Validation of mutated CEBPA bZIP as a distinct prognosis entity in acute myeloid leukemia: a study by the Spanish PETHEMA registry.

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Biological and clinical significance of dysplastic hematopoiesis in patients with newly diagnosed multiple myeloma.

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.

MYD88 Mutations: Transforming the Landscape of IgM Monoclonal Gammopathies.

The MYD88 gene has a physiological role in the innate immune system. Somatic mutations in MYD88, including the most common L265P, have been associated with the development of certain types of lymphoma. MYD88L265P is present in more than 90% of patients with Waldenström's macroglobulinemia (WM) and IgM monoclonal gammopathy of undetermined significance (IgM-MGUS). The absence of MYD88 mutations in WM patients has been associated with a higher risk of transformation into aggressive lymphoma, resistance to certain therapies (BTK inhibitors), and shorter overall survival. The MyD88 signaling pathway has also been used as a target for specific therapies. In this review, we summarize the clinical applications of MYD88 testing in the diagnosis, prognosis, follow-up, and treatment of patients. Although MYD88L265P is not specific to WM, few tumors present a single causative mutation in a recurrent position. The role of the oncogene in the pathogenesis of WM is still unclear, especially considering that the mutation can be found in normal B cells of patients, as recently reported. This may have important implications for early lymphoma detection in healthy elderly individuals and for the treatment response assessment based on a MYD88L265P analysis.

Networking for advanced molecular diagnosis in acute myeloid leukemia patients is possible: the PETHEMA NGS-AML project

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. This cooperative nationwide strategy provides advanced molecular diagnostic for acute myeloid leukemia patients of the PETHEMA group.

Identification of relapse-associated gene mutations by next-generation sequencing in low-risk acute myeloid leukaemia patients.

Recommended genetic categorization of acute myeloid leukaemias (AML) includes a favourable-risk category, but not all these patients have good prognosis. Here, we used next-generation sequencing to evaluate the mutational profile of 166 low-risk AML patients: 30 core-binding factor (CBF)-AMLs, 33 nucleophosmin (NPM1)-AMLs, 4 biCEBPα-AMLs and 101 acute promyelocytic leukaemias (APLs). Functional categories of mutated genes differed among subgroups. NPM1-AMLs showed frequent variations in DNA-methylation genes (DNMT3A, TET2, IDH1/2) (79%), although without prognostic impact. Within this group, splicing-gene mutations were an independent factor for relapse-free (RFS) and overall survival (OS). In CBF-AML, poor independent factors for RFS and OS were mutations in RAS pathway and cohesin genes, respectively. In APL, the mutational profile differed according to the risk groups. High-risk APLs showed a high mutation rate in cell-signalling genes (P = 0·002), highlighting an increased incidence of FLT3 internal tandem duplication (ITD) (65%, P < 0·0001). Remarkably, in low-risk APLs (n = 28), NRAS mutations were strongly correlated with a shorter five-year RFS (25% vs. 100%, P < 0·0001). Overall, a high number of mutations (≥3) was the worst prognostic factor RFS (HR = 2·6, P = 0·003). These results suggest that gene mutations may identify conventional low-risk AML patients with poor prognosis and might be useful for better risk stratification and treatment decisions.

Basophil-lineage commitment in acute promyelocytic leukemia predicts for severe bleeding after starting therapy.

Severe hemorrhagic events occur in a significant fraction of acute promyelocytic leukemia patients, either at presentation and/or early after starting therapy, leading to treatment failure and early deaths. However, identification of independent predictors for high-risk of severe bleeding at diagnosis, remains a challenge. Here, we investigated the immunophenotype of bone marrow leukemic cells from 109 newly diagnosed acute promyelocytic leukemia patients, particularly focusing on the identification of basophil-related features, and their potential association with severe bleeding episodes and patient overall survival.From all phenotypes investigated on leukemic cells, expression of the CD203c and/or CD22 basophil-associated markers showed the strongest association with the occurrence and severity of bleeding (p ≤ 0.007); moreover, aberrant expression of CD7, coexpression of CD34+/CD7+ and lack of CD71 was also more frequently found among patients with (mild and severe) bleeding at baseline and/or after starting treatment (p ≤ 0.009). Multivariate analysis showed that CD203c expression (hazard ratio: 26.4; p = 0.003) and older age (hazard ratio: 5.4; p = 0.03) were the best independent predictors for cumulative incidence of severe bleeding after starting therapy. In addition, CD203c expression on leukemic cells (hazard ratio: 4.4; p = 0.01), low fibrinogen levels (hazard ratio: 8.8; p = 0.001), older age (hazard ratio: 9.0; p = 0.002), and high leukocyte count (hazard ratio: 5.6; p = 0.02) were the most informative independent predictors for overall survival.In summary, our results show that the presence of basophil-associated phenotypic characteristics on leukemic cells from acute promyelocytic leukemia patients at diagnosis is a powerful independent predictor for severe bleeding and overall survival, which might contribute in the future to (early) risk-adapted therapy decisions.

