Comparing malignant monocytosis across the updated WHO and ICC classifications of 2022.

ABSTRACT: The World Health Organization (WHO) classification of hematolymphoid tumors and the International Consensus Classification (ICC) of 2022 introduced major changes to the definition of chronic myelomonocytic leukemia (CMML). To assess its qualitative and quantitative implications for patient care, we started with 3311 established CMML cases (according to WHO 2017 criteria) and included 2130 oligomonocytosis cases fulfilling the new CMML diagnostic criteria. Applying both 2022 classification systems, 356 and 241 of oligomonocytosis cases were newly classified as myelodysplastic (MD)-CMML (WHO and ICC 2022, respectively), most of which were diagnosed as myelodysplastic syndrome (MDS) according to the WHO 2017 classification. Importantly, 1.5 times more oligomonocytosis cases were classified as CMML according to WHO 2022 than based on ICC, because of different diagnostic criteria. Genetic analyses of the newly classified CMML cases showed a distinct mutational profile with strong enrichment of MDS-typical alterations, resulting in a transcriptional subgroup separated from established MD and myeloproliferative CMML. Despite a different cytogenetic, molecular, immunophenotypic, and transcriptional landscape, no differences in overall survival were found between newly classified and established MD-CMML cases. To the best of our knowledge, this study represents the most comprehensive analysis of routine CMML cases to date, both in terms of clinical characterization and transcriptomic analysis, placing newly classified CMML cases on a disease continuum between MDS and previously established CMML.

Tumor necrosis factor α promotes clonal dominance of KIT D816V+ cells in mastocytosis: role of survivin and impact on prognosis.

ABSTRACT: Systemic mastocytosis (SM) is defined by the expansion and accumulation of neoplastic mast cells (MCs) in the bone marrow (BM) and extracutaneous organs. Most patients harbor a somatic KIT D816V mutation, which leads to growth factor-independent KIT activation and accumulation of MC. Tumor necrosis factor α (TNF) is a proapoptotic and inflammatory cytokine that has been implicated in the clonal selection of neoplastic cells. We found that KIT D816V increases the expression and secretion of TNF. TNF expression in neoplastic MCs is reduced by KIT-targeting drugs. Similarly, knockdown of KIT or targeting the downstream signaling cascade of MAPK and NF-κB signaling reduced TNF expression levels. TNF reduces colony formation in human BM cells, whereas KIT D816V+ cells are less susceptible to the cytokine, potentially contributing to clonal selection. In line, knockout of TNF in neoplastic MC prolonged survival and reduced myelosuppression in a murine xenotransplantation model. Mechanistic studies revealed that the relative resistance of KIT D816V+ cells to TNF is mediated by the apoptosis-regulator BIRC5 (survivin). Expression of BIRC5 in neoplastic MC was confirmed by immunohistochemistry of samples from patients with SM. TNF serum levels are significantly elevated in patients with SM and high TNF levels were identified as a biomarker associated with inferior survival. We here characterized TNF as a KIT D816V-dependent cytokine that promotes clonal dominance. We propose TNF and apoptosis-associated proteins as potential therapeutic targets in SM.

Germ line DDX41 mutations define a unique subtype of myeloid neoplasms.

Germ line DDX41 variants have been implicated in late-onset myeloid neoplasms (MNs). Despite an increasing number of publications, many important features of DDX41-mutated MNs remain to be elucidated. Here we performed a comprehensive characterization of DDX41-mutated MNs, enrolling a total of 346 patients with DDX41 pathogenic/likely-pathogenic (P/LP) germ line variants and/or somatic mutations from 9082 MN patients, together with 525 first-degree relatives of DDX41-mutated and wild-type (WT) patients. P/LP DDX41 germ line variants explained ∼80% of known germ line predisposition to MNs in adults. These risk variants were 10-fold more enriched in Japanese MN cases (n = 4461) compared with the general population of Japan (n = 20 238). This enrichment of DDX41 risk alleles was much more prominent in male than female (20.7 vs 5.0). P/LP DDX41 variants conferred a large risk of developing MNs, which was negligible until 40 years of age but rapidly increased to 49% by 90 years of age. Patients with myelodysplastic syndromes (MDS) along with a DDX41-mutation rapidly progressed to acute myeloid leukemia (AML), which was however, confined to those having truncating variants. Comutation patterns at diagnosis and at progression to AML were substantially different between DDX41-mutated and WT cases, in which none of the comutations affected clinical outcomes. Even TP53 mutations made no exceptions and their dismal effect, including multihit allelic status, on survival was almost completely mitigated by the presence of DDX41 mutations. Finally, outcomes were not affected by the conventional risk stratifications including the revised/molecular International Prognostic Scoring System. Our findings establish that MDS with DDX41-mutation defines a unique subtype of MNs that is distinct from other MNs.

