By way of a Next-Generation Sequencing NGS high throughput approach, we defined the mutational profile in a cohort of 221 normal karyotype acute myeloid leukemia (NK-AML) enrolled into a prospective randomized clinical trial, designed to evaluate an intensified chemotherapy program for remission induction. NPM1, DNMT3A, and FLT3-ITD were the most frequently mutated genes while DNMT3A, FLT3, IDH1, PTPN11, and RAD21 mutations were more common in the NPM1 mutated patients (p < 0.05). IDH1 R132H mutation was strictly associated with NPM1 mutation and mutually exclusive with RUNX1 and ASXL1. In the whole cohort of NK-AML, no matter the induction chemotherapy used, by multivariate analysis, the achievement of complete remission was negatively affected by the SRSF2 mutation. Alterations of FLT3 (FLT3-ITD) and U2AF1 were associated with a worse overall and disease-free survival (p < 0.05). FLT3-ITD positive patients who proceeded to alloHSCT had a survival probability similar to FLT3-ITD negative patients and the transplant outcome was no different when comparing high and low-AR-FLT3-ITD subgroups in terms of both OS and DFS. In conclusion, a comprehensive molecular profile for NK-AML allows for the identification of genetic lesions associated to different clinical outcomes and the selection of the most appropriate and effective treatment strategies, including stem cell transplantation and targeted therapies.

Adult patients affected by acute myeloid leukemia who fail to achieve remission after two cycles of intensive chemotherapy based on a combination of anthracyclines and cytarabine are considered chemorefractory and are unlikely to benefit from further induction attempts. Characterized by a poor prognosis, they may still benefit from allogeneic hematopoietic stem cell transplantation, even if long-term survival rarely exceeds 20–30%. Still, the use of sequential high-dose chemotherapy followed by reduced-intensity conditioning, with transplantation performed during aplasia, and the optimization of the alloreactivity of donor leukocytes against leukemia (i.e., the graft-versus-leukemia effect) may ameliorate these results. Optimization of alloreactivity against leukemic cells can be achieved by proper donor selection, by the early withdrawal of immunosuppressive therapy, by post-transplant administration of donor lymphocyte infusions as prophylaxis of leukemia relapse, and by several other maintenance and preemptive therapies. Far from being the final stage of consolidation therapy, allogeneic hematopoietic stem cell transplantation is now considered as the moment when a unique immunological platform can be established in these patients, to be used for additional post-transplant measures. In this study we will critically review the different pre- and post-transplant strategies used in clinical trials to improve long-term survival in adult patients transplanted with chemorefractory leukemia.

Background: Graft versus host disease (GVHD) and the graft versus tumor (GVT) effect after allogeneic hematopoietic cell transplantation (allo-HCT) result from complex interactions between the donor immune system and the recipient environment. High-temporal longitudinal monitoring might be necessary to identify triggering events of GVHD and GVT and to intercept these events before their occurrence. But it would require an overall considerable amount of blood by venipuncture, which is unfeasible in such a fragile population. Methods: In this study, we implemented a targeted multiplex microfluidics q-PCR-based transcriptional fingerprint assay (TFA) on 50 µL of blood collected by a simple fingerstick to evaluate post-allo-HCT systemic immune perturbations associated with the development of GVHD. Fluctuations of a panel of 264 genes were measured in 31 allo-HCT patients by frequent (weekly or biweekly) analysis of 50 µL serial blood samples. Cross-sectional and longitudinal analyses correlated with detailed clinical annotations were performed. Results: Signatures of neutrophil activation and interferon (IFN) characterized the onset of acute GVHD, while an ongoing cytotoxic response was modulated in chronic mild GVHD and protein-synthesis and B-cell-related signatures characterized late acute/overlap GVHD. An unexpected erythroid signature distinguished patients with acute and mild chronic GVHD. Conclusions: Our micro-invasive approach unveiled the molecular heterogeneity of GVHD and identified hierarchically important biological processes conducive to different forms of GVHD. These findings increase our understanding of GVHD and reveal potentially targetable alterations. This approach might be implemented clinically to intercept GVHD before its occurrence and to modulate therapeutic interventions accordingly.