Assessment of Erythroid Chimerism in Sickle Cell Patients Undergoing HSCT By Digital Droplet PCR

Abstract Sickle cell disease (SCD) is an inherited autosomal recessive blood disorder associated with significant morbidity. Nonmyeloablative allogeneic hematopoietic stem cell transplantation (HSCT) is increasingly used in severely affected patients with SCD. These transplants result in mixed hematopoietic chimerism so accurate chimerism testing is important for monitoring the status of the transplant. Reconstitution of the erythroid compartment is essential. Since red cells lack a nucleus, DNA-based chimerism assays do not directly assess the chimerism in the erythroid compartment. Several studies have shown that the chimerism in the white cells and erythroid cells can be very different (W, C., etal. Exp. Hematol. 2003, 31:924; Andreanni, M., etal. Haematologica, 2011, 96:128). We developed a procedure for quantification of chimerism in the erythroid compartment of blood using RNA-based digital droplet PCR (ddPCR). The sensitivity and specificity of the assay was evaluated then tested post-transplant samples from sickle cell patients. Total RNA is reverse transcribed and amplified in a two-step RT-PCR approach. The PCR reaction containing allele-specific hydrolysis probes is partitioned into ~15,000 droplets then amplified. The copy number of HbA and HbS transcripts from cells of the erythroid lineage is determined with ddPCR (BioRad, QX-200). The %HbA and the %Donor can be calculated using the donor genotype (A/A or A/S). The assay is designed to have a sensitivity of 1% with donor genotype of A/A and 5% with donor genotype of A/S. Each post-transplant sample is tested in duplicate along with an AA control, SS control, AS control and 1% sensitivity controls (5 controls total). Thirty AA or SS samples were tested to assess assay specificity. The average %HbA detected in a SS sample was 0.03%; the average %HbS in a AA sample was as 0.02%. The background signal is significantly below the cutoff for 1% sensitivity. Using contrived samples with low, medium, and high %HbA, we demonstrate the assay is accurate and linear to 0-2%HbA across the reportable range of 0%HbA to 100%HbA. The % CV of the minor allele for samples in the range of 10%-90%HbA, were equal or below 15%. A total of 11 post-transplant samples from 7 transplanted sickle cell patients were tested, and the results were compared to DNA-based/FISH chimerism, if available. Our results were comparable with DNA-based chimerism (Table 1). Five of the samples had slightly higher %donor in the erythroid compartment compared to the white cell compartment. The erythroid chimerism reflected changes in chimerism status: the decrease then increase in chimerism (P2), stable chimerism (P5) and graft failure (P7). Abstract 563. Table 1: Erythroid chimerism status of post-transplant SCD patients Patient # Donor Genotype Sample # %HbA %Donor %Recipient %Donor by FISH/STR P1 AS 1 52 100 0 95 P2 AA 1 66 66 34 72 2 44 44 56 50 3 100 100 0 95 P3 AS 1 54 100 0 85 P4 AA 1 100 100 0 100 P5 AS 1 14 28 72 15 2 12 24 76 15 P6 AS 1 57 100 0 95 P7 AA 1 0 0 100 Rejecting graft 2 0 0 100 Rejecting graft Assessment of the chimerism in the erythroid lineage may be a better indicator of donor erythropoiesis. We describe an accurate and sensitive assay for monitoring erythroid chimerism and the effects of post-transplant therapies in sickle cell patients undergoing HSCT. This assay also demonstrates the feasibility measuring erythroid chimerism detection in other hematologic disorders, such as thalassemia. Disclosures No relevant conflicts of interest to declare. </jats:sec

Both ongoing suppression and clonal elimination contribute to graft-host tolerance after transplantation of HLA mismatched T cell-depleted marrow for severe combined immunodeficiency.

Abstract Lymphocytes from children with severe combined immunodeficiency who had been immunologically reconstituted with haploidentical T cell-depleted bone marrow were analyzed with regard to their immunologic recognition of donor, host, or third party alloantigens. When compared with freshly isolated donor lymphocytes, the engrafted donor cells exhibited markedly reduced to absent responses toward host Ag in primary or secondary MLC and cell-mediated lympholysis assays. However, under limiting dilution conditions, cytotoxic responses to host Ag could be demonstrated, indicating that small numbers of host reactive cells were present, although down-regulated at high responder cell doses. These results are consistent with prior observations in limiting dilution cultures that indicate that cells with the potential to lyse autologous target cells exist in the peripheral blood of all normal individuals. The number of host reactive cells present in these patients is significantly less than that present in cells isolated directly from the marrow donors, and is also less than the number of autocytotoxic cells normally seen in peripheral blood. Together, these observations indicate that two mechanisms contribute to donor host tolerance in these patients. The majority of host reactive cells appear to have undergone clonal deletion or inactivation, whereas the small residual host-reactive population appears to be under ongoing immunoregulatory control.</jats:p

Allogeneic bone marrow transplantation for patients with acute nonlymphocytic leukemia

Abstract Seventy patients with acute nonlymphocytic leukemia (ANLL) underwent allogeneic bone marrow transplantation following cytoreduction with total body irradiation and cyclophosphamide. Thirty patients underwent transplantation in first remission, 11 in second remission, 3 in third remission, and 26 in relapse. At a median follow-up of 30 mo, 17 of those in first remission and 7 of those in second remission survive in continuous remission, compared to 1 in third remission and 3 in relapse. The 3-yr Kaplan-Meier probability of disease-free survival among the various groups was 55% (+/- 9.2%) for the first remission transplants, 64% (+/- 14.5%) for second remission, 33% (+/- 20%) in third remission, and 10.3% (+/- 6.3%) in the relapse group. Statistical analysis showed a similar survival in the first and second remission groups that was significantly better than that seen in the third remission and relapse groups (p less than 0.01). The improved survival seen in the early remission groups was due to a significant decrease in the incidence of relapse posttransplant (p less than 0.01). These results confirm observations that a significant number of patients transplanted in first remission may achieve extended disease-free survival and document similar results for patients transplanted in second remission.</jats:p

Allogeneic bone marrow transplantation for patients with acute nonlymphocytic leukemia

Seventy patients with acute nonlymphocytic leukemia (ANLL) underwent allogeneic bone marrow transplantation following cytoreduction with total body irradiation and cyclophosphamide. Thirty patients underwent transplantation in first remission, 11 in second remission, 3 in third remission, and 26 in relapse. At a median follow-up of 30 mo, 17 of those in first remission and 7 of those in second remission survive in continuous remission, compared to 1 in third remission and 3 in relapse. The 3-yr Kaplan-Meier probability of disease-free survival among the various groups was 55% (+/- 9.2%) for the first remission transplants, 64% (+/- 14.5%) for second remission, 33% (+/- 20%) in third remission, and 10.3% (+/- 6.3%) in the relapse group. Statistical analysis showed a similar survival in the first and second remission groups that was significantly better than that seen in the third remission and relapse groups (p less than 0.01). The improved survival seen in the early remission groups was due to a significant decrease in the incidence of relapse posttransplant (p less than 0.01). These results confirm observations that a significant number of patients transplanted in first remission may achieve extended disease-free survival and document similar results for patients transplanted in second remission.</jats:p