Predicting the Outcome in Egyptian Patients with Severe Aplastic Anemia Following Allogeneic Hematopoietic Stem Cell Transplantation Using Matched Sibling Donors
Authors: Aly Mai , Awada Hassan , Sayed Douaa , ElBeih Esam , Abdel El Fattah Raafat , Naseer Refat Fathy Abdel-Aal , Alagooz Reem , Hafez Rania , Sultan Almetwaly M. , El-Sharkawy Nahla , Visconte Valeria , Mahfouz Reda Z. 
Published: November 2019

Patients presented with pancytopenia in the presence of bone marrow (BM) hypoplasia, aplastic anemia (AA), may get cured by specific medical care, if patients are under 40 years, mainly in the form of allogeneic hematopoietic stem cell transplantation (allo-HSCT) which is the treatment of choice for severe AA (SAA) in young age. In Egypt, SAA may get allo-HSCT only with availability of matched sibling (MSD). Success rate of allo HSCT-MSD is still suboptimal although it is a life challenging and expensive procedure with an outcome difficult to predict. Reports suggest that measurement of serum ferritin may help in predicting favorable MSD-alloHSCT outcome. Evidences showed that total infused CD34 cells and/ or measurements of hematogones (HGs) levels at the time of engraftment by flow cytometry may better predict the outcome of MSD-alloHSCT. HGs, normal B lymphocyte precursors, levels may be affected by the status of BM and humoral immunity. Elevated HGs have been noticed in healthy children, called reactive HGs. Regenerative HGs exist in non-malignant disorders such as autoimmune diseases, congenital cytopenia, acquired immunodeficiency and neoplastic disorders such as lymphoma. In addition they have been found in response to regeneration of humoral immunity post chemotherapy and post allo-HSCT. We aimed to examine predictive value of HGs in comparison to pre-transplant ferritin and/ or infused CD34⁺ counts for a favorable outcome of SAA patients treated with allo-HSCT from MSD. This is a prospective cross-sectional study, following institutional approval. In total 21 patients with SAA were consented and enrolled. All patients were treated with MSD allo-HSCT, between December 2013 and January 2016. Patients > 40years age, clinically unfit, with abnormal cytogenetic, diagnosed with inherited BM failure or paroxysmal nocturnal hemoglobinuria (PNH) were excluded. HGs were measured in BM samples at engraftment using flow cytometry where cells were stained with an anti-CD34, anti-CD38, anti- CD10, anti-CD19, and lineage (Lin) mixture (anti-CD3, -CD4, -CD8 -CD20, -CD14, and -CD56). Early HGs referred to both CD38ˉCD10⁺ CD19ˉCD34⁺ and CD38-CD10⁺CD19ˉCD34ˉ. Late HGs referred to CD38⁺CD10⁺CD19⁺CD34ˉ, and CD38⁺CD10ˉCD19⁺CD34ˉ cellss.
The mean ± SD of early HG was 0.8 ± 1.6 and median with range was 0.05 (0-7.8) while mean ± SD of late HG was 0.6 ± 0.5 (p=0.4) and median with range 0.4 (0-1.4). Total HG mean ± SD was 0.7±1.2 and median with range 0.2 (0 - 7.8).Interestingly the mean ± SD of HG levels were higher in survivors vs non-survivors (2.3 ± 3.4 vs. 0.2 ± 0.2; P=0.01). Infused CD34 count (10^6) were in survivors vs non-survivors (6.7 ± 3 vs 7.8 ± 4) while pre-transplant serum ferritin levels (pm/mL) were in survivors vs non-survivors (1493 ± 1025 vs 1213 ± 371) without showing significant differences. Using ROC curve analysis, it showed the following for engraftment HGs%, best cut-off value was 0.37%, with AUC of 0.88, likelihood ratio (LR) of 3.85, it was the most sensitive 76.9% (95% CI; 35.4-84.8) and specific 80% (95% CI; 28.4 - 99.5) predicting favorable MSD-alloHSCT outcome. Infused CD34⁺ count showed best cut-off level of 6.8 x 10⁶ cells/kg, 0.63 for AUC, 3.13 for LR; 62% sensitivity (95% CI; 46.2-95) and 80% specificity (95% CI; 28.4-99.5). Pre-transplant serum ferritin showed best cut-off level of 1337, 0.59 for AUC; 1.6 LR; 64% for sensitivity (95%CI; 35.14 - 87.24) and only 60% specific (95% CI, 14.66 - 94.73). Moreover, patients with high HG (>0.37%; 57% vs. <0.37%;43%) had significantly high white cell count (WBCs); (median; 5 x 10⁹ /L, range; 2-9 vs 3.5,1.3-5; 95% CI; -3.5 to -0.1; p=0.03), absolute neutrophil count (ANC); (0.7 ± 0.4 vs 2 ± 0.8; p=0.0004), platelet; (26 ± vs 89 ± 46; p=0.001), at engraftment time in addition to superior OS (P=0.005). There was no difference in incidence of acute and chronic GVHD, infections or transfusion frequency rate among survivors with HGs below or above 0.37%. Noticeably donor age of transplanted patients with high HGs tended to be younger. Neither pre-transplant ferritin level nor infused CD 34⁺ count showed any impact on the OS (P=0.9 and 0.1 respectively).
In sum HGs% FCM assay may be a highly sensitive and specific biomarker predicting HSCT outcome in SAA patients.. Estimating HGs% at engraftment time seem to be a promising predictor for the outcome of MSD-alloHSCT in SAA patients. SAA patients with HGs 0.37% or higher had better OS.

Disclosures
No relevant conflicts of interest to declare.