Genome-wide association study identifies novel susceptibility loci for KIT D816V positive mastocytosis.

Mastocytosis is a rare myeloid neoplasm characterized by uncontrolled expansion of mast cells, driven in >80% of affected individuals by acquisition of the KIT D816V mutation. To explore the hypothesis that inherited variation predisposes to mastocytosis, we performed a two-stage genome-wide association study, analyzing 1,035 individuals with KIT D816V positive disease and 17,960 healthy control individuals from five European populations. After quality control, we tested 592,007 SNPs at stage 1 and 75 SNPs at stage 2 for association by using logistic regression and performed a fixed effects meta-analysis to combine evidence across the two stages. From the meta-analysis, we identified three intergenic SNPs associated with mastocytosis that achieved genome-wide significance without heterogeneity between cohorts: rs4616402 (pmeta = 1.37 × 10-15, OR = 1.52), rs4662380 (pmeta = 2.11 × 10-12, OR = 1.46), and rs13077541 (pmeta = 2.10 × 10-9, OR = 1.33). Expression quantitative trait analyses demonstrated that rs4616402 is associated with the expression of CEBPA (peQTL = 2.3 × 10-14), a gene encoding a transcription factor known to play a critical role in myelopoiesis. The role of the other two SNPs is less clear: rs4662380 is associated with expression of the long non-coding RNA gene TEX41 (peQTL = 2.55 × 10-11), whereas rs13077541 is associated with the expression of TBL1XR1, which encodes transducin (ß)-like 1 X-linked receptor 1 (peQTL = 5.70 × 10-8). In individuals with available data and non-advanced disease, rs4616402 was associated with age at presentation (p = 0.009; beta = 4.41; n = 422). Additional focused analysis identified suggestive associations between mastocytosis and genetic variation at TERT, TPSAB1/TPSB2, and IL13. These findings demonstrate that multiple germline variants predispose to KIT D816V positive mastocytosis and provide novel avenues for functional investigation.

Enhancer retargeting of CDX2 and UBTF::ATXN7L3 define a subtype of high-risk B-progenitor acute lymphoblastic leukemia.

Transcriptome sequencing has identified multiple subtypes of B-progenitor acute lymphoblastic leukemia (B-ALL) of prognostic significance, but a minority of cases lack a known genetic driver. Here, we used integrated whole-genome (WGS) and -transcriptome sequencing (RNA-seq), enhancer mapping, and chromatin topology analysis to identify previously unrecognized genomic drivers in B-ALL. Newly diagnosed (n = 3221) and relapsed (n = 177) B-ALL cases with tumor RNA-seq were studied. WGS was performed to detect mutations, structural variants, and copy number alterations. Integrated analysis of histone 3 lysine 27 acetylation and chromatin looping was performed using HiChIP. We identified a subset of 17 newly diagnosed and 5 relapsed B-ALL cases with a distinct gene expression profile and 2 universal and unique genomic alterations resulting from aberrant recombination-activating gene activation: a focal deletion downstream of PAN3 at 13q12.2 resulting in CDX2 deregulation by the PAN3 enhancer and a focal deletion of exons 18-21 of UBTF at 17q21.31 resulting in a chimeric fusion, UBTF::ATXN7L3. A subset of cases also had rearrangement and increased expression of the PAX5 gene, which is otherwise uncommon in B-ALL. Patients were more commonly female and young adult with median age 35 (range,12-70 years). The immunophenotype was characterized by CD10 negativity and immunoglobulin M positivity. Among 16 patients with known clinical response, 9 (56.3%) had high-risk features including relapse (n = 4) or minimal residual disease >1% at the end of remission induction (n = 5). CDX2-deregulated, UBTF::ATXN7L3 rearranged (CDX2/UBTF) B-ALL is a high-risk subtype of leukemia in young adults for which novel therapeutic approaches are required.

Genomic profiling for clinical decision making in myeloid neoplasms and acute leukemia.

