Comparison of the 2022 and 2017 European LeukemiaNet risk classifications in a real-life cohort of the PETHEMA group.

Next-Generation Sequencing is needed for the accurate genetic risk stratification of acute myeloid leukemia according to European LeukemiaNet (ELN) guidelines. We validated and compared the 2022 ELN risk classification in a real-life cohort of 546 intensively and 379 non-intensively treated patients. Among fit patients, those aged ≥65 years old showed worse OS than younger regardless risk classification. Compared with the 2017 classification, 14.5% of fit patients changed the risk with the 2022 classification, increasing the high-risk group from 44.3% to 51.8%. 3.7% and 0.9% FLT3-ITD mutated patients were removed from the favorable and adverse 2017 categories respectively to 2022 intermediate risk group. We suggest that midostaurin therapy could be a predictor for 3 years OS (85.2% with vs. 54.8% without midostaurin, P = 0.04). Forty-seven (8.6%) patients from the 2017 intermediate group were assigned to the 2022 adverse-risk group as they harbored myelodysplasia (MDS)-related mutations. Patients with one MDS-related mutation did not reach median OS, while patients with ≥2 mutations had 13.6 months median OS (P = 0.002). Patients with TP53 ± complex karyotype or inv(3) had a dismal prognosis (7.1 months median OS). We validate the prognostic utility of the 2022 ELN classification in a real-life setting providing supportive evidences to improve risk stratification guidelines.

Preclinical models for prediction of immunotherapy outcomes and immune evasion mechanisms in genetically heterogeneous multiple myeloma.

The historical lack of preclinical models reflecting the genetic heterogeneity of multiple myeloma (MM) hampers the advance of therapeutic discoveries. To circumvent this limitation, we screened mice engineered to carry eight MM lesions (NF-κB, KRAS, MYC, TP53, BCL2, cyclin D1, MMSET/NSD2 and c-MAF) combinatorially activated in B lymphocytes following T cell-driven immunization. Fifteen genetically diverse models developed bone marrow (BM) tumors fulfilling MM pathogenesis. Integrative analyses of ∼500 mice and ∼1,000 patients revealed a common MAPK-MYC genetic pathway that accelerated time to progression from precursor states across genetically heterogeneous MM. MYC-dependent time to progression conditioned immune evasion mechanisms that remodeled the BM microenvironment differently. Rapid MYC-driven progressors exhibited a high number of activated/exhausted CD8+ T cells with reduced immunosuppressive regulatory T (Treg) cells, while late MYC acquisition in slow progressors was associated with lower CD8+ T cell infiltration and more abundant Treg cells. Single-cell transcriptomics and functional assays defined a high ratio of CD8+ T cells versus Treg cells as a predictor of response to immune checkpoint blockade (ICB). In clinical series, high CD8+ T/Treg cell ratios underlie early progression in untreated smoldering MM, and correlated with early relapse in newly diagnosed patients with MM under Len/Dex therapy. In ICB-refractory MM models, increasing CD8+ T cell cytotoxicity or depleting Treg cells reversed immunotherapy resistance and yielded prolonged MM control. Our experimental models enable the correlation of MM genetic and immunological traits with preclinical therapy responses, which may inform the next-generation immunotherapy trials.

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.

Prognostic significance of FLT3-ITD length in AML patients treated with intensive regimens.

FLT3-ITD mutations are detected in approximately 25% of newly diagnosed adult acute myeloid leukemia (AML) patients and confer an adverse prognosis. The FLT3-ITD allelic ratio has clear prognostic value. Nevertheless, there are numerous manuscripts with contradictory results regarding the prognostic relevance of the length and insertion site (IS) of the FLT3-ITD fragment. We aimed to assess the prognostic impact of these variables on the complete remission (CR) rates, overall survival (OS) and relapse-free survival (RFS) of AML patients with FLT3-ITDmutations. We studied the FLT3-ITD length of 362 adult AML patients included in the PETHEMA AML registry. We tried to validate the thresholds of ITD length previously published (i.e., 39 bp and 70 bp) in intensively treated AML patients (n = 161). We also analyzed the mutational profile of 118 FLT3-ITD AML patients with an NGS panel of 39 genes and correlated mutational status with the length and IS of ITD. The AUC of the ROC curve of the ITD length for OS prediction was 0.504, and no differences were found when applying any of the thresholds for OS, RFS or CR rate. Only four out of 106 patients had ITD IS in the TKD1 domain. Our results, alongside previous publications, confirm that FLT3-ITD length lacks prognostic value and clinical applicability.

Endogenous Retroelement Activation by Epigenetic Therapy Reverses the Warburg Effect and Elicits Mitochondrial-Mediated Cancer Cell Death.

