GMO. Genética Molecular en Oncohematología

Mepolizumab for hypereosinophilic syndrome: effectiveness and safety from real-world evidence

Wed, 01 Jan 2025 00:00:00 GMT

[EN]Hypereosinophilic syndrome (HES) is a rare condition characterized by elevated eosinophil levels and related symptoms of eosinophil-mediated organ damage. We reviewed the effectiveness and safety of mepolizumab for the treatment of HES. A scoping review was conducted following the PRISMA Scoping Reviews Checklist to identify real-world evidence of mepolizumab use in HES. In total, 36 references were identified as relevant and selected for review. Overall, 105 patients previously treated with glucocorticoids received mepolizumab at different dosages (range: 100-750 mg), routes of administration (subcutaneous/intravenous), and schedules (every 2-12 weeks). Remission rates were 57.1-76.0%. Most studies reported a range of 71.4-99.1% reduction in mean blood eosinophil counts with mepolizumab treatment. In addition, a glucocorticoid-sparing effect was observed; 85.7% of patients discontinued glucocorticoids after 12 months of mepolizumab administration. Mepolizumab was considered safe and well-tolerated and severe adverse events were rare. Mepolizumab provided clinically significant benefits in patients with HES in a real-world setting.

Adverse prognostic impact of complex karyotype (≥3 cytogenetic alterations) in adult T-cell acute lymphoblastic leukemia (T-ALL).

Fri, 01 Oct 2021 00:00:00 GMT

[EN]The potential prognostic value of conventional karyotyping in adult T-cell acute lymphoblastic leukemia (T-ALL) remains an open question. We hypothesized that a modified cytogenetic classification, based on the number and type of cytogenetic abnormalities, would allow the identification of high-risk adult T-ALL patients. Complex karyotype defined by the presence of ≥3 cytogenetic alterations identified T-ALL patients with poor prognosis in this study. Karyotypes with ≥3 abnormalities accounted for 16 % (22/139) of all evaluable karyotypes, corresponding to the largest poor prognosis cytogenetic subgroup of T-ALL identified so far. Patients carrying karyotypes with ≥3 cytogenetic alterations showed a significantly inferior response to therapy, and a poor outcome in terms of event-free survival (EFS), overall survival (OS) and cumulative incidence of relapse (CIR), independently of other baseline characteristics and the end-induction minimal residual disease (MRD) level. Additional molecular analyses of patients carrying ≥3 cytogenetic alterations showed a unique molecular profile that could contribute to understand the underlying molecular mechanisms of resistance and to evaluate novel targeted therapies (e.g. IL7R directed) with potential impact on outcome of adult T-ALL patients.

Machine learning risk stratification strategy for multiple myeloma: Insights from the EMN-HARMONY Alliance platform

Wed, 01 Oct 2025 00:00:00 GMT

[EN]Traditional risk stratification in multiple myeloma (MM) relies on clinical and cytogenetic parameters but has limited predictive accuracy. Machine learning (ML) offers a novel approach by leveraging large datasets and complex variable interactions. This study aimed to develop and validate novel ML-driven prognostic scores for newly diagnosed MM (NDMM), with the goal of improving upon existing ones. To this end, we analyzed data from the EMN-HARMONY MM cohort, comprising 14,345 patients, including 10,843 NDMM patients enrolled across 16 clinical trials. Three ML models were developed: (1) a comprehensive model incorporating 20 variables, (2) a reduced model including six key variables (age, hemoglobin, β2-microglobulin, albumin, 1q gain, and 17p deletion), and (3) a cytogenetics-free model. All models were internally validated using out-of-bag cross-validation and externally validated with data from the Myeloma XI trial. Model performance was evaluated using the concordance index (C-index) and time-dependent area under the receiver operating characteristic curve (ROC-AUC). The comprehensive model achieved C-index values of 0.666 (training) and 0.667 (test) for overall survival (OS) and 0.620/0.627 for progression-free survival (PFS). The reduced model maintained accuracy (OS: 0.658/0.657; PFS: 0.608/0.614). The cytogenetics-free model showed C-index values of 0.636/0.643 for OS and 0.600/0.610 for PFS. Incorporating treatment type and best response to first-line treatment further improved performance. The new prognostic models improved over the International Staging System (ISS), Revised International Staging System (R-ISS), and Second Revision of the International Staging System (R2-ISS) and were reproducible in real-world and relapsed/refractory MM, including daratumumab-treated patients. This ML-based risk stratification strategy provides individualized risk predictions, surpassing traditional group-based methods and demonstrating broad applicability across patient subgroups. An online calculator is available at https://taxonomy.harmony-platform.eu/riskcalculator/.