Myeloid neoplasms and acute leukemias derive from the clonal expansion of hematopoietic cells driven by somatic gene mutations. Although assessment of morphology plays a crucial role in the diagnostic evaluation of patients with these malignancies, genomic characterization has become increasingly important for accurate diagnosis, risk assessment, and therapeutic decision making. Conventional cytogenetics, a comprehensive and unbiased method for assessing chromosomal abnormalities, has been the mainstay of genomic testing over the past several decades and remains relevant today. However, more recent advances in sequencing technology have increased our ability to detect somatic mutations through the use of targeted gene panels, whole-exome sequencing, whole-genome sequencing, and whole-transcriptome sequencing or RNA sequencing. In patients with myeloid neoplasms, whole-genome sequencing represents a potential replacement for both conventional cytogenetic and sequencing approaches, providing rapid and accurate comprehensive genomic profiling. DNA sequencing methods are used not only for detecting somatically acquired gene mutations but also for identifying germline gene mutations associated with inherited predisposition to hematologic neoplasms. The 2022 International Consensus Classification of myeloid neoplasms and acute leukemias makes extensive use of genomic data. The aim of this report is to help physicians and laboratorians implement genomic testing for diagnosis, risk stratification, and clinical decision making and illustrates the potential of genomic profiling for enabling personalized medicine in patients with hematologic neoplasms.

Classical meets malignant hematology: a case of acquired εγδß-thalassemia in clonal hematopoiesis.

Hemoglobinopathies including thalassemias are among the most frequent genetic disorders worldwide. Primarily, these entities result from germline variants in the globin gene clusters and their cis-acting regulatory elements, and thus the WHO classifies thalassemias as inherited diseases. Non-inherited disorders of globin chain synthesis mimicking the phenotype of thalassemias have also been described and are referred to as acquired thalassemias. These forms mainly affect the alpha-globin genes and are observed at much lower frequencies...

A case of familial donor-derived acute myeloid leukemia with underlying pre-leukemic mutations.

Not available.

Next-generation diagnostics for precision oncology: Preanalytical considerations, technical challenges, and available technologies.

Molecular diagnostics as the centrepiece of precision oncology has gone through revolutionary developments over the last decade, becoming tremendously broad, deep and precise with still ongoing advancements. In the majority of scenarios, treatment selection for cancer patients without any type of molecular characterization is no longer conceivable. Considering the impact of sample quality on the reliability of molecular analyses and the importance of the results for the fate of an individual patient, it is surprising how sparsely preanalytical and analytical requirements are addressed scientifically. Standardization and rigorous quality assessment continue to play only a marginal role in the field. Within this review, we will systematically discuss influencing preanalytic parameters and technology setups affecting molecular test results. We will shed light on the specifics of different analytes, technical modalities, and analysis pipelines. The review will have a certain focus on broad molecular genetic tumour testing with next generation sequencing but will go beyond that including other molecular diagnostic modalities and will give a glimpse into the future of molecular testing.

Clinical interpretation of whole-genome and whole-transcriptome sequencing for precision oncology.

Whole-genome sequencing either alone or in combination with whole-transcriptome sequencing has started to be used to analyze clinical tumor samples to improve diagnosis, provide risk stratification, and select patient-specific therapies. Compared with current genomic testing strategies, largely focused on small number of genes tested individually or targeted panels, whole-genome and transcriptome sequencing (WGTS) provides novel opportunities to identify and report a potentially much larger number of actionable alterations with diagnostic, prognostic, and/or predictive impact. Such alterations include point mutations, indels, copy- number aberrations and structural variants, but also germline variants, fusion genes, noncoding alterations and mutational signatures. Nevertheless, these comprehensive tests are accompanied by many challenges ranging from the extent and diversity of sequence alterations detected by these methods to the complexity and limited existing standardization in interpreting them. We describe the challenges of WGTS interpretation and the opportunities with comprehensive genomic testing.

Clinical utility of whole-genome sequencing in precision oncology.

Precision diagnostics is one of the two pillars of precision medicine. Sequencing efforts in the past decade have firmly established cancer as a primarily genetically driven disease. This concept is supported by therapeutic successes aimed at particular pathways that are perturbed by specific driver mutations in protein-coding domains and reflected in three recent FDA tissue agnostic cancer drug approvals. In addition, there is increasing evidence from studies that interrogate the entire genome by whole-genome sequencing that acquired global and complex genomic aberrations including those in non-coding regions of the genome might also reflect clinical outcome. After addressing technical, logistical, financial and ethical challenges, national initiatives now aim to introduce clinical whole-genome sequencing into real-world diagnostics as a rational and potentially cost-effective tool for response prediction in cancer and to identify patients who would benefit most from 'expensive' targeted therapies and recruitment into clinical trials. However, so far, this has not been accompanied by a systematic and prospective evaluation of the clinical utility of whole-genome sequencing within clinical trials of uniformly treated patients of defined clinical outcome. This approach would also greatly facilitate novel predictive biomarker discovery and validation, ultimately reducing size and duration of clinical trials and cost of drug development. This manuscript is the third in a series of three to review and critically appraise the potential and challenges of clinical whole-genome sequencing in solid tumors and hematological malignancies.

