Hematopoietic stem cells with granulo-monocytic differentiation state overcome venetoclax sensitivity in patients with myelodysplastic syndromes.

The molecular mechanisms of venetoclax-based therapy failure in patients with acute myeloid leukemia were recently clarified, but the mechanisms by which patients with myelodysplastic syndromes (MDS) acquire secondary resistance to venetoclax after an initial response remain to be elucidated. Here, we show an expansion of MDS hematopoietic stem cells (HSCs) with a granulo-monocytic-biased transcriptional differentiation state in MDS patients who initially responded to venetoclax but eventually relapsed. While MDS HSCs in an undifferentiated cellular state are sensitive to venetoclax treatment, differentiation towards a granulo-monocytic-biased transcriptional state, through the acquisition or expansion of clones with STAG2 or RUNX1 mutations, affects HSCs' survival dependence from BCL2-mediated anti-apoptotic pathways to TNFα-induced pro-survival NF-κB signaling and drives resistance to venetoclax-mediated cytotoxicity. Our findings reveal how hematopoietic stem and progenitor cell (HSPC) can eventually overcome therapy-induced depletion and underscore the importance of using close molecular monitoring to prevent HSPC hierarchical change in MDS patients enrolled in clinical trials of venetoclax.

Single agent subcutaneous blinatumomab for advanced acute lymphoblastic leukemia.

Blinatumomab is a BiTE® (bispecific T-cell engager) molecule that redirects CD3+ T-cells to engage and lyse CD19+ target cells. Here we demonstrate that subcutaneous (SC) blinatumomab can provide high efficacy and greater convenience of administration. In the expansion phase of a multi-institutional phase 1b trial (ClinicalTrials.gov, NCT04521231), heavily pretreated adults with relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL) received SC blinatumomab at two doses: (1) 250 µg once daily (QD) for week 1 and 500 µg three times weekly (TIW) thereafter (250 µg/500 µg) or (2) 500 µg QD for week 1 and 1000 µg TIW thereafter (500 µg/1000 µg). The primary endpoint was complete remission/complete remission with partial hematologic recovery (CR/CRh) within two cycles. At the data cutoff of September 15, 2023, 29 patients were treated: 14 at the 250 µg/500 µg dose and 13 at 500 µg/1000 µg dose. Data from two ineligible patients were excluded. At the end of two cycles, 12 of 14 patients (85.7%) from the 250 µg/500 µg dose achieved CR/CRh of which nine patients (75.0%) were negative for measurable residual disease (MRD; <10-4 leukemic blasts). At the 500 µg/1000 µg dose, 12 of 13 patients (92.3%) achieved CR/CRh; all 12 patients (100.0%) were MRD-negative. No treatment-related grade 4 cytokine release syndrome (CRS) or neurologic events (NEs) were reported. SC injections were well tolerated and all treatment-related grade 3 CRS and NEs responded to standard-of-care management, interruption, or discontinuation. Treatment with SC blinatumomab resulted in high efficacy, with high MRD-negativity rates and acceptable safety profile in heavily pretreated adults with R/R B-ALL.

Covering Hierarchical Dirichlet Mixture Models on binary data to enhance genomic stratifications in onco-hematology.

Onco-hematological studies are increasingly adopting statistical mixture models to support the advancement of the genomically-driven classification systems for blood cancer. Targeting enhanced patients stratification based on the sole role of molecular biology attracted much interest and contributes to bring personalized medicine closer to reality. In onco-hematology, Hierarchical Dirichlet Mixture Models (HDMM) have become one of the preferred method to cluster the genomics data, that include the presence or absence of gene mutations and cytogenetics anomalies, into components. This work unfolds the standard workflow used in onco-hematology to improve patient stratification and proposes alternative approaches to characterize the components and to assign patient to them, as they are crucial tasks usually supported by a priori clinical knowledge. We propose (a) to compute the parameters of the multinomial components of the HDMM or (b) to estimate the parameters of the HDMM components as if they were Multivariate Fisher's Non-Central Hypergeometric (MFNCH) distributions. Then, our approach to perform patients assignments to the HDMM components is designed to essentially determine for each patient its most likely component. We show on simulated data that the patients assignment using the MFNCH-based approach can be superior, if not comparable, to using the multinomial-based approach. Lastly, we illustrate on real Acute Myeloid Leukemia data how the utilization of MFNCH-based approach emerges as a good trade-off between the rigorous multinomial-based characterization of the HDMM components and the common refinement of them based on a priori clinical knowledge.

