A sensitive and inexpensive high-resolution melting-based testing algorithm for diagnosis of transient abnormal myelopoiesis and myeloid leukemia of Down syndrome.

Patients with Down syndrome (DS) are commonly affected by a pre-leukemic disorder known as transient abnormal myelopoiesis (TAM). This condition usually undergoes spontaneous remission within the first 2 months after birth; however, in children under 5, 20%-30% of cases evolve to myeloid leukemia of Down syndrome (ML-DS). TAM and ML-DS are caused by co-operation between trisomy 21 and acquired mutations in the GATA1 gene. Currently, only next-generation sequencing (NGS)-based methodologies are sufficiently sensitive for diagnosis in samples with small GATA1 mutant clones (≤10% blasts). Alternatively, this study presents research on a new, fast, sensitive, and inexpensive high-resolution melting (HRM)-based diagnostic approach that allows the detection of most cases of GATA1 mutations, including silent TAM. The algorithm first uses flow cytometry for blast count, followed by HRM and Sanger sequencing to search for mutations on exons 2 and 3 of GATA1. We analyzed 138 samples of DS patients: 110 of asymptomatic neonates, 10 suspected of having TAM, and 18 suspected of having ML-DS. Our algorithm enabled the identification of 33 mutant samples, among them five cases of silent TAM (5/110) and seven cases of ML-DS (7/18) with blast count ≤10%, in which GATA1 alterations were easily detected by HRM. Depending on the type of genetic variation and its location, our methodology reached sensitivity similar to that obtained by NGS (0.3%) at a considerably reduced time and cost, thus making it accessible worldwide.

B-Cell Regeneration Profile and Minimal Residual Disease Status in Bone Marrow of Treated Multiple Myeloma Patients.

B-cell regeneration during therapy has been considered as a strong prognostic factor in multiple myeloma (MM). However, the effects of therapy and hemodilution in bone marrow (BM) B-cell recovery have not been systematically evaluated during follow-up. MM (n = 177) and adult (≥50y) healthy donor (HD; n = 14) BM samples were studied by next-generation flow (NGF) to simultaneously assess measurable residual disease (MRD) and residual normal B-cell populations. BM hemodilution was detected in 41 out of 177 (23%) patient samples, leading to lower total B-cell, B-cell precursor (BCP) and normal plasma cell (nPC) counts. Among MM BM, decreased percentages (vs. HD) of BCP, transitional/naïve B-cell (TBC/NBC) and nPC populations were observed at diagnosis. BM BCP increased after induction therapy, whereas TBC/NBC counts remained abnormally low. At day+100 postautologous stem cell transplantation, a greater increase in BCP with recovered TBC/NBC cell numbers but persistently low memory B-cell and nPC counts were found. At the end of therapy, complete response (CR) BM samples showed higher CD19- nPC counts vs. non-CR specimens. MRD positivity was associated with higher BCP and nPC percentages. Hemodilution showed a negative impact on BM B-cell distribution. Different BM B-cell regeneration profiles are present in MM at diagnosis and after therapy with no significant association with patient outcome.