Analytical demands to use whole-genome sequencing in precision oncology.

Interrogating the tumor genome in its entirety by whole-genome sequencing (WGS) offers an unprecedented insight into the biology and pathogenesis of cancer, with potential impact on diagnostics, prognostication and therapy selection. WGS is able to detect sequence as well as structural variants and thereby combines central domains of cytogenetics and molecular genetics. Given the potential of WGS in directing targeted therapeutics and clinical decision-making, we envision a gradual transition of the method from research to clinical routine. This review is one out of three within this issue aimed at facilitating this effort, by discussing in-depth analytical validation, clinical interpretation and clinical utility of WGS. The review highlights the requirements for implementing, validating and maintaining a clinical WGS pipeline to obtain high-quality patient-specific data in accordance with the local regulatory landscape. Every step of the WGS pipeline, which includes DNA extraction, library preparation, sequencing, bioinformatics analysis, and data storage, is considered with respect to its logistics, necessities, potential pitfalls, and the required quality management. WGS is likely to drive clinical diagnostics and patient care forward, if requirements and challenges of the technique are recognized and met.

Prevalence and Therapeutic Implications of Clonal Hematopoiesis of Indeterminate Potential in Young Patients With Stroke.

BACKGROUND: Undetermined stroke etiology hampers optimal secondary prevention in a large proportion of young patients. We explored whether genetic screening for clonal hematopoiesis of indetermined potential (CHIP), a novel risk factor for stroke, could identify patients with myeloid precursor lesions or covert myeloid neoplasm requiring specific treatment. METHODS: We performed targeted sequencing on 56 genes recurrently mutated in hematologic neoplasms in a prospective cohort of patients with acute brain ischemia between 18 and 60 years. CHIP prevalence was compared with age-matched healthy controls from the Nijmegen Biomedical Study (n=1604) and the UK Biobank (n=101 678). Patients with suspicion of high-risk CHIP or myeloid neoplasm were invited for further hematologic evaluation. RESULTS: We included 248 consecutive patients (39% women) of whom 176 (71%) had cryptogenic stroke etiology. Fifty-one (21%) patients had CHIP, 3-fold more than in the general population (7.7% versus 2.6% for the Nijmegen Biomedical Study and 11.9% versus 4.1% for UK Biobank; P<0.001 for both). Patients with CHIP were older (median [interquartile range], 53 [50-59] versus 51 [41-56] years; P<0.001), had higher carotid intima-media thickness (0.68 [0.58-0.80] versus 0.59 [0.51-0.73] mm; P=0.009), and had higher burden of atherosclerosis (29.4% versus 16.7%; P=0.04). We invited 11 patients (4.4%) for further hematologic assessment, which in 7 led to the diagnosis of high-risk CHIP and in 2 to the new diagnosis of a myeloproliferative neoplasm with indication for cytoreductive therapy. CONCLUSIONS: Using genetic screening for myeloid disorders in patients with stroke of predominantly undetermined etiology, we found a 3-fold higher CHIP prevalence than in the general population. We identified high-risk CHIP and previously covert myeloproliferative neoplasms as potential stroke etiologies in 4.4% and 1% of patients, respectively. Our findings demonstrate the diagnostic and therapeutic yield of genetic screening in young patients with stroke. Future studies should investigate the role of CHIP for stroke recurrence and optimal secondary prevention.

Highly accurate differentiation of bone marrow cell morphologies using deep neural networks on a large image data set.