The prognostic impact of non-driver gene mutations and variant allele frequency in primary myelofibrosis.

Prognostic impact of non-MPN driver gene mutations in primary myelofibrosis. MIPSS70: Mutation-Enhanced International Prognostic Score System.

Outcomes and effect of somatic mutations after erythropoiesis stimulating agents in patients with lower-risk myelodysplastic syndromes.

Background: Erythropoiesis stimulating agents (ESAs) are the first-line therapy in patients with lower-risk myelodysplastic syndromes (LR-MDS). Some predictive factors for ESAs response have been identified. Type and number of somatic mutations have been associated with prognosis and response to therapies in MDS patients. Objectives: The objective was to evaluate the outcomes after ESAs in patients with LR-MDS and to address the potential predictive value of somatic mutations in ESAs-treated patients. Design: Multi-center retrospective study of a cohort of 722 patients with LR-MDS included in the SPRESAS (Spanish Registry of Erythropoietic Stimulating Agents Study) study. Retrospective analysis of 65 patients with next generation sequencing (NGS) data from diagnosis. Methods: ESAs' efficacy and safety were evaluated in patients receiving ESAs and best supportive care (BSC). To assess the potential prognostic value of somatic mutations in erythroid response (ER) rate and outcome, NGS was performed in responders and non-responders. Results: ER rate for ESAs-treated patients was 65%. Serum erythropoietin (EPO) level <200 U/l was the only variable significantly associated with a higher ER rate (odds ratio, 2.45; p = 0.036). Median overall survival (OS) in patients treated with ESAs was 6.7 versus 3.1 years in patients receiving BSC (p < 0.001). From 65 patients with NGS data, 57 (87.7%) have at least one mutation. We observed a trend to a higher frequency of ER among patients with a lower number of mutated genes (40.4% in <3 mutated genes versus 22.2% in ⩾3; p = 0.170). The presence of ⩾3 mutated genes was also significantly associated with worse OS (hazard ratio, 2.8; p = 0.015), even in responders. A higher cumulative incidence of acute myeloid leukemia progression at 5 years was also observed in patients with ⩾3 mutated genes versus <3 (33.3% and 10.7%, respectively; p < 0.001). Conclusion: This large study confirms the beneficial effect of ESAs and the adverse effect of somatic mutations in patients with LR-MDS.

Feasibility and outcomes after dose reduction of immunochemotherapy in young adults with Burkitt lymphoma and leukemia: results of the BURKIMAB14 trial.

High dose-intensive or infusional intermediate-dose immunochemotherapy is highly effective treatment for Burkitt lymphoma irrespective of human immunodeficiency virus (HIV) infection. However, toxicities of these regimens are relevant, especially in older adults and elderly patients. The prospective multicenter BURKIMAB14 trial included four to six blocks of immunochemotherapy according to stage (localized: 1 and 2 non-bulky; advanced: 2 bulky, 3, 4) and age, with dose reduction in patients >55 years old. Dose-intensity of chemotherapy was reduced in patients ≤55 years old after achieving complete metabolic response (CMR). Their outcomes were compared with those of similar patients included in the former BURKIMAB08 trial, in which there was no dose reduction. CMR was attained in 86 of 107 (80%) patients (17/19 in localized stages and 69/88 in advanced stages). Patients from the BURKIMAB14 trial ≤55 years old showed similar overall survival (OS), fewer infections and cytopenias than patients from the BURKIMAB08 trial. Patients >55 years old had a significantly higher treatment- related mortality despite dose reduction of chemotherapy. With a median follow-up of 3.61 years the 4-year OS probability was 73% (range, 63-81%). Age (≤55 vs. >55 years) and stage (localized vs. advanced) had prognostic significance. No significant differences in OS were observed in HIV-positive versus HIV-negative patients. The results of BURKIMAB14 are similar to those of other dose-intensive immunochemotherapy trials. Age >55 years and advanced stage, but not HIV infection, were associated with poor survival. Dose reduction of chemotherapy in young adults in CMR is safe and does not impact outcomes (clinicaltrials gov. Identifier: NCT05049473).

Real life data: follow-up assessment on Spanish Gaucher disease patients treated with eliglustat. TRAZELGA project.