Prediction of peripheral neuropathy in multiple myeloma patients receiving bortezomib and thalidomide: a genetic study based on a single nucleotide polymorphism array.

Bortezomib- and thalidomide-based therapies have significantly contributed to improved survival of multiple myeloma (MM) patients. However, treatment-induced peripheral neuropathy (TiPN) is a common adverse event associated with them. Risk factors for TiPN in MM patients include advanced age, prior neuropathy, and other drugs, but there are conflicting results about the role of genetics in predicting the risk of TiPN. Thus, we carried out a genome-wide association study based on more than 300 000 exome single nucleotide polymorphisms in 172 MM patients receiving therapy involving bortezomib and thalidomide. We compared patients developing and not developing TiPN under similar treatment conditions (GEM05MAS65, NCT00443235). The highest-ranking single nucleotide polymorphism was rs45443101, located in the PLCG2 gene, but no significant differences were found after multiple comparison correction (adjusted P = .1708). Prediction analyses, cytoband enrichment, and pathway analyses were also performed, but none yielded any significant findings. A copy number approach was also explored, but this gave no significant results either. In summary, our study did not find a consistent genetic component associated with TiPN under bortezomib and thalidomide therapies that could be used for prediction, which makes clinical judgment essential in the practical management of MM treatment.

Panobinostat as part of induction and maintenance for elderly patients with newly diagnosed acute myeloid leukemia: phase Ib/II panobidara study.

This phase Ib/II trial combined the pan-deacetylase inhibitor panobinostat with chemotherapy followed by panobinostat maintenance in elderly patients with newly diagnosed acute myeloid leukemia. Patients with prior history of myelodysplastic syndrome were excluded and 38 evaluable patients were included in the study (median age: 71 years; range: 65-83). Study patients received an induction with idarubicin (8 mg/m(2) iv days 1-3) plus cytarabine (100 mg/m(2) iv days 1-7) plus panobinostat po at escalating doses (days 8, 10, 12, 15, 17 and 19) that could be repeated in non-responding patients. Patients achieving complete remission received a consolidation cycle with the same schema, followed by panobinostat maintenance (40 mg po 3 days/week) every other week until progression. Thirty-one patients were treated at the maximum tolerated dose of panobinostat in the combination (10 mg) with good tolerability. Complete remission rate was 64% with a time to relapse of 17.0 months (12.8-21.1). Median overall survival for the whole series was 17 months (5.5-28.4). Moreover, in 4 of 5 patients with persistent minimal residual disease before maintenance, panobinostat monotherapy reduced its levels, with complete negativization in two of them. Maintenance phase was well tolerated. The most frequent adverse events were thrombocytopenia (25% grades 3/4), and gastrointestinal toxicity, asthenia and anorexia (mainly grades 1/2). Five patients required dose reduction during this phase, but only one discontinued therapy due to toxicity. These results suggest that panobinostat is one of the first novel agents with activity in elderly acute myeloid leukemia patients, and suggest further investigation is warranted, particularly in the context of maintenance therapy. This trial is registered at clinicaltrials.gov identifier: 00840346.

Clonal architecture and evolutionary history of Waldenström's macroglobulinemia at the single-cell level.

To provide insight into the subclonal architecture and co-dependency patterns of the alterations in Waldenström's macroglobulinemia (WM), we performed single-cell mutational and protein profiling of eight patients. A custom panel was designed to screen for mutations and copy number alterations at the single-cell level in samples taken from patients at diagnosis (n=5) or at disease progression (n=3). Results showed that in asymptomatic WM at diagnosis, MYD88L265P was the predominant clonal alteration; other events, if present, were secondary and subclonal to MYD88L265P. In symptomatic WM, clonal diversity was more evident, uncovering combinations of alterations that synergized to promote clonal expansion and dominance. At disease progression, a dominant clone was observed, sometimes accompanied by other less complex minor clones, which could be consistent with a clonal selection process. Clonal diversity was also reduced, probably due to the effect of treatment. Finally, we combined protein expression with mutational analysis to map somatic genotype with the immunophenotype. Our findings provide a comprehensive view of the clonality of tumor populations in WM and how clonal complexity can evolve and impact disease progression.

Single-Cell DNA Sequencing and Immunophenotypic Profiling to Track Clonal Evolution in an Acute Myeloid Leukemia Patient.