Biomedical applications of deep learning algorithms rely on large expert annotated data sets. The classification of bone marrow (BM) cell cytomorphology, an important cornerstone of hematological diagnosis, is still done manually thousands of times every day because of a lack of data sets and trained models. We applied convolutional neural networks (CNNs) to a large data set of 171 374 microscopic cytological images taken from BM smears from 945 patients diagnosed with a variety of hematological diseases. The data set is the largest expert-annotated pool of BM cytology images available in the literature. It allows us to train high-quality classifiers of leukocyte cytomorphology that identify a wide range of diagnostically relevant cell species with high precision and recall. Our CNNs outcompete previous feature-based approaches and provide a proof-of-concept for the classification problem of single BM cells. This study is a step toward automated evaluation of BM cell morphology using state-of-the-art image-classification algorithms. The underlying data set represents an educational resource, as well as a reference for future artificial intelligence-based approaches to BM cytomorphology.

Machine learning integrates genomic signatures for subclassification beyond primary and secondary acute myeloid leukemia.

Although genomic alterations drive the pathogenesis of acute myeloid leukemia (AML), traditional classifications are largely based on morphology, and prototypic genetic founder lesions define only a small proportion of AML patients. The historical subdivision of primary/de novo AML and secondary AML has shown to variably correlate with genetic patterns. The combinatorial complexity and heterogeneity of AML genomic architecture may have thus far precluded genomic-based subclassification to identify distinct molecularly defined subtypes more reflective of shared pathogenesis. We integrated cytogenetic and gene sequencing data from a multicenter cohort of 6788 AML patients that were analyzed using standard and machine learning methods to generate a novel AML molecular subclassification with biologic correlates corresponding to underlying pathogenesis. Standard supervised analyses resulted in modest cross-validation accuracy when attempting to use molecular patterns to predict traditional pathomorphologic AML classifications. We performed unsupervised analysis by applying the Bayesian latent class method that identified 4 unique genomic clusters of distinct prognoses. Invariant genomic features driving each cluster were extracted and resulted in 97% cross-validation accuracy when used for genomic subclassification. Subclasses of AML defined by molecular signatures overlapped current pathomorphologic and clinically defined AML subtypes. We internally and externally validated our results and share an open-access molecular classification scheme for AML patients. Although the heterogeneity inherent in the genomic changes across nearly 7000 AML patients was too vast for traditional prediction methods, machine learning methods allowed for the definition of novel genomic AML subclasses, indicating that traditional pathomorphologic definitions may be less reflective of overlapping pathogenesis.

A JAK/STAT-mediated inflammatory signaling cascade drives oncogenesis in AF10-rearranged AML.

Leukemias bearing fusions of the AF10/MLLT10 gene are associated with poor prognosis, and therapies targeting these fusion proteins (FPs) are lacking. To understand mechanisms underlying AF10 fusion-mediated leukemogenesis, we generated inducible mouse models of acute myeloid leukemia (AML) driven by the most common AF10 FPs, PICALM/CALM-AF10 and KMT2A/MLL-AF10, and performed comprehensive characterization of the disease using transcriptomic, epigenomic, proteomic, and functional genomic approaches. Our studies provide a detailed map of gene networks and protein interactors associated with key AF10 fusions involved in leukemia. Specifically, we report that AF10 fusions activate a cascade of JAK/STAT-mediated inflammatory signaling through direct recruitment of JAK1 kinase. Inhibition of the JAK/STAT signaling by genetic Jak1 deletion or through pharmacological JAK/STAT inhibition elicited potent antioncogenic effects in mouse and human models of AF10 fusion AML. Collectively, our study identifies JAK1 as a tractable therapeutic target in AF10-rearranged leukemias.

Novel NPM1 exon 5 mutations and gene fusions leading to aberrant cytoplasmic nucleophosmin in AML.

Nucleophosmin (NPM1) mutations in acute myeloid leukemia (AML) affect exon 12, but also sporadically affect exons 9 and 11, causing changes at the protein C-terminal end (tryptophan loss, nuclear export signal [NES] motif creation) that lead to aberrant cytoplasmic NPM1 (NPM1c+), detectable by immunohistochemistry. Combining immunohistochemistry and molecular analyses in 929 patients with AML, we found non-exon 12 NPM1 mutations in 5 (1.3%) of 387 NPM1c+ cases. Besides mutations in exons 9 (n = 1) and 11 (n = 1), novel exon 5 mutations were discovered (n = 3). Another exon 5 mutation was identified in an additional 141 patients with AML selected for wild-type NPM1 exon 12. Three NPM1 rearrangements (NPM1/RPP30, NPM1/SETBP1, NPM1/CCDC28A) were detected and characterized among 13 979 AML samples screened by cytogenetic/fluorescence in situ hybridization and RNA sequencing. Functional studies demonstrated that in AML cases, new NPM1 proteins harbored an efficient extra NES, either newly created or already present in the fusion partner, ensuring its cytoplasmic accumulation. Our findings support NPM1 cytoplasmic relocation as critical for leukemogenesis and reinforce the role of immunohistochemistry in predicting AML-associated NPM1 genetic lesions. This study highlights the need to develop new assays for molecular diagnosis and monitoring of NPM1-mutated AML.

