Recent evidence suggests that the prognostic impact of gene mutations in patients with chronic lymphocytic leukemia (CLL) may differ depending on the immunoglobulin heavy variable (IGHV) gene somatic hypermutation (SHM) status. In this study, we assessed the impact of nine recurrently mutated genes (BIRC3, EGR2, MYD88, NFKBIE, NOTCH1, POT1, SF3B1, TP53, and XPO1) in pre-treatment samples from 4580 patients with CLL, using time-to-first-treatment (TTFT) as the primary end-point in relation to IGHV gene SHM status. Mutations were detected in 1588 (34.7%) patients at frequencies ranging from 2.3-9.8% with mutations in NOTCH1 being the most frequent. In both univariate and multivariate analyses, mutations in all genes except MYD88 were associated with a significantly shorter TTFT. In multivariate analysis of Binet stage A patients, performed separately for IGHV-mutated (M-CLL) and unmutated CLL (U-CLL), a different spectrum of gene alterations independently predicted short TTFT within the two subgroups. While SF3B1 and XPO1 mutations were independent prognostic variables in both U-CLL and M-CLL, TP53, BIRC3 and EGR2 aberrations were significant predictors only in U-CLL, and NOTCH1 and NFKBIE only in M-CLL. Our findings underscore the need for a compartmentalized approach to identify high-risk patients, particularly among M-CLL patients, with potential implications for stratified management.

Simple Summary Next-Generation Sequencing (NGS) has provided a deeper genetic understanding of acute myeloid leukemia (AML) that has been recently incorporated into AML classification and risk-stratification guidelines. Single molecular analysis has become inefficient and molecular testing based on NGS is emerging as an irreplaceable diagnostic tool in clinical settings. The PETHEMA cooperative group has constituted a nationwide NGS network with centralized analysis in seven high-skilled laboratories. The study of molecular profiles in the real-life PETHEMA cohort supports the increasing role of NGS on the clinical management of AML patients. 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.

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-kappa B, 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 similar to 500 mice and similar to 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 (T-reg) cells, while late MYC acquisition in slow progressors was associated with lower CD8(+) T cell infiltration and more abundant T-reg cells. Single-cell transcriptomics and functional assays defined a high ratio of CD8(+) T cells versus T-reg cells as a predictor of response to immune checkpoint blockade (ICB). In clinical series, high CD8(+) T/T-reg 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 T-reg 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. New experimental models provide much-needed tools for understanding how genetically diverse multiple myeloma progresses and evolves in response to therapy.

Genome complexity has been associated with poor outcome in patients with chronic lymphocytic leukemia (CLL). Previous cooperative studies established five abnormalities as the cut-off that best predicts an adverse evolution by chromosome banding analysis (CBA) and genomic microarrays (GM). However, data comparing risk stratification by both methods are scarce. Herein, we assessed a cohort of 340 untreated CLL patients highly enriched in cases with complex karyotype (CK) (46.5%) with parallel CBA and GM studies. Abnormalities found by both techniques were compared. Prognostic stratification in three risk groups based on genomic complexity (0-2, 3-4 >= 5 and abnormalities) was also analyzed. No significant differences in the percentage of patients in each group were detected, but only a moderate agreement was observed between methods when focusing on individual cases (kappa=0.507; P<0.001). Discordant classification was obtained in 100 patients (29.4%), including 3% classified in opposite risk groups. Most discrepancies were technique-dependent and no greater correlation in the number of abnormalities was achieved when different filtering strategies were applied for GM. Nonetheless, both methods showed a similar concordance index for prediction of time to first treatment (TTFT) (CBA: 0.67 vs. GM: 0.65) and overall survival (CBA: 0.55 vs. GM: 0.57). High complexity maintained its significance in the multivariate analysis for TTFT including TP53 and IGHV status when defined by CBA (hazard ratio [HR] 3.23; P<0.001) and GM (HR 2.74; P<0.001). Our findings suggest that both methods are useful but not equivalent for risk stratification of CLL patients. Validation studies are needed to establish the prognostic value of genome complexity based on GM data in future prospective studies.

