Donor Lymphocyte Infusion (DLI) post allogeneic stem cell transplant (allo-SCT) in Acute Myeloid Leukemia (AML) and High-Grade Myelodysplastic Syndrome (MDS). A longitudinal retrospective study using peripheral blood (PB) CD34+ and CD3+ donor chimerism (DC) monitoring.

INTRODUCTION: This longitudinal study was based on the outcomes of Donor Lymphocyte Infusion (DLI) for falling peripheral blood (PB) CD34+ and CD3+ donor chimerism (DC). METHODS: From 2012 to 2018, data was collected from the BMT database and electronic medical records (EMR). The primary objective was to compare the indication for DLI based on falling PB CD34+ or CD3+ DC in patients post allo-SCT for AML and MDS and their overall survival (OS). RESULTS: 18/70 patients met the inclusion criteria. Indications for DLI were i) falling PB CD34+ DC ≤ 80 % with morphological relapse, ii) falling PB CD34+ DC ≤ 80 % without morphological relapse and iii) falling PB CD3+ DC ≤ 80 % without falling PB CD34+ DC. Log rank analysis showed falling PB CD34+ DC and morphological relapse had significantly lower OS. Linear regression demonstrated better OS post DLI if there was PB CD34+ and CD3+ chimerism response at 30 days (p = 0.029), GVHD (p = 0.032) and tapering immunosuppression at the time of falling DC (p = 0.042). CONCLUSION: DLI for PB CD34+ DC values ≤ 80 % and morphological relapse had the lowest OS. In this study, full DC was achieved after DLI even with a PB CD3+DC value as low as 13 %, provided the PB CD34+ DC remained > 80 %. Further research is vital in CD34+ DC as a biomarker for disease relapse and loss of engraftment.

Peripheral Blood CD34 Donor Chimerism has Greater Clinical Utility Than CD3 for Detecting Relapse after Allogeneic Stem Cell Transplantation for Acute Myeloid Leukemia or Myelodysplastic Syndrome.

Monitoring of donor chimerism (DC) may detect early relapse following allogeneic hematopoietic stem cell transplantation (allo-SCT) for acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Most centers use unfractionated peripheral blood or T-cells to monitor DC, although CD34+ DC may be more predictive. The limited adoption of CD34+ DC may be due to the lack of detailed, comparative studies. To address this knowledge gap, we compared peripheral blood CD34+ and CD3+ DC in 134 patients who underwent allo-SCT for AML or MDS. In July 2011, the Alfred Hospital Bone Marrow Transplantation Service adopted routine monitoring of DC in the lineage-specific CD34+ and CD3+ cell subsets from peripheral blood at 1, 2, 3, 4, 6, 9, and 12 months post-transplantation for AML or MDS. Immunologic interventions, including rapid withdrawal of immunosuppression, azacitidine, and donor lymphocyte infusion, were prespecified for CD34+ DC ≤80%. Overall, CD34+ DC ≤80% detected 32 of 40 relapses (positive predictive value [PPV], 68%; negative predictive value [NPV], 91%), compared with 13 of 40 relapses for CD3+ DC ≤80% (PPV, 52%; NPV, 75%). Receiver operating characteristic analysis showed the superiority of CD34+ DC, with the greatest value at day 120 post-transplantation. CD3+ DC provided additional value in only 3 cases, preceding CD34+ DC ≤80% by 1 month. We further show that the CD34+ DC sample can be used to detect NPM1mut, with the combination of CD34+ DC ≤80% and NPM1mut identifying the highest risk of relapse. Among the 24 patients in morphologic remission at the time of CD34+ DC ≤80%, 15 (62.5%) responded to immunologic interventions (rapid withdrawal of immunosuppression, azacitidine, or donor lymphocyte infusion) with recovery of CD34+ DC >80%, and 11 of these patients remained in complete remission for a median of 34 months (range, 28 to 97 months). In contrast, the other 9 patients did not respond to the clinical intervention and relapsed within a median of 59 days after detecting CD34+ DC ≤80%. The CD34+ DC was significantly higher in responders than in nonresponders (median, 72% versus 56%; P = .015, Mann-Whitney U test). Overall, monitoring of CD34+ DC was considered clinically useful (early diagnosis of relapse enabling preemptive therapy or predicting low risk of relapse) in 107 of 125 evaluable patients (86%). Our findings show that peripheral blood CD34+ DC is feasible and superior to CD3+ DC for predicting relapse. It also provides a source of DNA for measurable residual disease testing, which may further stratify the risk of relapse. If validated by an independent cohort, our results suggest that CD34+ should be used in preference to CD3+ DC for detecting early relapse and guiding immunologic interventions following allo-SCT for AML or MDS.

Cellular distribution of IDH mutations in AML during morphologic remission.