2021 Update on MRD in acute myeloid leukemia: a consensus document from the European LeukemiaNet MRD Working Party.

Measurable residual disease (MRD) is an important biomarker in acute myeloid leukemia (AML) that is used for prognostic, predictive, monitoring, and efficacy-response assessments. The European LeukemiaNet (ELN) MRD Working Party evaluated standardization and harmonization of MRD in an ongoing manner and has updated the 2018 ELN MRD recommendations based on significant developments in the field. New and revised recommendations were established during in-person and online meetings, and a 2-stage Delphi poll was conducted to optimize consensus. All recommendations are graded by levels of evidence and agreement. Major changes include technical specifications for next-generation sequencing-based MRD testing and integrative assessments of MRD irrespective of technology. Other topics include use of MRD as a prognostic and surrogate end point for drug testing; selection of the technique, material, and appropriate time points for MRD assessment; and clinical implications of MRD assessment. In addition to technical recommendations for flow- and molecular-MRD analysis, we provide MRD thresholds and define MRD response, and detail how MRD results should be reported and combined if several techniques are used. MRD assessment in AML is complex and clinically relevant, and standardized approaches to application, interpretation, technical conduct, and reporting are of critical importance.

Massive parallel sequencing unveils homologous recombination deficiency in follicular dendritic cell sarcoma.

Standardized treatment options are lacking for patients with unresectable or multifocal follicular dendritic cell sarcoma (FDCS) and disease-related mortality is as high as 20%. Applying whole genome sequencing (WGS) in one case and whole exome sequencing (WES) in additional twelve, this study adds information on the molecular landscape of FDCS, expanding knowledge on pathobiological mechanisms and identifying novel markers of potential theragnostic significance. Massive parallel sequencing showed high frequency of mutations on oncosuppressor genes, particularly in RB1, CARS and BRCA2 and unveiled alterations on homologous recombination DNA damage repair related genes in 70% (9/13) of cases. This indicates that patients with high stage FDCS may be eligible for poly ADP ribose polymerase inhibition protocols. Low tumor mutational burden was confirmed in this study despite common PDL1 expression in FDCS arguing on the efficacy of immune checkpoint inhibitors. CDKN2A deletion, detected by WGS and confirmed by FISH in 41% of cases (9/22) indicates that impairment of cell cycle regulation may sustain oncogenesis in FDCS. Absence of mutations in the RAS/RAF/MAPK pathway and lack of clonal hematopoiesis related mutations in FDCS sanction its differences from dendritic cell-derived neoplasms of haematopoietic derivation. WGS and WES in FDCS provides additional information on the molecular landscape of this rare tumor, proposing novel candidate genes for innovative therapeutical approaches to improve survival of patients with multifocal disease.

Characterization of genetic variants in the EGLN1/PHD2 gene identified in a European collection of patients with erythrocytosis.

Hereditary erythrocytosis is a rare hematologic disorder characterized by an excess of red blood cell production. Here we describe a European collaborative study involving a collection of 2,160 patients with erythrocytosis sequenced in ten different laboratories. We focused our study on the EGLN1 gene and identified 39 germline missense variants including one gene deletion in 47 probands. EGLN1 encodes the PHD2 prolyl 4-hydroxylase, a major inhibitor of hypoxia-inducible factor. We performed a comprehensive study to evaluate the causal role of the identified PHD2 variants: (i) in silico studies of localization, conservation, and deleterious effects; (ii) analysis of hematologic parameters of carriers identified in the UK Biobank; (iii) functional studies of the protein activity and stability; and (iv) a comprehensive study of PHD2 splicing. Altogether, these studies allowed the classification of 16 pathogenic or likely pathogenic mutants in a total of 48 patients and relatives. The in silico studies extended to the variants described in the literature showed that a minority of PHD2 variants can be classified as pathogenic (36/96), without any differences from the variants of unknown significance regarding the severity of the developed disease (hematologic parameters and complications). Here, we demonstrated the great value of federating laboratories working on such rare disorders in order to implement the criteria required for genetic classification, a strategy that should be extended to all hereditary hematologic diseases.