Clonal evolution in acute myeloid leukemia (AML) originates long before diagnosis and is a dynamic process that may affect survival. However, it remains uninvestigated during routine diagnostic workups. We hypothesized that the mutational status of bone marrow dysplastic cells and leukemic blasts, analyzed at the onset of AML using integrated multidimensional flow cytometry (MFC) immunophenotyping and fluorescence-activated cell sorting (FACS) with next-generation sequencing (NGS), could reconstruct leukemogenesis. Dysplastic cells were detected by MFC in 285 of 348 (82%) newly diagnosed patients with AML. Presence of dysplasia according to MFC and World Health Organization criteria had no prognostic value in older adults. NGS of dysplastic cells and blasts isolated at diagnosis identified 3 evolutionary patterns: stable (n= 12 of 21), branching (n= 4 of 21), and clonal evolution (n= 5 of 21). In patients achieving complete response (CR), integrated MFC and FACS with NGS showed persistent measurable residual disease (MRD) in phenotypically normal cell types, as well as the acquisition of genetic traits associated with treatment resistance. Furthermore, whole-exome sequencing of dysplastic and leukemic cells at diagnosis and of MRD uncovered different clonal involvement in dysplastic myelo-erythropoiesis, leukemic transformation, and chemoresistance. Altogether, we showed that it is possible to reconstruct leukemogenesis in 80% of patients with newly diagnosed AML, using techniques other than single-cell multiomics.

Simple Summary The prognostic impact of FLT3-ITD allele ratio (AR) is a matter of controversy. We analyzed 2901 AML patients with long-term follow-up treated with PETHEMA protocols in the pre-FLT3 inhibitors era, with 579 of them harboring the FLT3-ITD mutation. We found that FLT3-ITD AR > 0.5 was associated with lower complete remission and rate and overall survival, while AR > 0.8 was associated with lower RFS. An AR of 0.44 was the best cutoff for OS and 0.8 for RFS. Overall, allo- and auto-hematopoietic stem cell transplant (HSCT) in first CR offered similar OS in patients with AR < 0.44, while allo-HSCT improved OS for those with higher AR. However, allo-HSCT resulted in better OS and RFS as compared to auto-HSCT in NPM1/FLT3-ITD-mutated AML regardless of pre-established AR cutoff (<= 0.5 vs. >0.5), supporting the use of other risk stratification tools, such as NPM1 MRD monitoring, in this setting. FLT3-ITD results in a poor prognosis in terms of overall survival (OS) and relapse-free survival (RFS) in acute myeloid leukemia (AML). However, the prognostic usefulness of the allelic ratio (AR) to select post-remission therapy remains controversial. Our study focuses on the prognostic impact of FLT3-ITD and its ratio in a series of 2901 adult patients treated intensively in the pre-FLT3 inhibitor era and reported in the PETHEMA registry. A total of 579 of these patients (20%) harbored FLT3-ITD mutations. In multivariate analyses, patients with an FLT3-ITD allele ratio (AR) of >0.5 showed a lower complete remission (CR rate) and OS (HR 1.47, p = 0.009), while AR > 0.8 was associated with poorer RFS (HR 2.1; p < 0.001). Among NPM1/FLT3-ITD-mutated patients, median OS gradually decreased according to FLT3-ITD status and ratio (34.3 months FLT3-ITD-negative, 25.3 months up to 0.25, 14.5 months up to 0.5, and 10 months >= 0.5, p < 0.001). Post-remission allogeneic transplant (allo-HSCT) resulted in better OS and RFS as compared to auto-HSCT in NPM1/FLT3-ITD-mutated AML regardless of pre-established AR cutoff (<= 0.5 vs. >0.5). Using the maximally selected log-rank statistics, we established an optimal cutoff of FLT3-ITD AR of 0.44 for OS, and 0.8 for RFS. We analyzed the OS and RFS according to FLT3-ITD status in all patients, and we found that the group of FLT3-ITD-positive patients with AR < 0.44 had similar 5-year OS after allo-HSCT or auto-HSCT (52% and 41%, respectively, p = 0.86), but worse RFS after auto-HSCT (p = 0.01). Among patients with FLT3-ITD AR > 0.44, allo-HSCT was superior to auto-HSCT in terms of OS and RFS. This study provides more evidence for a better characterization of patients with AML harboring FLT3-ITD mutations.

