Importance of genetic clarification in cytopenia syndromes (childhood myelodysplastic syndrome forms)

Childhood myelodysplastic syndrome (cMDS), also referred to as myelodysplastic neoplasm, is a clonal hematopoietic disorder, resulting in peripheral cytopenia due to abnormal maturation of hematopoietic stem cells and presenting a risk of advancing to acute myeloid leukemia (AML) [1]. The incidence is low, typically 1–4 cases per million children, with only 10–25% of affected individuals showing increased blast cell counts [1, 2]. The 5th edition of the World Health Organization (WHO) classification of hematolymphoid tumors categorizes cMDS into two groups: MDS with a low blast count and MDS with an increased blast count [2]. Other clonal disorders, namely juvenile myelomonocytic leukemia (JMML) or myeloid proliferations associated with Down syndrome, are not included within these two categories. Most children with cMDS display a distinctive histomorphologic pattern, recognized as refractory cytopenia of childhood (or now classified as cMDS with low blasts).

Among children presenting with pancytopenia and a significant reduction in bone marrow cellularity, refractory cytopenia of childhood (RCC) as well as severe aplastic anemia (SAA) and inherited bone marrow failure syndromes (IBMFS) stand out as the most prevalent hematopoietic conditions [3]. Their differential diagnosis is crucial as it significantly influences treatment decisions.

In 2022, two distinct classifications featuring divergent views on the landscape of childhood MDS emerged: the WHO 5th edition and the international consensus classification (ICC) [2, 4]. In the revision of the WHO 4th edition classification [5], RCC was designated as a provisional entity. It was characterized by a histopathologic pattern featuring hypocellular marrow (in 80% of cases) with patchy erythroid islands, often accompanied by marked myeloid hypoplasia and decreased megakaryocytes. Diagnosis of RCC requires dysplasia in more than 10% of cells in at least one lineage or a lesser degree of dysplasia in two cell lineages [6]. Immunohistochemical staining of the megakaryocytic antigens CD61 or CD41 is essential for identifying micromegakaryocytes and for differentiating between RCC and SAA. Notably, distinguishing between SAA and RCC primarily relies on identifying the presence or absence of dysplasia.

However, in the WHO 5th edition classification, the term RCC has been replaced with childhood MDS with low blasts (cMDS-LB), defined by having less than 5% blasts in the bone marrow and less than 2% in the peripheral blood. This classification now includes two different subtypes: cMDS-LB, hypocellular and cMDS-LB, not otherwise specified (NOS). Childhood MDS with increased blasts (cMDS-IB) is characterized by 5–19% of blasts in the bone marrow or 2–19% in peripheral blood.

The second recently published classification by the ICC retains the term RCC and broadens its scope to include children with germline predisposition. While one of the main features distinguishing MDS from other causes of cytopenia is morphological dysplasia, this observation is not mandatory for diagnosis. MDS can also be defined by molecular and cytogenetic abnormalities including monosomy 7, del (7q), monosomy 5, del (5q), multi-hit TP53 (variant allele frequency [VAF] ≥ 10%), and SF3B1 mutation (VAF ≥ 10%). It is crucial to note that MDS is not a static disease, and regular monitoring is mandatory. Karyotypic evolution is usually accompanied by progression to more advanced forms of MDS [7].

RCC, IBMFS, and SAA exhibit significant overlap and may all present with pancytopenia and a severe decrease in bone marrow cellularity [3]. While RCC represents the most prevalent form of cMDS [1], not all RCC cases are of clonal origin and, therefore, not all are bona fide MDS [4]. Baumann et al. [3] demonstrated that the vast majority of cases of SAA and RCC can be accurately distinguished solely through morphological analysis. Further studies, such as those by Karow et al. [8], Yoshimi et al. [9], Behrens et al. [10], Wlodarski et al. [11], and Schwartz et al. [12], have revealed that the RCC pattern also exists in patients with constitutional disorders like classical IBMFS, which include newly described disorders such as germline GATA2 deficiency. However, discerning hypocellular RCC from immune-mediated SAA can also be challenging, with studies questioning pathomorphological reproducibility [13], which may lead to a significant clinical dilemma. In cases of nonconstitutional SAA, timely initiation of immunosuppressive therapy (IST) is crucial, as delayed treatment is strongly associated with adverse outcomes [14]. IST is ideally initiated within 2 weeks and should not commence later than 4 weeks following the initial presentation as suggested in the guidelines of the European Working Group (EWOG) for the management of childhood SAA. On the contrary, comprehensive, time-consuming genomic analysis can help discover patients with constitutional diseases who may probably not benefit from IST.

The 4th WHO classification included three different groups that took germline predisposition into account: predisposition without pre-existing disorders or organ dysfunction (CEBPA, DDX41), predisposition with pre-existing platelet disorder (RUNX1, ANKRD26, ETV6), and predisposition with potential organ dysfunction (e.g., GATA2). While the WHO 5th edition classification now categorizes these germline predispositions as secondary myeloid malignancies (with the addition of SAMD9/SAMD9L and TP 53), the ICC classifies them under hematologic neoplasms with germline predisposition, as pathogenic variants in certain genes predispose individuals to both lymphoid and myeloid neoplasms (e.g., ETV6 and RUNX 1).