Single-cell DNA sequencing can address the sequence of somatic genetic events during myeloid transformation in relapsed acute myeloid leukemia (AML). We present an NPM1-mutated AML patient with an initial low ratio of FLT3-ITD (low-risk ELN-2017), treated with midostaurin combined with standard chemotherapy as front-line treatment, and with salvage therapy plus gilteritinib following allogenic stem cell transplantation after relapse. Simultaneous single-cell DNA sequencing and cell-surface immunophenotyping was used in diagnostic and relapse samples to understand the clinical scenario of this patient and to reconstruct the clonal composition of both tumors. Four independent clones were present before treatment: DNMT3A/DNMT3A/NPM1 (63.9%), DNMT3A/DNMT3A (13.9%), DNMT3A/DNMT3A/NPM1/FLT3 (13.8%), as well as a wild-type clone (8.3%), but only the minor clone with FLT3-ITD survived and expanded after therapy, being the most represented one (58.6%) at relapse. FLT3-ITD was subclonal and was found only in the myeloid blast population (CD38/CD117/CD123). Our study shows the usefulness of this approach to reveal the clonal architecture of the leukemia and the identification of small subclones at diagnosis and relapse that may explain how the neoplastic cells can escape from the activity of different treatments in a stepwise process that impedes the disease cure despite different stages of complete remission.

Integrated flow cytometry and sequencing to reconstruct evolutionary patterns from dysplasia to acute myeloid leukemia.

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.

Molecular Landscape and Validation of New Genomic Classification in 2668 Adult AML Patients: Real Life Data from the PETHEMA Registry.

Next-Generation Sequencing (NGS) implementation to perform accurate diagnosis in acute myeloid leukemia (AML) represents a major challenge for molecular laboratories in terms of specialization, standardization, costs and logistical support. In this context, the PETHEMA cooperative group has established the first nationwide diagnostic network of seven reference laboratories to provide standardized NGS studies for AML patients. Cross-validation (CV) rounds are regularly performed to ensure the quality of NGS studies and to keep updated clinically relevant genes recommended for NGS study. The molecular characterization of 2856 samples (1631 derived from the NGS-AML project; NCT03311815) with standardized NGS of consensus genes (ABL1, ASXL1, BRAF, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA2, HRAS, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1 and WT1) showed 97% of patients having at least one mutation. The mutational profile was highly variable according to moment of disease, age and sex, and several co-occurring and exclusion relations were detected. Molecular testing based on NGS allowed accurate diagnosis and reliable prognosis stratification of 954 AML patients according to new genomic classification proposed by Tazi et al. Novel molecular subgroups, such as mutated WT1 and mutations in at least two myelodysplasia-related genes, have been associated with an adverse prognosis in our cohort. In this way, the PETHEMA cooperative group efficiently provides an extensive molecular characterization for AML diagnosis and risk stratification, ensuring technical quality and equity in access to NGS studies.

Identification of the novel HLA-A*23:01:01:27 allele in an acute myeloid patient and related donor.

A single nucleotide polymorphism in intron 1 determines the new HLA-A*23:01:01:27 allele.

Impact of FLT3-ITD Mutation Status and Its Ratio in a Cohort of 2901 Patients Undergoing Upfront Intensive Chemotherapy: A PETHEMA Registry Study.

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.

The novel HLA-DQB1*06:03:27 allele characterised by sequence-based typing in a European bone marrow donor.

A single nucleotide substitution in exon 3 of HLA-DQB1*06:03:01 results in a new allele, HLA-DQB1*06:03:27.

Long FLT3 internal tandem duplications and reduced PML-RARα expression at diagnosis characterize a high-risk subgroup of acute promyelocytic leukemia patients.

BACKGROUND: Internal tandem duplications of the FLT3 gene (FLT3-ITDs) are frequent in patients with acute promyelocytic leukemia (APL), however its clinical impact remains controversial. DESIGN AND METHODS: We analyzed the prognostic significance of FLT3-ITD mutant level and size, as well as FLT3-D835 point mutations, PML-RARalpha expression and other predictive factors in 129 APL patients at diagnosis enrolled on the Spanish LPA96 (n=43) or LPA99 (n=86) PETHEMA trials. RESULTS: FLT3-ITDs and D835 mutations were detected in 21% and 9% of patients, respectively. Patients with increased ITD mutant/wild-type ratio or longer ITD size displayed shorter 5-year relapse-free survival (RFS) (P=0.048 and P<0.0001, respectively). However, patients with D835 mutations did not show differences in RFS or overall survival (OS). Moreover, patients with initial normalized copy number (NCN) of PML-RARalpha transcripts less than the 25(th) percentile had adverse clinical features and shorter 5-year RFS (P<0.0001) and OS (P=0.004) compared to patients with higher NCN. Patients with low NCN showed increased incidence of ITDs (P=0.001), with higher ratios (P<0.0001) and/or longer sizes (P=0.007). Multivariate analysis showed that long FLT3-ITD (P=0.001), low PML-RARalpha levels (P=0.004) and elevated WBC counts (>10x10(9)/L) (P=0.018) were independent predictors for shorter RFS. We identified a subgroup of patients with high WBC, long FLT3-ITD and low NCN of transcripts that showed an extremely bad prognosis (5-year RFS 23.4%, P<0.0001). CONCLUSIONS: In conclusion, FLT3-ITD size and PML-RARalpha transcript levels at diagnosis could contribute to improve the risk stratification in APL.