BACKGROUND: The availability of multiple treatments for type 1 Gaucher disease increases the need for real-life studies to evaluate treatment efficacy and safety and provide clinicians with more information to choose the best personalized therapy for their patients. AIMS: To determine whether treatment with eliglustat produces, in adult GD1 patients, ans optimal response in daily clinical practice. METHODS: We designed a real-life study with 2 years of follow-up (TRAZELGA [GEE-ELI-2017-01]) to uniformly evaluate the response and adverse events to eliglustat treatment. This study, conducted in 30 patients across Spain and previously treated with other therapies, included the evaluation of safety and efficacy by assessing visceral enlargement, bone disease (DEXA and T and Z scores), concomitant treatments and adverse events, as well as a quality of life evaluation (SF-36). In addition, the quantification of classical biomarkers (chitotriosidase activity, CCL18/PARC and glucosylsphingosine (GluSph)) and new candidates for GD biomarkers (YKL-40, cathepsin S, hepcidin and lipocalin-2 determined by immunoassay) were also assessed. Non-parametric statistical analysis was performed and p < 0.05 was considered statistically significant. MAIN RESULTS: Thirty patients were enrolled in the study. The median age was 41.5 years and the male-female ratio was 1.1:1. 84% of the patients had received ERT and 16% SRT as previous treatment. The most common symptoms at baseline were fatigue (42%) and bone pain (38%), no patient had a bone crisis during the study, and two years after switching, 37% had reduced their use of analgesics. Patient-reported outcomes showed a significant increase in physical function scores (p = 0.027) and physical pain scores (p = 0.010). None of the enrolled patients discontinued treatment due to adverse events, which were mild and transient in nature, mainly gastrointestinal and skin dryness. None of the biomarkers show a significant increase or decompensation after switching. CCL18/PARC (p = 0.0012), YKL-40 (p = 0.00004) and lipocalin-2 (p = 0.0155) improved after two years and GluSph after one year (p = 0.0008) and two years (p = 0.0245) of oral therapy. CONCLUSION: In summary, this real-life study, showed that eliglustat maintains stability and can improve quality of life with few side effects. Significant reductions in classic and other novel biomarkers were observed after two years of therapy.

Mutational Profile Enables the Identification of a High-Risk Subgroup in Myelodysplastic Syndromes with Isolated Trisomy 8.

Trisomy 8 (+8) is the most frequent trisomy in myelodysplastic syndromes (MDS) and is associated with clinical heterogeneity and intermediate cytogenetic risk when found in isolation. The presence of gene mutations in this group of patients and the prognostic significance has not been extensively analyzed. Targeted deep sequencing was performed in a cohort of 79 MDS patients showing isolated +8. The most frequently mutated genes were: TET2 (38%), STAG2 (34.2%), SRSF2 (29.1%) and RUNX1 (26.6%). The mutational profile identified a high-risk subgroup with mutations in STAG2, SRSF2 and/or RUNX1, resulting in shorter time to acute myeloid leukemia progression (14 months while not reached in patients without these mutations, p < 0.0001) and shorter overall survival (23.7 vs. 46.3 months, p = 0.001). Multivariate analyses revealed the presence of mutations in these genes as an independent prognostic factor in MDS showing +8 isolated (HR: 3.1; p < 0.01). Moreover, 39.5% and 15.4% of patients classified as low/intermediate risk by the IPSS-R and IPSS-M, respectively, were re-stratified as a high-risk subgroup based on the mutational status of STAG2, SRSF2 and RUNX1. Results were validated in an external cohort (n = 2494). In summary, this study validates the prognosis significance of somatic mutations shown in IPSS-M and adds STAG2 as an important mutated gene to consider in this specific subgroup of patients. The mutational profile in isolated +8 MDS patients could, therefore, offer new insights for the correct management of patients with a higher risk of leukemic transformation.

Results of the compassionate program of inotuzumab ozogamicin for adult patients with relapsed or refractory acute lymphoblastic leukemia in Spain.