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.

Gemtuzumab ozogamicin (GO) is a conjugate of a monoclonal antibody and calicheamicin, which has been reapproved for the treatment of acute myeloid leukemia (AML). AML patients with the CD33 rs12459419 CC genotype might benefit from the addition of GO to intensive treatment in contrast to patients with CT/TT genotypes. Nevertheless, contradictory results have been reported. We sought to shed light on the prediction of GO response in AML patients with rs12459419 polymorphism who were treated with GO in the consolidation (n=70) or reinduction (n=20) phase. The frequency distribution of the rs12459419 polymorphism in the complete cohort of patients was 44.4% (n=40), 50% (n=45), and 5.6% (n=5) for CC, CT, and TT genotypes, respectively. Regarding the patients treated with GO for consolidation, we performed a Kaplan-Meier analysis of overall survival and relapse-free survival according to the rs12459419 polymorphism (CC vs. CT/TT patients) and genetic risk using the European Leukemia Net (ELN) 2010 risk score. We also carried out a Cox regression analysis for the prediction of overall survival, with age and ELN 2010 as covariates. We found no statistical significance in the univariate or multivariate analysis. Additionally, we performed a global Kaplan-Meier analysis for the patients treated with GO for reinduction and did not find significant differences; however, our cohort was too small to draw any conclusion from this analysis. The use of GO in consolidation treatment is included in the approval of the compound; however, evidence regarding its efficacy in this setting is lacking. Rs12459419 polymorphism could help in the selection of patients who might benefit from GO. Regrettably, in our cohort, the rs12459419 polymorphism does not seem to be an adequate tool for the selection of patients who might benefit from the addition of GO in consolidation cycles.

Normal cell counterparts of solid and myeloid tumors accumulate mutations years before disease onset; whether this occurs in B lymphocytes before lymphoma remains uncertain. We sequenced multiple stages of the B lineage in elderly individuals and patients with lymphoplasmacytic lymphoma, a singular disease for studying lymphomagenesis because of the high prevalence of mutated MYD88. We observed similar accumulation of random mutations in B lineages from both cohorts and unexpectedly found MYD88(L265P) in normal precursor and mature B lymphocytes from patients with lymphoma. We uncovered genetic and transcriptional pathways driving malignant transformation and leveraged these to model lymphoplasmacytic lymphoma in mice, based on mutated MYD88 in B cell precursors and BCL2 overexpression. Thus, MYD88(L265P) is a preneoplastic event, which challenges the current understanding of lymphomagenesis and may have implications for early detection of B cell lymphomas.

Simple Summary Chromothripsis, a genomic event that generates massive chromosomal rearrangements, has been described in 1-3% of CLL patients and is associated with poor prognostic factors (e.g., TP53 abnormalities and genomic complexity). However, previous studies have not assessed its role in CLL patients with complex karyotypes. Herein, we aimed to describe the genetic characteristics of 33 CLL patients with high genomic complexity and chromothripsis. Moreover, we analyzed the clinical impact of chromothripsis, comparing these patients against a cohort of 129 patients with complex karyotypes not presenting this catastrophic event. Nine cases were also assessed via the novel cytogenomic methodology known as optical genome mapping. We confirmed that this phenomenon is heterogeneous and associated with a shorter time to first treatment. Nonetheless, our findings suggested that TP53 abnormalities, rather than chromothripsis itself, underlie the dismal outcome. Chromothripsis (cth) has been associated with a dismal outcome and poor prognosis factors in patients with chronic lymphocytic leukemia (CLL). Despite being correlated with high genome instability, previous studies have not assessed the role of cth in the context of genomic complexity. Herein, we analyzed a cohort of 33 CLL patients with cth and compared them against a cohort of 129 non-cth cases with complex karyotypes. Nine cth cases were analyzed using optical genome mapping (OGM). Patterns detected by genomic ...

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...

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.