For millions of years, endogenous retroelements have remained transcriptionally silent within mammalian genomes by epigenetic mechanisms. Modern anticancer therapies targeting the epigenetic machinery awaken retroelement expression, inducing antiviral responses that eliminate tumors through mechanisms not completely understood. Here, we find that massive binding of epigenetically activated retroelements by RIG-I and MDA5 viral sensors promotes ATP hydrolysis and depletes intracellular energy, driving tumor killing independently of immune signaling. Energy depletion boosts compensatory ATP production by switching glycolysis to mitochondrial oxidative phosphorylation, thereby reversing the Warburg effect. However, hyperfunctional succinate dehydrogenase in mitochondrial electron transport chain generates excessive oxidative stress that unleashes RIP1-mediated necroptosis. To maintain ATP generation, hyperactive mitochondrial membrane blocks intrinsic apoptosis by increasing BCL2 dependency. Accordingly, drugs targeting BCL2 family proteins and epigenetic inhibitors yield synergistic responses in multiple cancer types. Thus, epigenetic therapy kills cancer cells by rewiring mitochondrial metabolism upon retroelement activation, which primes mitochondria to apoptosis by BH3-mimetics. SIGNIFICANCE: The state of viral mimicry induced by epigenetic therapies in cancer cells remodels mitochondrial metabolism and drives caspase-independent tumor cell death, which sensitizes to BCL2 inhibitor drugs. This novel mechanism underlies clinical efficacy of hypomethylating agents and venetoclax in acute myeloid leukemia, suggesting similar combination therapies for other incurable cancers.This article is highlighted in the In This Issue feature, p. 995.

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.

Spanish Guidelines for the use of targeted deep sequencing in myelodysplastic syndromes and chronic myelomonocytic leukaemia.

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.

Richter transformation driven by Epstein-Barr virus reactivation during therapy-related immunosuppression in chronic lymphocytic leukaemia.

The increased risk of Richter transformation (RT) in patients with chronic lymphocytic leukaemia (CLL) due to Epstein-Barr virus (EBV) reactivation during immunosuppressive therapy with fludarabine other targeted agents remains controversial. Among 31 RT cases classified as diffuse large B-cell lymphoma (DLBCL), seven (23%) showed EBV expression. In contrast to EBV- tumours, EBV+ DLBCLs derived predominantly from IGVH-hypermutated CLL, and they also showed CLL-unrelated IGVH sequences more frequently. Intriguingly, despite having different cellular origins, clonally related and unrelated EBV+ DLBCLs shared a previous history of immunosuppressive chemo-immunotherapy, a non-germinal centre DLBCL phenotype, EBV latency programme type II or III, and very short survival. These data suggested that EBV reactivation during therapy-related immunosuppression can transform either CLL cells or non-tumoural B lymphocytes into EBV+ DLBCL. To investigate this hypothesis, xenogeneic transplantation of blood cells from 31 patients with CLL and monoclonal B-cell lymphocytosis (MBL) was performed in Rag2-/- IL2γc-/- mice. Remarkably, the recipients' impaired immunosurveillance favoured the spontaneous outgrowth of EBV+ B-cell clones from 95% of CLL and 64% of MBL patients samples, but not from healthy donors. Eventually, these cells generated monoclonal tumours (mostly CLL-unrelated but also CLL-related), recapitulating the principal features of EBV+ DLBCL in patients. Accordingly, clonally related and unrelated EBV+ DLBCL xenografts showed indistinguishable cellular, virological and molecular features, and synergistically responded to combined inhibition of EBV replication with ganciclovir and B-cell receptor signalling with ibrutinib in vivo. Our study underscores the risk of RT driven by EBV in CLL patients receiving immunosuppressive therapies, and provides the scientific rationale for testing ganciclovir and ibrutinib in EBV+ DLBCL. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Prognostic value of FLT3 mutations in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline monochemotherapy.

BACKGROUND: Fms-like tyrosine kinase-3 (FLT3) gene mutations are frequent in acute promyelocytic leukemia but their prognostic value is not well established. DESIGN AND METHODS: We evaluated FLT3-internal tandem duplication and FLT3-D835 mutations in patients treated with all-trans retinoic acid and anthracycline-based chemotherapy enrolled in two subsequent trials of the Programa de Estudio y Tratamiento de las Hemopatías Malignas (PETHEMA) and Hemato-Oncologie voor Volwassenen Nederland (HOVON) groups between 1996 and 2005. RESULTS: FLT3-internal tandem duplication and FLT3-D835 mutation status was available for 306 (41%) and 213 (29%) patients, respectively. Sixty-eight (22%) and 20 (9%) patients had internal tandem duplication and D835 mutations, respectively. Internal tandem duplication was correlated with higher white blood cell and blast counts, lactate dehydrogenase, relapse-risk score, fever, hemorrhage, coagulopathy, BCR3 isoform, M3 variant subtype, and expression of CD2, CD34, human leukocyte antigen-DR, and CD11b surface antigens. The FLT3-D835 mutation was not significantly associated with any clinical or biological characteristic. Univariate analysis showed higher relapse and lower survival rates in patients with a FLT3-internal tandem duplication, while no impact was observed in relation to FLT3-D835. The prognostic value of the FLT3-internal tandem duplication was not retained in the multivariate analysis. CONCLUSIONS: FLT3-internal tandem duplication mutations are associated with several hematologic features in acute promyelocytic leukemia, in particular with high white blood cell counts, but we were unable to demonstrate an independent prognostic value in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline-based regimens.

