Dottorato in INNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Titolo della tesi: Donor specific anti-HLA antibodies in Hematopoietic Stem Cell Transplantation. A stepwise project to establish a better definition of their role and a desensitization strategy.

ABSTRACT Introduction: In the setting of mismatched-hematopoietic stem cells transplantation (mmHSCT), considering the risk of graft failure (GF), the detection of antibodies directed against donor specific HLA allele(s) or antigen(s) (DSA) represents a contraindication to proceed with the same donor. In many cases, it is necessary to plan an immunosuppressive strategy to decrease DSA levels, thus reducing the risk of GF. Aim: The aim of the project was to evaluate our strategy for testing/monitoring DSAs and to define the best desensitization strategy (DS), with a prospective unicentric study, including all consecutive patients candidates for a mmHSCT. The main endpoints were as follows: 1. To determine the incidence of anti-HLA antibodies (Abs) and DSAs; 2. To define the role of DSAs against each of the HLA loci; 3. To determine the critical DSAs mean fluorescence intensity (MFI) cut-off for engraftment; 4. To determine the efficacy of the employed DS; 5. To define the correct timeframe for monitoring DSAs; 6. To determine how DSAs influence outcome in haploHSCT. In addition, we evaluated the results obtained in a retrospective, multicenter study GITMO/AIBT, coordinated by our center, involving 34 transplant centers (TC) in Italy. Methods: Anti-HLA Abs research was carried out using Luminex bead assays (Lifecode ID and LSA I/II-Immucor). The results were expressed as MFI; MFI >1000 was considered positive. If the patient had DSAs and only 1 available donor, a DS was employed, scheduled with Rituximab on day -15, two single-volume plasmapheresis procedures (PP) on days -9 and -8, intravenous immunoglobulin on day -7 and an infusion of HLA selected platelets for DSA absorption, in case of persistent Abs directed against class I HLA antigens. The DS employed was defined according to DSAs MFI and FCXM (flow cytometry cross match) results; in case of MFI >1000 and < 5000, and a negative FCXM, we employed a DS without PP. In addition, the results of the GITMO/AIBT study concerning the practice of DSA monitoring and DS employed in each TC were analyzed. Preliminary data: From 2014 to 2017, 20 out of 65 patients (30.77%) showed anti-HLA Abs; 4 of them were DSAs (20%, 6.15% of total). In 2 cases, an alternative donor was selected. Two patients were treated with the DS; both obtained neutrophil engraftment. Neither DSA rebound nor other complications were observed. Results-Prospective study: Since October 2017, 22/126 patients (17.46%) showed anti-HLA Abs, 5 of them DSAs (22%, 3.9% of total); 3 of them received DS following our protocol obtaining engraftment; in 1 of the 4 patients with DSAs, both the antibodies detection and the crossmatch, before starting the conditioning regimen, showed a negative result, so a DS was not necessary. One patient died before receiving HSCT, due to disease progression. DSA detection was performed every 7 days after HSCT for the 1st month, and then 60, 180 and 365 days following HSCT. Female sex (p=0.033) and a history of previous pregnancies or abortions (p=0.009) showed a statistically significant impact on alloimmunization. Factors associated with delayed PMN engraftment were patient’s female gender (p=0.039), bone marrow as stem cell source (p=0.025), and a high HSCT-specific comorbidity index (p=0.028). In our experience, none of the analyzed variables, including the detection of anti-HLA antibodies or DSAs, influenced the risk of developing GF or PGF. Multicenter study (GITMO/AIBT): Data obtained from 24/34 Italian TC showed that 382 out of 896 patients (43%) candidates for a mmHSCT had been screened. Our analysis was conducted on 771/896 patients with complete data. Of them, 322 (42%) were screened and 82 (25%) showed anti-HLA antibodies. Eighteen had DSAs (22%, 5.5% of patients screened). In 9 cases various DS were employed. In 3 cases an alternative donor was selected. Six patients were not treated, according to center policy. We found a statistically significant correlation between female sex (p=0.018) and the number of previous pregnancies (p=0.012) with anti-HLA antibodies detection. Pre-transplant blood transfusions (leucodepleted only in 87% of patients) had a statistical impact on a delayed full donor engraftment (p=0.001). Conclusions: Our analyses confirm that anti-HLA Abs are frequent, with a high probability of identifying DSAs. Our DS proved successful in reducing DSAs. Currently, in our country, there is no shared policy for anti-HLA Abs management in mmHSCT. We propose a prospective multicenter study to define and validate a consensus on DSAs management and DS to be shared with others Italian TC, GITMO and AIBT.