Integrating AIPSS-MF and molecular predictors: A comparative analysis of prognostic models for myelofibrosis

Mon, 01 Jan 2024 00:00:00 GMT

[EN]Myelofibrosis (MF) is a chronic myeloproliferative neoplasm that can manifest as a primary condition (primary myelofibrosis [PMF]) or after progression from a polycythemia vera or essential thrombocythemia (secondary myelofibrosis [SMF]). The aberrant activation of the JAK‐STAT pathway is central to MF pathogenesis which is caused by driver mutations in JAK2, CALR, and MPL genes. These mutations, along with additional somatic variants that mainly impact epigenetic modifiers or spliceosome components, shape the clinical features of the disease. Although the median overall survival (OS) is around 6 years, the clinical course of MF is heterogeneous. The only curative strategy, allogeneic hematopoietic cell transplantation, carries a significant risk of early mortality.2 It is therefore critical to accurately assess transplantation risk and estimate survival with medical therapies to determine the most appropriate treatment approach for each individual.

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

Fri, 01 Sep 2023 00:00:00 GMT

[EN]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. 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). 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. 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.

Single agent subcutaneous blinatumomab for advanced acute lymphoblastic leukemia

Mon, 01 Jan 2024 00:00:00 GMT

[EN]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.

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

Thu, 01 Feb 2024 00:00:00 GMT

[EN]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).

Mutational profile enables the identification of a high risk subgroup in myelodysplastic syndromes with isolated trisomy 8

Thu, 27 Jul 2023 00:00:00 GMT

[EN]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.

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

Mon, 01 Apr 2024 00:00:00 GMT

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

Covering hierarchical Dirichlet mixture models on binary data to enhance genomic stratifications in onco-hematology

Thu, 01 Feb 2024 00:00:00 GMT

[EN]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.

Impact of center-related characteristics and macroeconomic factors on the outcome of adult patients with acute lymphoblastic leukemia treated with pediatric inspired protocols

Sun, 01 Jan 2023 00:00:00 GMT

[EN]Diagnosis and treatment of hematological cancers is usually provided in many healthcare facilities including large but also middle size centers.1 Providing cancer care in local institutions might be advantageous for patients and caregivers in terms of financial burden and quality of life. However, it might carry potential risks derived of the limited experience of smaller centers and differences in accessibility to complex therapies including allogeneic hematopoietic cell transplantation (allo-HCT) and chimeric antigen receptor (CAR) T-cells. These risks might be especially relevant in infrequent cancers as adult acute lymphoblastic leukemia (ALL). In most European countries, ALL treatment protocols are based on pediatric-inspired regimens which include a large number of immune-chemotherapeutic agents and several key decision points to allocate patients to distinctive treatment arms based on genetics and treatment response.3,4 Several patient and disease characteristics have been identified as prognostic factors for outcomes including age, white blood cell count (WBC) at diagnosis, central nervous system (CNS) infiltration, clearance of measurable residual disease (MRD),4 and disease genetics.5–7 The outcome of patients with ALL may also depend on external factors including center experience, access to cellular therapies and economic variables.8–10 The impact of these center-related and macroeconomic variables on the outcome of patients has been scarcely studied. Thus, the aim of this study was to analyze the potential impact of center-related and macroeconomic variables on the outcome of newly diagnosed adult ALL patients included in 4 consecutive trials of the Spanish Program for Treatment of Hematological Malignancies (PETHEMA) Group. Patients with Philadelphia chromosome (Ph) positive or negative ALL enrolled in one of the 4 consecutive protocols of the PETHEMA group by Spanish institutions adhered to the public health system from 2003 to 2018 were included in this study. The 4 protocols have been closed and reported elsewhere.4,11–13 Centralized analysis of MRD was performed in 5 centers in the ALL-AR-03 and in a single institution in the ALL-HR-11 trial. The PH-08 protocol included adults with newly diagnosed Ph-positive ALL up to the age of 60 years. Patients received an induction therapy with daunorubicin, vincristine, and steroids in combination with imatinib 600 mg/d followed by a 12-week consolidation chemotherapy based on alternated cycles of high-dose methotrexate and cytarabine in combination with imatinib. Allo-HCT was offered to all fit patients with a suitable donor. Treatment protocols used in the ALL-RE-2008, ALL-AR-03, and ALL-HR-11 trials were pediatric-inspired.4,11–13 The ALL-RE-2008 trial included intermediate risk patients based on age (<30 years), WBC count (<25,000 cells/μL), and cytogenetics. The ALL-AR-03 and ALL-HR-11 protocols included high-risk patients up to the age of 60 years diagnosed from 2003 to 2011 and 2011 to 2019, respectively. In both trials, bone marrow MRD assessment by flow cytometry was performed at the end of induction (week 5) and at the end of the third consolidation cycle (weeks 16–18). Only patients with slow clearance of MRD in both trials were allocated to allo-HCT, while patients with good MRD clearance continued with chemotherapy for up to 2 years. Clinical variables analyzed in this study included age, gender, ECOG performance status, WBC, CNS infiltration, precursor lineage (B or T) presence or absence of Ph and treatment period (2003–2010 versus 2011–2018). The Allo-HCT center was defined as centers having authorization by the Spanish government to perform allo-HCT in the same institution where the patient was treated for the ALL. The Allo-HCT center in the same province was defined as having a designated allo-HCT center in the same province where the patient was treated. Reported ALL referred to the number of ALL patients reported to the PETHEMA database by a particular center and served as a surrogate marker of center experience in treating ALL. Nine centers reporting at least 30 ALL patients each and around half of the patients together in this data set were considered as “experienced centers.” Protocol deviation center referred to centers with identified protocol deviations in key treatment decisions (allo-HCT versus chemotherapy allocation or autologous HCT instead of allo-HCT when not indicated in the protocol) in at least 5% of the patients.