The relevance of preferentially expressed antigen of melanoma (PRAME) as a marker of disease activity and prognosis in acute promyelocytic leukemia.

BACKGROUND: The gene for preferentially expressed antigen of melanoma (PRAME) has been shown to be over-expressed in acute promyelocytic leukemia, but its actual incidence and clinical impact are still unknown. DESIGN AND METHODS: We studied PRAME expression at diagnosis using real-time quantitative polymerase chain reaction in 125 patients with acute promyelocytic leukemia enrolled in the Spanish PETHEMA-96 (n=45) and PETHEMA-99 (n=80) clinical trials. In addition, PRAME expression was evaluated as a marker of disease activity in 225 follow-up samples from 67 patients with acute promyelocytic leukemia. RESULTS: At diagnosis, PRAME expression in patients with acute promyelocytic leukemia was significantly higher (p<0.001) than in patients with non-M3 acute myeloid leukemia (n=213) and in healthy controls (n=10). Furthermore, patients with acute promyelocytic leukemia with high PRAME expression had a favorable outcome. Thus, the 5-year relapse-free survival was better in patients with >100-fold PRAME expression (86% vs. 74%; p=0.03), and this cut-off established two sub-groups with different relapse-free survival rates among patients with a white cell count <10(9)/L (5-year relapse-free survival 94% vs. 80%, p=0.01). This effect was similar in patients with a white cell count >10(9)/L, although differences were not statistically significant. In multivariate analysis, white cell count >10(9)/L (p<0.001), bone marrow blasts >90% (p=0.001), and PRAME expression <100-fold (p=0.009) were associated with short relapse-free survival. Samples at remission showed PRAME levels similar to those in normal controls while samples at relapse over-expressed PRAME again. Furthermore, 12/13 samples collected within the 6-month period preceding relapse showed a >10-fold increase in PRAME expression levels. CONCLUSIONS: Low PRAME expression defines a subgroup of patients with acute promyelocytic leukemia with a short relapse-free survival. This marker could be useful as a secondary marker for monitoring patients with acute promyelocytic leukemia.

Using quantification of the PML-RARalpha transcript to stratify the risk of relapse in patients with acute promyelocytic leukemia.

BACKGROUND AND OBJECTIVES: The detection of PML-RARalpha by real-time polymerase chain reaction (RQ-PCR) is becoming an important tool for monitoring minimal residual disease (MRD) in patients with acute promyelocytic leukemia (APL). However, its clinical value remains to be determined. Our aim was to analyze any associations between the risk of relapse and RQ-PCR results in different phases of treatment, comparing these data with those yielded by conventional qualitative reverse transcriptase-PCR. DESIGN AND METHODS: Follow-up samples from 145 APL patients treated with the PETHEMA protocols were evaluated by the RQ-PCR protocol (Europe Against Cancer program) and by the RT-PCR method (BIOMED-1 Concerted Action). Hematologic and molecular relapses and relapse-free survival were recorded. We then looked for associations between relapse risk and RQ-PCR results. RESULTS: After induction therapy, no association was found between positive RQ-PCR results and relapse. The PCR result here did not imply any change in the scheduled therapy. After the third consolidation course, two out of three cases with positive RQ-PCR relapsed in contrast to 16 out of 119 (13%) patients with negative RQ-PCR. During maintenance therapy and out-of treatment, all patients with >10 PML-RARalpha normalized copy number (NCN) (n=19) relapsed while all patients with <1 NCN at the end of the study remained in hematologic remission (p<0.0001). In the intermediate group (NCN 1-10) (n=18), the relapse-free survival at 5 years was 60%. Hematologic relapses were predicted if a positive RQ-PCR result had been obtained in a follow-up sample within the previous 4 months. INTERPRETATION AND CONCLUSIONS: Based on the information provided by RQ-PCR in samples obtained after the end of consolidation and subsequently, a relapse risk stratification could be established for APL patients. This stratification divides patients into three groups: those at high risk of relapse, those with an intermediate risk and those with a low risk of relapse.