respectively (p 0.01). ATG treatment was not related to CD4+ reconstitution. CD4+CD45RA+ cells, more likely naïve cells, significatively increase at each time point, peaking at 37.7/uL at M+18. Conversely, CD3-CD56+/CD16+ NK cells significatively decreased from M+3 to M+24.
Pts not reconstituting CD4+ and ALC at M+9 were associated with a higher relapse rate (p 0.05) and showed a trend towards worse overall survival. At M+24, the cumulative incidence of relapse was 33% in pts with less than 200 CD4+/uL at M+9, compared to 0% in those with recovered CD4+ counts (p 0.05). Similarly, pts not recovering ALC at M+9 had relapse CI of 40% at M+24, compared to 0% in those with recovered ALC. ALC and CD4+ recovery at M+9 also impacted the maintenance of chimerism (p 0.05). CD4+ recovery at M+3 and M+6 was associated with a trend towards fewer infections. Additionally, not achieving CD4+ recovery at M+3 was linked to a higher risk of chronic GVHD.
Conclusion
MF pts exhibit slow immune reconstitution after alloHSCT, with median CD4+ count exceeding 200/uL only after 9 months, and CD4CD45RA+ counts peaking at 18 months. ALC and CD4+ recovery at 9 months is associated with lower late-relapse rates and lower chimerism loss. Despite being influenced by various factors (e.g., donor type), IR after alloHSCT in MF pts can be a valuable tool for assessing the risk of infections, GVHD development, and relapse.