Optimizing surgical approaches for patients with inherited factor VII deficiency

Tue, 06 Jan 2026 00:00:00 GMT

[EN]Inherited factor VII deficiency (FVIID) presents a highly variable bleeding phenotype. The weak correlation between plasma FVII levels (FVII:C) and bleeding severity results in diverse management strategies and complicates surgical decision-making.To describe surgical management and bleeding outcomes in patients with FVIID, and to identify key decision-making variables and predictors of surgical bleeding.We conducted a multicenter, retrospective study of 380 surgeries performed in 215 patients with FVIID. Patients were classified by FVII:C levels as mild, moderate, or severe deficiency. Bleeding score (BS) was defined according to ISTH-BAT. Surgeries were categorized as low-moderate risk (LR) or high risk (HR) for bleeding. A decision-tree simulation was performed.Most patients had mild FVIID (76%), and 68% of surgeries were classified as LR. Prophylactic treatment with tranexamic acid (TA) and/or rFVIIa was administered in 42.8% of LR and 62.8% of HR surgeries. Prophylaxis was given to 73.9% of moderate/severe and 41% of mild FVIID patients, especially for HR procedures. FVII:C levels and surgical bleeding risk were key factors that influenced the selection of treatment. The overall bleeding rate was 3.1% (HR: 9%; LR: 0.4%). Most bleeding events occurred in mild FVIID patients with BS ≥3. Our algorithm recommends hemostatic treatment for all moderate/severe, and for mild patients HR surgeries and LR procedures when BS is ≥3.FVII:C levels and surgery type influence prophylactic hemostatic treatment strategies. Patients with mild FVIID, higher BS, and no hemostatic treatment had a greater risk of bleeding. Bleeding score and procedural risk were identified as predictors of surgical bleeding.

Insights into the clinical, platelet and genetic landscape of inherited thrombocytopenia with malignancy risk

Wed, 01 Oct 2025 00:00:00 GMT

[EN]Inherited thrombocytopenia (IT) with germline variants in RUNX1, ETV6 or ANKRD26 carries a high risk (10%-45%) of developing haematological malignancy (IT-HM). We evaluated the clinical, platelet and molecular characteristics in 37 patients with RUNX1-related thrombocytopenia (RT), 9 with ETV6-RT and 20 with ANRKD26-RT. Genetic diagnosis was delayed by about 20 years from the identification of thrombocytopenia. Bleeding tendency was present in 25%-30% of RUNX1-RT and ANKRD26-RT patients. Platelet aggregation was impaired in 90% of all patients, while reduced activation and granule secretion were heterogeneous. Most RUNX1-RT patients had low glycoprotein Ia (GPIa) levels, which may be a useful disease biomarker. Sixteen distinct genetic variants in RUNX1, four in ETV6 and four in ANKRD26 were identified in patients. The clinical profile showed immune, skin, gastrointestinal and other comorbidities in many patients. One third of the cases developed a malignancy: This included eight RUNX1-RT patients with myelodysplastic syndrome (MDS), five with acute myeloid leukaemia (AML), and one with chronic myeloid leukaemia (CML) Ph+. One patient with ETV6-RT subsequently developed B-cell acute lymphoblastic leukaemia (B-ALL) during childhood. Three cases with ANKRD26-RT demonstrated a multifaceted clinical presentation, including B-ALL Ph+, MDS and breast cancer. The high incidence of HM development highlights the importance of early diagnosis in life.