Simple Summary Multiple Myeloma (MM) is considered an incurable chronic disease, which prognosis depends on the presence of different genomic alterations. To accomplish a complete molecular diagnosis in a single essay, we have designed and validated a capture-based NGS approach to reliably identify pathogenic mutations (SNVs and indels), genomic alterations (CNVs and chromosomic translocations), and IGH rearrangements. We have observed a good correlation of the results obtained using our capture panel with data obtained by both FISH and WES techniques. In this study, the molecular classification performed using our approach was significantly associated with the stratification and outcome of MM patients. Additionally, this panel has been proven to detect specific IGH rearrangements that could be used as biomarkers in patient follow-ups through minimal residual disease (MRD) assays. In conclusion, we think that MM patients could benefit from the use of this capture-based NGS approach with a more accurate, single-essay molecular diagnosis. Next-generation sequencing (NGS) has greatly improved our ability to detect the genomic aberrations occurring in multiple myeloma (MM); however, its transfer to routine clinical labs and its validation in clinical trials remains to be established. We designed a capture-based NGS targeted panel to identify, in a single assay, known genetic alterations for the prognostic stratification of MM. The NGS panel was designed for the simultaneous study of single nucleotide and copy number variations, insertions and deletions, chromosomal translocations and V(D)J rearrangements. The panel was validated using a cohort of 149 MM patients enrolled in the GEM2012MENOS65 clinical trial. The results showed great global accuracy, with positive and negative predictive values close to 90% when compared with available data from fluorescence in situ hybridization and whole-exome sequencing. While the treatments used in the clinical trial showed high efficacy, patients defined as high-risk by the panel had shorter progression-free survival (p = 0.0015). As expected, the mutational status of TP53 was significant in predicting patient outcomes (p = 0.021). The NGS panel also efficiently detected clonal IGH rearrangements in 81% of patients. In conclusion, molecular karyotyping using a targeted NGS panel can identify relevant prognostic chromosomal abnormalities and translocations for the clinical management of MM patients.

Next-Generation Sequencing has recently been introduced to efficiently and simultaneously detect genetic variations in acute myeloid leukemia. However, its implementation in the clinical routine raises new challenges focused on the diversity of assays and variant reporting criteria. To overcome this challenge, the PETHEMA group established a nationwide network of reference laboratories aimed to deliver molecular results in the clinics. We report the technical cross-validation results for next-generation sequencing panel genes during the standardization process and the clinical validation in 823 samples of 751 patients with newly diagnosed or refractory/relapse acute myeloid leukemia. Two cross-validation rounds were performed in seven nationwide reference laboratories in order to reach a consensus regarding quality metrics criteria and variant reporting. In the pre-standardization cross-validation round, an overall concordance of 60.98% was obtained with a great variability in selected genes and conditions across laboratories. After consensus of relevant genes and optimization of quality parameters the overall concordance rose to 85.57% in the second cross-validation round. We show that a diagnostic network with harmonized next-generation sequencing analysis and reporting in seven experienced laboratories is feasible in the context of a scientific group.

Patients with myeloid neoplasms who relapsed after allogenic hematopoietic stem cell transplant (HSCT) have poor prognosis. Monitoring of chimerism and specific molecular markers as a surrogate measure of relapse is not always helpful; therefore, improved systems to detect early relapse are needed. We hypothesized that the use of next generation sequencing (NGS) could be a suitable approach for personalized follow-up post-HSCT. To validate our hypothesis, we analyzed by NGS, a retrospective set of peripheral blood (PB) DNA samples previously evaluated by high-sensitive quantitative PCR analysis using insertion/deletion polymorphisms (indel-qPCR) chimerism engraftment. Post-HCST allelic burdens assessed by NGS and chimerism status showed a similar time-course pattern. At time of clinical relapse in 8/12 patients, we detected positive NGS-based minimal residual disease (NGS-MRD). Importantly, in 6/8 patients, we were able to detect NGS-MRD at time points collected prior to clinical relapse. We also confirmed the disappearance of post-HCST allelic burden in non-relapsed patients, indicating true clinical specificity. This study highlights the clinical utility of NGS-based post-HCST monitoring in myeloid neoplasia as a complementary specific analysis to high-sensitive engraftment testing. Overall, NGS-MRD testing in PB is widely applicable for the evaluation of patients following HSCT and highly valuable to personalized early treatment intervention when mixed chimerism is detecte

The diagnosis of myeloid neoplasms (MN) has significantly evolved through the last few decades. Next Generation Sequencing (NGS) is gradually becoming an essential tool to help clinicians with disease management. To this end, most specialized genetic laboratories have implemented NGS panels targeting a number of different genes relevant to MN. The aim of the present study is to evaluate the performance of four different targeted NGS gene panels based on their technical features and clinical utility. A total of 32 patient bone marrow samples were accrued and sequenced with 3 commercially available panels and 1 custom panel. Variants were classified by two geneticists based on their clinical relevance in MN. There was a difference in panel¿s depth of coverage. We found 11 discordant clinically relevant variants between panels, with a trend to miss long insertions. Our data show that there is a high risk of finding different mutations depending on the panel of choice, due both to the panel design and the data analysis method. Of note, CEBPA, CALR and FLT3 genes, remains challenging the use of NGS for diagnosis of MN in compliance with current guidelines. Therefore, conventional molecular testing might need to be kept in place for the correct diagnosis of MN for now.