INTRODUCTION: The prognosis of relapsed B cell precursor acute lymphoblastic leukemia (B-ALL) is poor and few patients can be successfully rescued with conventional therapies. Inotuzumab ozogamicin (IO), an antibody against the CD22 antigen linked to calicheamicin, has been approved as a rescue treatment in relapsed/refractory (R/R) B-ALL. PATIENTS AND METHODS: This was an observational, retrospective, multicenter study of adult patients included in the Spanish program of compassionate use of IO in centers from the PETHEMA group (Programa Español de Tratamientos en Hematología). RESULTS: Thirty-four patients with a median age of 43 years (range, 19-73) were included. Twenty patients (59%) were refractory to the last treatment, IO treatment was given as ≥3rd salvage treatment in 25 patients (73%) and 20 patients (59%) received allogeneic hematopoietic stem cell transplantation before IO treatment. After a median of 2 cycles of IO, 64% of patients achieved complete response (CR)/complete response with incomplete recovery. The median response duration, progression-free survival and overall survival (OS) were 4.7 (95%CI, 2.4-7.0 months), 3.5 (95%CI, 1.0-5.0 months) and 4 months (95%CI, 1.9-6.1 months) respectively, with better OS for patients with relapsed B-ALL versus refractory disease (10.4 vs. 2.5 months, respectively) (p = .01). There was a trend for better OS for patients with first CR duration >12 months (7.2 months [95%CI, 3.2-11.2] vs. 3 months [95% CI, 1.8-4.2] respectively) (p = .054). There was no sinusoidal obstruction syndrome (SOS) event during IO treatment, but three patients (9%) developed grade 3-4 SOS during alloHSCT after IO treatment. CONCLUSIONS: Our study showed slightly inferior outcomes of the pivotal trial probably due to poorer risk factors and late onset of IO therapy of recruited patients. Our results support early use of IO in relapsed/refractory ALL patients.

A t(4;13)(q21;q14) translocation in B-cell chronic lymphocytic leukemia causing concomitant homozygous DLEU2/miR15a/miR16-1 and heterozygous ARHGAP24 deletions.

13q14 deletion is the most recurrent chromosomal aberration reported in B-CLL, having a favorable prognostic significance when occurring as the sole cytogenetic alteration. However, its clinical outcome is also related to the deletion size and number of cells with the del(13)(q14) deletion. In 10% of cases, 13q14 deletion arises following a translocation event with multiple partner chromosomes, whose oncogenic impact has not been investigated so far due to the assumption of a possible role as a passenger mutation. Here, we describe a t(4;13)(q21;q14) translocation occurring in a B-CLL case from the diagnosis to spontaneous regression. FISH and SNP-array analyses revealed a heterozygous deletion at 4q21, leading to the loss of the Rho GTPase Activating Protein 24 (ARHGAP24) tumor suppressor gene, down-regulated in the patient RNA, in addition to the homozygous deletion at 13q14 involving DLEU2/miR15a/miR16-1 genes. Interestingly, targeted Next Generation Sequencing analysis of 54 genes related to B-CLL indicated no additional somatic mutation in the patient, underlining the relevance of this t(4;13)(q21;q14) aberration in the leukemogenic process. In all tested RNA samples, RT-qPCR experiments assessed the downregulation of the PCNA, MKI67, and TOP2A proliferation factor genes, and the BCL2 anti-apoptotic gene as well as the up-regulation of TP53 and CDKN1A tumor suppressors, indicating a low proliferation potential of the cells harboring the aberration. In addition, RNA-seq analyses identified four chimeric transcripts (ATG4B::PTMA, OAZ1::PTMA, ZFP36::PTMA, and PIM3::BRD1), two of which (ATG4B::PTMA and ZFP36::PTMA) failed to be detected at the remission, suggesting a possible transcriptional remodeling during the disease course. Overall, our results indicate a favorable prognostic impact of the described chromosomal aberration, as it arises a permissive molecular landscape to the spontaneous B-CLL regression in the patient, highlighting ARHGAP24 as a potentially relevant concurrent alteration to the 13q14 deletion in delineating B-CLL disease evolution.

Machine Learning Improves Risk Stratification in Myelofibrosis: An Analysis of the Spanish Registry of Myelofibrosis.