Platelet transcriptome analysis in patients with germline RUNX1 mutations

Mon, 01 May 2023 00:00:00 GMT

[EN]Germline mutations in RUNX1 can cause a familial platelet disorder that may lead to acute myeloid leukemia, an autosomal dominant disorder characterized by moderate thrombocytopenia, platelet dysfunction, and a high risk of developing acute myeloid leukemia or myelodysplastic syndrome. Discerning the pathogenicity of novel RUNX1 variants is critical for patient management. To extend the characterization of RUNX1 variants and evaluate their effects by transcriptome analysis. Three unrelated patients with long-standing thrombocytopenia carrying heterozygous RUNX1 variants were included: P1, who is a subject with recent development of myelodysplastic syndrome, with c.802 C>T[p.Gln268∗] de novo; P2 with c.586A>G[p.Thr196Ala], a variant that segregates with thrombocytopenia and myeloid neoplasia in the family; and P3 with c.476A>G[p.Asn159Ser], which did not segregate with thrombocytopenia or neoplasia. Baseline platelet evaluations were performed. Ultrapure platelets were prepared for platelet transcriptome analysis. In P1 and P2, but not in P3, transcriptome analysis confirmed aberrant expression of genes recognized as RUNX1 targets. Data allowed grouping patients by distinct gene expression profiles, which were partitioned with clinical parameters. Functional studies and platelet mRNA expression identified alterations in the actin cytoskeleton, downregulation of GFI1B, defective GPVI downstream signaling, and reduction of alpha granule proteins, such as thrombospondin-1, as features likely implicated in thrombocytopenia and platelet dysfunction. Platelet phenotype, familial segregation, and platelet transcriptomics support the pathogenicity of RUNX1 variants p.Gln268∗ and p.Thr196Ala, but not p.Asn159Ser. This study is an additional proof of concept that platelet RNA analysis could be a tool to help classify pathogenic RUNX1 variants and identify novel RUNX1 targets.

Inherited Thrombocytopenia Caused by Variants in Crucial Genes for Glycosylation

Tue, 07 Mar 2023 00:00:00 GMT

[EN]Protein glycosylation, including sialylation, involves complex and frequent post-translational modifications, which play a critical role in different biological processes. The conjugation of carbohydrate residues to specific molecules and receptors is critical for normal hematopoiesis, as it favors the proliferation and clearance of hematopoietic precursors. Through this mechanism, the circulating platelet count is controlled by the appropriate platelet production by megakaryocytes, and the kinetics of platelet clearance. Platelets have a half-life in blood ranging from 8 to 11 days, after which they lose the final sialic acid and are recognized by receptors in the liver and eliminated from the bloodstream. This favors the transduction of thrombopoietin, which induces megakaryopoiesis to produce new platelets. More than two hundred enzymes are responsible for proper glycosylation and sialylation. In recent years, novel disorders of glycosylation caused by molecular variants in multiple genes have been described. The phenotype of the patients with genetic alterations in GNE, SLC35A1, GALE and B4GALT is consistent with syndromic manifestations, severe inherited thrombocytopenia, and hemorrhagic complications.

Expanding the genetic spectrum of TUBB1-related thrombocytopenia

Fri, 01 Jan 2021 00:00:00 GMT

[EN]β1-Tubulin plays a major role in proplatelet formation and platelet shape maintenance, and pathogenic variants in TUBB1 lead to thrombocytopenia and platelet anisocytosis (TUBB1-RT). To date, the reported number of pedigrees with TUBB1-RT and of rare TUBB1 variants with experimental demonstration of pathogenicity is limited. Here, we report 9 unrelated families presenting with thrombocytopenia carrying 6 β1-tubulin variants, p.Cys12LeufsTer12, p.Thr107Pro, p.Gln423*, p.Arg359Trp, p.Gly109Glu, and p.Gly269Asp, the last of which novel. Segregation studies showed incomplete penetrance of these variants for platelet traits. Indeed, most carriers showed macrothrombocytopenia, some only increased platelet size, and a minority had no abnormalities. Moreover, only homozygous carriers of the p.Gly109Glu variant displayed macrothrombocytopenia, highlighting the importance of allele burden in the phenotypic expression of TUBB1-RT. The p.Arg359Trp, p.Gly269Asp, and p.Gly109Glu variants deranged β1-tubulin incorporation into the microtubular marginal ring in platelets but had a negligible effect on platelet activation, secretion, or spreading, suggesting that β1-tubulin is dispensable for these processes. Transfection of TUBB1 missense variants in CHO cells altered β1-tubulin incorporation into the microtubular network. In addition, TUBB1 variants markedly impaired proplatelet formation from peripheral blood CD34+ cell-derived megakaryocytes. Our study, using in vitro modeling, molecular characterization, and clinical investigations provides a deeper insight into the pathogenicity of rare TUBB1 variants. These novel data expand the genetic spectrum of TUBB1-RT and highlight a remarkable heterogeneity in its clinical presentation, indicating that allelic burden or combination with other genetic or environmental factors modulate the phenotypic impact of rare TUBB1 variants.