Limited information exists about the cellular distribution of mutations which persist in remission in acute myeloid leukemia (AML) (variably considered pre-leukemic mutations). We hypothesized that mutations detectable in all cell compartments may be less pathogenic than those that are myeloid-restricted. Here, we describe the cellular compartments that have IDH mutations in five patients with IDH-mutated AML in morphologic remission. Unlike pre-leukemic clones harboring the more common DNMT3A, TET2 and ASXL1 (DTA) mutations, we show that IDH mutations are myeloid-restricted. This finding provides an explanation for the reports that IDH mutations carry a higher risk for relapse than DTA mutations. Detailed analysis of one case also shows acquisition of additional mutations in distinct cellular compartments, illustrating subclonal complexity associated with therapeutics.

Evaluation of EuroFlow minimal residual disease measurement and donor chimerism monitoring following tandem auto-allogeneic transplantation for multiple myeloma.

Prognostic factors for multiple myeloma (MM) after allogeneic haemopoietic stem cell transplantation (alloHSCT) are poorly characterised. Two potential factors include minimal residual disease (MRD) and CD3+ donor-specific chimerism. We retrospectively examined 93 consecutive patients who received upfront or deferred tandem auto-alloHSCT. Bone marrow (Euroflow) MRD was assessed pre-alloHSCT and 3-monthly post-alloHSCT. CD3+ donor chimerism was assessed at D30, D60, D90, 6 m and 12 m post-alloHSCT. There was no statistical difference between upfront and deferred transplants in progression free survival (PFS) (34 m vs. 15 m respectively, p = 0.20) and overall survival (OS) (75.5 m vs. 62.7 m respectively, p = 0.56). Patients who were MRD-positive post-alloHSCT had inferior PFS to MRD-negative patients from 6 m (6 m HR 3.32, p = 0.02; 9 m HR 4.08, p = 0.003; 12 m HR 4.47, p = 0.008). Attainment or maintenance of MRD-negativity predicted reduced relapse risk (23.5% vs. 62.5%, p = 0.04). However, there was no significant difference in OS between the MRD-negative and positive groups. Full CD3+ donor chimerism at early time points (D30 and D90) was associated with increased risk of acute GVHD (D30 p < 0.001, D90 p = 0.006) and extensive chronic GVHD (D90 p = 0.04), but not PFS or OS. These data support the use of sequential MRD evaluation post-alloHSCT to inform intervention to eradicate persistent or emergent MRD-positive disease.

B-Cell Epitope Mapping Using a Library of Overlapping Synthetic Peptides in an Enzyme-Linked Immunosorbent Assay.

This chapter describes a strategy for mapping linear B-cell epitopes of proteins using synthetic biotinylated peptides in an ELISA.A set of overlapping peptides were designed based upon a known amino acid sequence of the target protein, VapA (Virulence-associated Protein A) of the bacterium Rhodococcus equi, an important pulmonary pathogen in foals. The peptides synthesized as biotinylated peptides were coated directly onto micro titer plates which had been pre-coated with NeutrAvidin™ and used to screen sera from foals confirmed to have R. equi disease. A linear B-cell epitope was identified which corresponded to a 20 mer sequence of the VapA protein.

Comparison of biosimilar filgrastim with originator filgrastim for peripheral blood stem cell mobilization and engraftment in patients with multiple myeloma undergoing autologous stem cell transplantation.

BACKGROUND: Nivestim is a biosimilar approved for the same indications as Neupogen including the mobilization of autologous peripheral blood stem cells (PBSCs). The clinical efficacy and safety of Nivestim for this use have not been formally assessed in clinical trials. STUDY DESIGN AND METHODS: In our retrospective single-center study we compared variables of PBSC mobilization and engraftment of 60 patients mobilized with Nivestim to that of 38 patients mobilized with Neupogen. RESULTS: We found no difference between Nivestim and Neupogen in peripheral blood CD34+ at first leukapheresis (47 × 10(6) cells/L vs. 60 × 10(6) cells/L, p = 0.48) nor the total CD34+ collected (5.37 × 10(6)/kg vs. 4.59 × 10(6) /kg, p = 0.22). However, a difference in the median number of leukapheresis procedures (one vs. two, p = 0.0007) was observed. Eighty-one patients (51 Nivestim and 30 Neupogen mobilized) went on to transplantation. Median time to neutrophil engraftment (15 days vs. 13.5 days, p = 0.09) and platelet (PLT) engraftment (20 days vs. 18 days, p = 0.01) was longer in the Nivestim group. The significant delay in PLT engraftment did not, however, translate to increased PLT transfusions (two vs. three, p = 0.2) or impact significantly on hospitalization time for admissions within 30 days posttransplant (20 days vs. 18 days, p = .17). CONCLUSION: Nivestim is as effective as Neupogen for PBSC mobilization; however, its use was associated with a delay in PLT recovery. A prospective study should be conducted to confirm our findings.