Myelofibrosis (MF) is a myeloproliferative neoplasm (MPN) with heterogeneous clinical course. Allogeneic hematopoietic cell transplantation remains the only curative therapy, but its morbidity and mortality require careful candidate selection. Therefore, accurate disease risk prognostication is critical for treatment decision-making. We obtained registry data from patients diagnosed with MF in 60 Spanish institutions (N = 1386). These were randomly divided into a training set (80%) and a test set (20%). A machine learning (ML) technique (random forest) was used to model overall survival (OS) and leukemia-free survival (LFS) in the training set, and the results were validated in the test set. We derived the AIPSS-MF (Artificial Intelligence Prognostic Scoring System for Myelofibrosis) model, which was based on 8 clinical variables at diagnosis and achieved high accuracy in predicting OS (training set c-index, 0.750; test set c-index, 0.744) and LFS (training set c-index, 0.697; test set c-index, 0.703). No improvement was obtained with the inclusion of MPN driver mutations in the model. We were unable to adequately assess the potential benefit of including adverse cytogenetics or high-risk mutations due to the lack of these data in many patients. AIPSS-MF was superior to the IPSS regardless of MF subtype and age range and outperformed the MYSEC-PM in patients with secondary MF. In conclusion, we have developed a prediction model based exclusively on clinical variables that provides individualized prognostic estimates in patients with primary and secondary MF. The use of AIPSS-MF in combination with predictive models that incorporate genetic information may improve disease risk stratification.

Novel variants in GALE cause syndromic macrothrombocytopenia by disrupting glycosylation and thrombopoiesis.

Glycosylation is recognized as a key process for proper megakaryopoiesis and platelet formation. The enzyme uridine diphosphate (UDP)-galactose-4-epimerase, encoded by GALE, is involved in galactose metabolism and protein glycosylation. Here, we studied 3 patients from 2 unrelated families who showed lifelong severe thrombocytopenia, bleeding diathesis, mental retardation, mitral valve prolapse, and jaundice. Whole-exome sequencing revealed 4 variants that affect GALE, 3 of those previously unreported (Pedigree A, p.Lys78ValfsX32 and p.Thr150Met; Pedigree B, p.Val128Met; and p.Leu223Pro). Platelet phenotype analysis showed giant and/or grey platelets, impaired platelet aggregation, and severely reduced alpha and dense granule secretion. Enzymatic activity of the UDP-galactose-4-epimerase enzyme was severely decreased in all patients. Immunoblotting of platelet lysates revealed reduced GALE protein levels, a significant decrease in N-acetyl-lactosamine (LacNAc), showing a hypoglycosylation pattern, reduced surface expression of gylcoprotein Ibα-IX-V (GPIbα-IX-V) complex and mature ß1 integrin, and increased apoptosis. In vitro studies performed with patients-derived megakaryocytes showed normal ploidy and maturation but decreased proplatelet formation because of the impaired glycosylation of the GPIbα and ß1 integrin, and reduced externalization to megakaryocyte and platelet membranes. Altered distribution of filamin A and actin and delocalization of the von Willebrand factor were also shown. Overall, this study expands our knowledge of GALE-related thrombocytopenia and emphasizes the critical role of GALE in the physiological glycosylation of key proteins involved in platelet production and function.

Expert consensus on the use of systemic glucocorticoids for managing eosinophil-related diseases.

Eosinophil-related diseases represent a group of pathologic conditions with highly heterogeneous clinical presentation and symptoms ranging from mild to critical. Both systemic and localized forms of disease are typically treated with glucocorticoids. The approval of novel biologic therapies targeting the interleukin-5 pathway can help reduce the use of systemic glucocorticoids (SGC) in eosinophilic diseases and reduce the risk of SGC-related adverse effects (AEs). In this article, a panel of experts from different medical specialties reviewed current evidence on the use of SGC in two systemic eosinophilic diseases: Eosinophilic Granulomatosis with PolyAngiitis (EGPA) and HyperEosinophilic Syndrome (HES); and in two single-organ (respiratory) eosinophilic diseases: Chronic RhinoSinusitis with Nasal Polyps (CRSwNP) and Severe Asthma with Eosinophil Phenotype (SA-EP), and contrasted it with their experience in clinical practice. Using nominal group technique, they reached consensus on key aspects related to the dose and tapering of SGC as well as on the initiation of biologics as SGC-sparing agents. Early treatment with biologics could help prevent AEs associated with medium and long-term use of SGC.

Predictors of thrombosis and bleeding in 1613 myelofibrosis patients from the Spanish Registry of Myelofibrosis.