Simultaneous translocations of FGFR3/MMSET and CCND1 into two different IGH alleles in multiple myeloma: lack of concurrent activation of both proto-oncogenes.

The simultaneous occurrence of two different translocations affecting both alleles of the IGH gene has rarely been reported in multiple myeloma. In such a case, two different oncogenes might become transcriptionally deregulated. To investigate this hypothesis, we have characterized the plasma cell leukemia cell line SK-MM2 and a primary myeloma both carrying simultaneous IGH-FGFR3/MMSET and IGH-CCND1 fusions as shown by multicolor fluorescence in situ hybridization. Remarkably, quantitative real-time polymerase chain reaction demonstrated that only one of the oncogene loci was transcriptionally upregulated in both instances. Moreover, the upregulated oncogenes differed between both samples. Thus, biallelic IGH translocations might exert different pathogenetic effects in plasma cell disorders.

Coexistence of different clonal populations harboring the b3a2 (p210) and e1a2 (p190) BCR-ABL1 fusion transcripts in chronic myelogenous leukemia resistant to imatinib.

In this study, we report the case of a Philadelphia (Ph) positive chronic myelogenous leukemia (CML) patient with the presence of p190 and p210 BCR-ABL1 mRNA fusion transcripts derived from e1a2 and b3a2 BCR-ABL1 genomic rearrangements, respectively. The presence of e1a2 BCR-ABL1 genomic rearrangement was seen in 2 different clones, one with the rearrangement and another one with the rearrangement and deletion of the BCR gene of the non-rearranged chromosome 22. After treatment with imatinib, the p210 transcript could not be detected, whereas p190 was still present 6 months after initiation of imatinib therapy and progression to blast phase. The absence of p210 transcript post treatment indicates that the clone with b3a2 responded to imatinib and that the observed resistance was associated to cells harboring the e1a2 genomic rearrangement. Despite resistance of this patient to imatinib, no evidence of mutations in the kinase domain of ABL1 was found. Loss of normal BCR in one cell clone may contribute to the resistance to imatinib due to the lack of BCR mediated inhibition of BCR-ABL1.

p53 Aberrations do not predict individual response to fludarabine in patients with B-cell chronic lymphocytic leukaemia in advanced stages Rai III/IV.

Abnormalities of p53 have been associated with short survival and non-response to therapy in chronic lymphocytic leukaemia (CLL). We have evaluated the rate of response to fludarabine as first-line therapy in 54 patients with advanced stage CLL, analysing the cytogenetic profile, aberrations in p53, including the methylation status of its promoter, and the immunoglobulin heavy-chain variable-region (IGVH) mutation status. According to the advanced stage of the disease in this series, 75% of patients presented genetic aberrations associated with poor prognosis: del(17p) and/or del(11q), and no-mutated IGVH genes. Ten patients (18.5%) had methylation in the promoter region of p53. Eighty-three per cent of patients treated achieved a response, with a high rate of complete remission (47.6%). Although we found a significant correlation between failures and the presence of p53 aberrations (P = 0.0065), either with methylation (P = 0.018) or deletion (P = 0.015), 64% of the patients with aberrations in this gene responded to treatment (11/17), suggesting that fludarabine induces high remission rates, even in these patients. This is the first time that the significance of p53 promoter methylation status is described in this pathology, and our data support that this epigenetic phenomenon could be involved in the pathogenesis and clinical evolution of CLL.

NUP98 is fused to HOXA9 in a variant complex t(7;11;13;17) in a patient with AML-M2.