Risk of developing myelodysplastic syndrome (MDS) is significantly increased in both multiple myeloma (MM) and monoclonal gammopathy of undetermined significance, suggesting that it is therapy independent. However, the incidence and sequelae of dysplastic hematopoiesis at diagnosis are unknown. Here, we used multidimensional flow cytometry (MFC) to prospectively screen for the presence of MDS-associated phenotypic alterations (MDS-PA) in the bone marrow of 285 patients with MM enrolled in the PETHEMA/GEM2012MENOS65 trial (#NCT01916252). We investigated the clinical significance of monocytic MDS-PA in a larger series of 1252 patients enrolled in 4 PETHEMA/GEM protocols. At diagnosis, 33 (11.6%) of 285 cases displayed MDS-PA. Bulk and single-cell-targeted sequencing of MDS recurrently mutated genes in CD34+ progenitors (and dysplastic lineages) from 67 patients revealed clonal hematopoiesis in 13 (50%) of 26 cases with MDS-PA vs 9 (22%) of 41 without MDS-PA; TET2 and NRAS were the most frequently mutated genes. Dynamics of MDS-PA at diagnosis and after autologous transplant were evaluated in 86 of 285 patients and showed that in most cases (69 of 86 [80%]), MDS-PA either persisted or remained absent in patients with or without MDS-PA at diagnosis, respectively. Noteworthy, MDS-associated mutations infrequently emerged after high-dose therapy. Based on MFC profiling, patients with MDS-PA have altered hematopoiesis and T regulatory cell distribution in the tumor microenvironment. Importantly, the presence of monocytic MDS-PA at diagnosis anticipated greater risk of hematologic toxicity and was independently associated with inferior progression-free survival (hazard ratio, 1.5; P = .02) and overall survival (hazard ratio, 1.7; P = .01). This study reveals the biological and clinical significance of dysplastic hematopoiesis in newly diagnosed MM, which can be screened with moderate sensitivity using cost-effective MFC.

The landscape of medical sequencing has rapidly changed with the evolution of next generation sequencing (NGS). These technologies have contributed to the molecular characterization of the myelodysplastic syndromes (MDS) and chronic myelomonocytic leukaemia (CMML), through the identification of recurrent gene mutations, which are present in >80% of patients. These mutations contribute to a better classification and risk stratification of the patients. Currently, clinical laboratories include NGS genomic analyses in their routine clinical practice, in an effort to personalize the diagnosis, prognosis and treatment of MDS and CMML. NGS technologies have reduced the cost of large-scale sequencing, but there are additional challenges involving the clinical validation of these technologies, as continuous advances are constantly being made. In this context, it is of major importance to standardize the generation, analysis, clinical interpretation and reporting of NGS data. To that end, the Spanish MDS Group (GESMD) has expanded the present set of guidelines, aiming to establish common quality standards for the adequate implementation of NGS and clinical interpretation of the results, hoping that this effort will ultimately contribute to the benefit of patients with myeloid malignancies.

Myeloid neoplasms (MN) are usually sporadic late-onset cancers; nevertheless, growing evidence suggests that similar to 5% of the cases could emerge as a consequence of inherited predisposition. Distinguishing somatic from germline variants is of vital importance, in order to establish an appropriate individualized management and counsel the patients and their relatives. Since many of the genes associated with myeloid neoplasm germline predisposition (MNGP) are also affected in sporadic MN, we intended to design a strategy to identify potentially inherited variants in a tumor only NGS panel in a cohort of 299 patients with a variety of MN. We considered as indicative of potential inherited origin, variants detected in BM sample at a similar to 50% VAF classified as pathogenic, likely pathogenic or of unknown significance detected in MNGP-related genes. A total of 104 suspicious variants from 90 patients were filtered-in in tumor samples. Mutational patterns, follow-up data, and sequencing of a range of non-myeloid tissues were used for narrowing down the list of suspicious variants, and ultimately discriminate their nature. Our data supports the importance of considering variants found upon tumor-only sequencing as potentially of germline origin, and we offer a pipeline to define the nature of the variants.

Transcriptional activation of the EVI1 oncogene (3q26) leads to aggressive forms of human acute myeloid leukemia (AML). However, the mechanism of EVI1-mediated leukemogenesis has not been fully elucidated. Previously, by characterizing the EVI1 promoter, we have shown that RUNX1 and ELK1 directly regulate EVI1 transcription. Intriguingly, bioinformatic analysis of the EVI1 promoter region identified the presence of several EVI1 potential binding sites. Thus, we hypothesized that EVI1 could bind to these sites regulating its own transcription. In this study, we show that there is a functional interaction between EVI1 and its promoter, and that the different EVI1 isoforms (EVI1-145 kDa, EVI1-Delta 324 and MDS1-EVI1) regulate the transcription of EVI1 transcripts through distinct promoter regions. Moreover, we determine that the EVI1-145 kDa isoform activates EVI1 transcription, whereas EVI1-Delta 324 and MDS1-EVI1 act as repressors. Finally, we demonstrate that these EVI1 isoforms are involved in cell transformation; functional experiments show that EVI1-145 kDa prolongs the maintenance of hematopoietic stem and progenitor cells; conversely, MDS1-EVI1 repressed hematopoietic stem and progenitor colony replating capacity. We demonstrate for the first time that EVI1 acts as a regulator of its own expression, highlighting the complex regulation of EVI1, and open new directions to better understand the mechanisms of EVI1 overexpressing leukemias.