Available data have proved insufficient to develop consensus recommendations on the prevention of thrombosis and bleeding in myelofibrosis (MF). We evaluated the incidence and risk factors of vascular complications in 1613 patients from the Spanish Myelofibrosis Registry. Over a total of 6981 patient-years at risk, 6.4% of the study population had at least one thrombotic event after MF diagnosis, amounting to an incidence rate of 1.65 per 100 patient-years. Prior history of thrombosis, the JAK2 mutation, and the intermediate-2/high-risk International Prognostic Scoring System (IPSS) categories conferred an increased thrombotic risk after adjustment for the risk-modifying effect of anti-thrombotic and cytoreductive treatments. History of thrombosis and the JAK2 mutation allowed us to pinpoint a group of patients at higher risk of early thrombosis. No decreased incidence of thrombosis was observed while patients were on anti-thrombotic or cytoreductive treatment. An increased risk of venous thrombosis was found during treatment with immunomodulatory agents. A total of 5.3% of patients had at least one episode of major bleeding, resulting in an incidence rate of 1.5 events per 100 patient-years. Patients in the intermediate-2/high-risk IPSS categories treated with anti-coagulants had an almost sevenfold increased risk of major bleeding. These findings should prove useful for guiding decision-making in clinical practice.

Second Revision of the International Staging System (R2-ISS) for Overall Survival in Multiple Myeloma: A European Myeloma Network (EMN) Report Within the HARMONY Project.

PURPOSE: Patients with newly diagnosed multiple myeloma (NDMM) show heterogeneous outcomes, and approximately 60% of them are at intermediate-risk according to the Revised International Staging system (R-ISS), the standard-of-care risk stratification model. Moreover, chromosome 1q gain/amplification (1q+) recently proved to be a poor prognostic factor. In this study, we revised the R-ISS by analyzing the additive value of each single risk feature, including 1q+. PATIENTS AND METHODS: The European Myeloma Network, within the HARMONY project, collected individual data from 10,843 patients with NDMM enrolled in 16 clinical trials. An additive scoring system on the basis of top features predicting progression-free survival (PFS) and overall survival (OS) was developed and validated. RESULTS: In the training set (N = 7,072), at a median follow-up of 75 months, ISS, del(17p), lactate dehydrogenase, t(4;14), and 1q+ had the highest impact on PFS and OS. These variables were all simultaneously present in 2,226 patients. A value was assigned to each risk feature according to their OS impact (ISS-III 1.5, ISS-II 1, del(17p) 1, high lactate dehydrogenase 1, and 1q+ 0.5 points). Patients were stratified into four risk groups according to the total additive score: low (Second Revision of the International Staging System [R2-ISS]-I, 19.2%, 0 points), low-intermediate (II, 30.8%, 0.5-1 points), intermediate-high (III, 41.2%, 1.5-2.5 points), high (IV, 8.8%, 3-5 points). Median OS was not reached versus 109.2 versus 68.5 versus 37.9 months, and median PFS was 68 versus 45.5 versus 30.2 versus 19.9 months, respectively. The score was validated in an independent validation set (N = 3,771, of whom 1,214 were with complete data to calculate R2-ISS) maintaining its prognostic value. CONCLUSION: The R2-ISS is a simple prognostic staging system allowing a better stratification of patients with intermediate-risk NDMM. The additive nature of this score fosters its future implementation with new prognostic variables.

Building a Healthcare Alliance for Resourceful Medicine Offensive Against Neoplasms in Hematology Added Value Framework for Hematologic Malignancies: A Comparative Analysis of Existing Tools.

OBJECTIVES: The Innovative Medicines Initiative-funded, multistakeholders project Healthcare Alliance for Resourceful Medicine Offensive Against Neoplasms in Hematology (HARMONY) created a task force involving patient organizations, medical associations, pharmaceutical companies, and health technology assessment/regulator agencies' representatives to evaluate the suitability of previously established value frameworks (VFs) for assessing the clinical and societal impact of new interventions for hematologic malignancies (HMs). METHODS: Since the HARMONY stakeholders identified the inclusion of patients' points of view on evaluating VFs as a priority, surveys were conducted with the patient organizations active in HMs and part of the HARMONY network, together with key opinion leaders, pharmaceutical companies, and regulators, to establish which outcomes were important for each HM. Next, to evaluate VFs against the sources of information taken into account (randomized clinical trials, registries, real-world data), structured questionnaires were created and filled by HARMONY health professionals to specify preferred data sources per malignancy. Finally, a framework evaluation module was built to analyze existing clinical VFs (American Society of Clinical Oncology, European Society of Medical Oncology, Magnitude of Clinical Benefit Scale, Institut für Qualität und Wirtschaftlichkeit im Gesundheitswesen, Institute for Clinical and Economic Review, National Comprehensive Cancer Network Evidence Blocks, and patient-perspective VF). RESULTS: The comparative analysis describes challenges and opportunities for the use of each framework in the context of HMs and drafts possible lines of action for creating or integrating a more specific, patient-focused clinical VF for HMs. CONCLUSIONS: None of the frameworks meets the HARMONY goals for a tool that applies to HMs and assesses in a transparent, reproducible, and systematic way the therapeutic value of innovative health technologies versus available alternatives, taking a patient-centered approach and using real-world evidence.