The t(7;11)(p15;p15.4) has been reported to fuse the NUP98 gene (11p15), a component of the nuclear pore complex, with the class-1 homeobox gene HOXA9 at 7p15. This translocation has been associated with myeloid leukemias, predominantly acute myeloid leukemia (AML) M2 subtype with trilineage myelodysplastic features, and with a poor prognosis. The derived fusion protein retains the FG repeat motif of NUP98 N-terminus and the homeodomain shared by the HOX genes, acting as an oncogenic transcription factor critical for leukemogenesis. We report here a new complex t(7;11)-variant, i.e., t(7;11;13;17)(p15;p15;p?;p1?2) in a patient with AML-M2 and poor prognosis. The NUP98-HOXA9 fusion transcript was detected by RT-PCR, suggesting its role in the malignant transformation as it has been postulated for other t(7;11)-associated leukemias. No other fusion transcripts involving the NUP98 or HOXA9 genes were present, although other mechanisms involving several genes on chromosomes 13 and 17 may also be involved. To our knowledge, this is the first t(7;11) variant involving NUP98 described in hematological malignancies.

Clinical variability of patients with the t(6;8)(q27;p12) and FGFR1OP-FGFR1 fusion: two further cases.

We report two new cases with the t(6;8)(q27;p12) and FGFR1OP-FGFR1 fusion. Case 1 presented with polycythaemia vera (PV) and evolved over 4 years to a myeloproliferative disorder (MPD) resembling the 8p11 myeloproliferative syndrome (EMS). Case 2 presented with B-cell lymphoblastic leukaemia (B-ALL). These cases illustrate the clinical heterogeneity observed in patients with FGFR1 rearrangements and suggest that constitutively activated tyrosine kinases may be more widespread in MPDs.

Molecular heterogeneity in AML/MDS patients with 3q21q26 rearrangements.

Patients with 3q21q26 rearrangements seem to share similar clinicopathologic features and a common molecular mechanism, leading to myelodysplasia or acute myeloid leukemia (AML). The ectopic expression of EVI1 (3q26) has been implicated in the dysplasia that characterizes this subset of myeloid neoplasias. However, lack of EVI1 expression has been reported in several cases, and overexpression of EVI1 was detected in 9% of AML cases without 3q26 abnormalities. We report the molecular characterization of seven patients with inv(3)(q21q26), t(3;3)(q21;q26) or related abnormalities. EVI1 expression was detected in only one case, and thus ectopic expression of this gene failed to explain all of these cases. GATA2 (3q21) was found to be overexpressed in 5 of the 7 patients. GATA2 is highly expressed in stem cells, and its expression dramatically decreases when erythroid and megakaryocytic differentiation proceeds. No mutations in GATA1 were found in any patient, excluding loss of function of GATA1 as the cause of GATA2 overexpression. We report finding molecular heterogeneity in patients with 3q21q26 rearrangements in both breakpoints and in the expression pattern of the genes near these breakpoints. Our data suggest that a unique mechanism is not likely to be involved in 3q21q26 rearrangements.

NIN, a gene encoding a CEP110-like centrosomal protein, is fused to PDGFRB in a patient with a t(5;14)(q33;q24) and an imatinib-responsive myeloproliferative disorder.

We describe a new PDGFRB fusion associated with a t(5;14)(q33;q24) in a patient with a longstanding chronic myeloproliferative disorder with eosinophilia. After confirmation of PDGFRB involvement and definition of the chromosome 14 breakpoint by fluorescence in situ hybridization, candidate partner genes were selected on the basis of the presence of predicted oligomerization domains believed to be an essential feature of tyrosine kinase fusion proteins. We demonstrate that the t(5;14) fuses PDGFRB to NIN, a gene encoding a centrosomal protein with CEP110-like function. After treatment with imatinib, the patient achieved hematological and cytogenetical remission, but NIN-PDGFRB mRNA remained detectable by reverse transcription-PCR.

Molecular characterization of a t(1;3)(p36;q21) in a patient with MDS. MEL1 is widely expressed in normal tissues, including bone marrow, and it is not overexpressed in the t(1;3) cells.

Patients with myeloid malignancies and either the 3q21q26 syndrome or t(1;3)(p36;q21) have been reported to share similar clinicopathological features and a common molecular mechanism for leukemogenesis. Overexpression of MDS1/EVI1 (3q26) or MEL1/PRDM16 (1p36), both members of the PR-domain family, has been directly implicated in the malignant transformation of this subset of neoplasias. The breakpoints in both entities are outside the genes, and the 3q21 region, where RPN1 is located, seems to act as an enhancer. MEL1 has been reported to be expressed in leukemia cells with t(1;3) and in the normal uterus and fetal kidney, but neither in bone marrow (BM) nor in other tissues, suggesting that this gene is specific to t(1;3)-positive MDS/AML. We report the molecular characterization of a t(1;3)(p36;q21) in a patient with MDS (RAEB-2). In contrast to previous studies, we demonstrate that MEL1, the PR-containing form, and MEL1S, the PR-lacking form, are widely expressed in normal tissues, including BM. The clinicopathological features and the breakpoint on 1p36 are different from cases previously described, and MEL1 is not overexpressed, suggesting a heterogeneity in myeloid neoplasias with t(1;3).