TRAF3 alterations are frequent in del-3'IGH chronic lymphocytic leukemia patients and define a specific subgroup with adverse clinical features.

Interstitial 14q32 deletions involving IGH gene are infrequent events in chronic lymphocytic leukemia (CLL), affecting less than 5% of patients. To date, little is known about their clinical impact and molecular underpinnings, and its mutational landscape is currently unknown. In this work, a total of 871 CLLs were tested for the IGH break-apart probe, and 54 (6.2%) had a 300 kb deletion of 3'IGH (del-3'IGH CLLs), which contributed to a shorter time to first treatment (TFT). The mutational analysis by next-generation sequencing of 317 untreated CLLs (54 del-3'IGH and 263 as the control group) showed high mutational frequencies of NOTCH1 (30%), ATM (20%), genes involved in the RAS signaling pathway (BRAF, KRAS, NRAS, and MAP2K1) (15%), and TRAF3 (13%) within del-3'IGH CLLs. Notably, the incidence of TRAF3 mutations was significantly higher in del-3'IGH CLLs than in the control group (p < .001). Copy number analysis also revealed that TRAF3 loss was highly enriched in CLLs with 14q deletion (p < .001), indicating a complete biallelic inactivation of this gene through deletion and mutation. Interestingly, the presence of mutations in the aforementioned genes negatively refined the prognosis of del-3'IGH CLLs in terms of overall survival (NOTCH1, ATM, and RAS signaling pathway genes) and TFT (TRAF3). Furthermore, TRAF3 biallelic inactivation constituted an independent risk factor for TFT in the entire CLL cohort. Altogether, our work demonstrates the distinct genetic landscape of del-3'IGH CLL with multiple molecular pathways affected, characterized by a TRAF3 biallelic inactivation that contributes to a marked poor outcome in this subgroup of patients.

A novel nonsense variant in TPM4 caused dominant macrothrombocytopenia, mild bleeding tendency and disrupted cytoskeleton remodeling.

BACKGROUND: Rare inherited thrombocytopenias are caused by alterations in genes involved in megakaryopoiesis, thrombopoiesis and/or platelet release. Diagnosis is challenging due to poor specificity of platelet laboratory assays, large numbers of culprit genes, and difficult assessment of the pathogenicity of novel variants. OBJECTIVES: To characterize the clinical and laboratory phenotype, and identifying the underlying molecular alteration, in a pedigree with thrombocytopenia of uncertain etiology. PATIENTS/METHODS: Index case was enrolled in our Spanish multicentric project of inherited platelet disorders due to lifelong thrombocytopenia and bleeding. Bleeding score was recorded by ISTH-BAT. Laboratory phenotyping consisted of blood cells count, blood film, platelet aggregation and flow cytometric analysis. Genotyping was made by whole-exome sequencing (WES). Cytoskeleton proteins were analyzed in resting/spreading platelets by immunofluorescence and immunoblotting. RESULTS: Five family members displayed lifelong mild thrombocytopenia with a high number of enlarged platelets in blood film, and mild bleeding tendency. Patient's platelets showed normal aggregation and granule secretion response to several agonists. WES revealed a novel nonsense variant (c.322C>T; p.Gln108*) in TPM4 (NM_003290.3), the gene encoding for tropomyosin-4 (TPM4). This variant led to impairment of platelet spreading capacity after stimulation with TRAP-6 and CRP, delocalization of TPM4 in activated platelets, and significantly reduced TPM4 levels in platelet lysates. Moreover, the index case displayed up-regulation of TPM2 and TPM3 mRNA levels. CONCLUSIONS: This study identifies a novel TPM4 nonsense variant segregating with macrothrombocytopenia and impaired platelet cytoskeletal remodeling and spreading. These findings support the relevant role of TPM4 in thrombopoiesis and further expand our knowledge of TPM4-related thrombocytopenia.