Approximately 100 genes have currently been identified as linked to germline predisposition to both IBMFS and MDS [15]. The spectrum of these diseases is continually broadening alongside the classical IBMFS, which include Fanconi anemia, Diamond–Blackfan anemia, severe congenital neutropenia, dyskeratosis congenita, Shwachman–Diamond syndrome, and congenital thrombocytopenias. One of these congenital thrombocytopenias is congenital amegakaryocytic thrombocytopenia (CAMT), characterized by severe thrombocytopenia progressing to pancytopenia and bone marrow failure in early childhood [16]. This condition results from mutations in either the MPL gene (CAMT-MPL), responsible for encoding the thrombopoietin receptor, or from mutations in the thrombopoietin gene itself (CAMT-THPO). While patients with CAMT-MPL should undergo hematopoietic stem cell transplantation (HSCT), those with CAMT-THPO respond effectively to thrombopoietin receptor agonists [17].

Genetic analyses in patients suspected of childhood MDS range from the targeted testing for specific genetic alterations, such as mutations in GATA2, SAMD9/SAMD9L and RUNX1, which are most commonly associated with childhood MDS, to more comprehensive yet time- and cost-intensive methods like whole-exome sequencing (WES) or even whole-genome analysis in suspected predisposition syndromes.

GATA2 deficiency stands out as the most prevalent germline predisposition for pediatric MDS, particularly prominent among adolescents with monosomy 7. As a zinc finger transcription factor, GATA2 plays a crucial role in hematopoiesis, which is reflected by the complex disorder, including immunodeficiency and autoimmunity, caused by its deficiency [18]. In a study on MDS in childhood conducted by EWOG [11] involving 508 pediatric MDS cases, germline GATA2 mutations were detected in 57 patients. Despite their high rate in advanced MDS cases (15%) and adolescents with monosomy 7, GATA2 mutations do not inherently predict a poor prognosis in childhood MDS. The management of patients with hematologic symptoms in combination with GATA2 deficiency depends on the severity of bone marrow failure and the presence of karyotypic abnormalities. In cases with a normal karyotype, no transfusion requirement and a neutrophil count above 1.0 G/L, regular monitoring of the patients is recommended. For more severe cytopenia, certain karyotypic abnormalities, or the presence of blasts, HSCT is indicated (EWOG MDS 2017 guidelines).

Recent research has underscored the benefits of comprehensive genomic analysis in diagnosing IBMFS and conditional MDS, facilitating the resolution of yet unresolved cases and yielding effective outcomes in treatment and family monitoring [15]. Blombery et al. [19] conducted an extensive analysis of 115 pediatric and adult patients (median age 24 years, range 3 months–81 years), utilizing various genomic techniques including whole exome sequencing, targeted gene panels, RNA sequencing, and droplet digital PCR (ddPCR). This analysis led to a change of the diagnostic category for 26% of the cohort, notably identifying germline causes in 3 of 47 patients initially diagnosed with SAA/acquired MDS and 16 of 45 patients with clinically unclassifiable BMF. An Israeli nationwide study of 189 children with refractory cytopenias identified pathogenic or likely pathogenic germline variants in almost one-third of the patients of whom 80% had germline predisposition to leukemia [20]. Similar results were found in studies on young adults, with causative germline mutations identified in 19% (13/68) of patients with MDS or AML [21]. Regarding the genomic profile of somatic mutations, pediatric MDS patients differ from adult patients, notably in the absence of mutations linked to epigenetic regulation or RNA splicing. Instead, somatic abnormalities in the RAS/MAPK pathway or somatic driver mutations in SETBP1, ASXL1, and RUNX1 are prevalent in cMDS [12]. This spectrum is continuously broadening, as highlighted by the recent identification of UBTF-tandem duplications in one-third of patients with advanced primary cMDS within a cohort of 104 individuals [22].

As demonstrated in the following two cases presented below, the differentiation between the disorders IBMFS, SAA, and cMDS presents a well-recognized challenge, with significant implications for treatment.

A 7-year-old Ukrainian refugee was transferred to our hospital with severe pancytopenia that had prevailed for several weeks. On admission, the patient had transfusion-dependent anemia and thrombocytopenia, with an absolute neutrophil count (ANC) < 0.2 G/L. Histopathology results indicated SAA without signs of dysplasia, and karyotyping revealed no cytogenetic abnormalities. As a matched sibling donor was not available, we swiftly initiated IST according to the EWOG-SAA 2010 guidance. However, genetic testing results concurrently revealed a rare heterozygous pathogenic GATA2 splice-site variant (NM_032638.5:c.871 + 2T > C). Interestingly, the patient responded well to IST, achieving transfusion independence and an ANC > 1.0 G/L after 30 days of treatment. On day 360, the patient exhibited continued improvement (according to criteria good partial response [GPR]) with normal ANC count, hemoglobin approx. 10 g/dL and platelet count approx. 100 G/L. Trilinear hematopoiesis was evident without significant dysplasia in the bone marrow examination. Clinically, there were no infection-related complications, which is a major concern of IST (Fig. 1).

We performed WES in addition to bone marrow examination and flow cytometry in a girl born to consanguineous parents who presented with severe pancytopenia at the age of 6 years (hemoglobin 6.5 g/dL, MCV 106 fl, platelet count 5 G/L, ANC 0.85 G/L). Bone marrow examination suggested RCC with severe hypocellularity, aplasia of megakaryopoiesis, and signs of erythropoietic dysplasia. Flow cytometry analysis showed a significantly reduced number of CD34-positive progenitor cells with a skewed maturation profile depleted of multipotent progenitors (MPPs), erythroid–myeloid progenitors (EMPs), and common myeloid progenitors (CMPs), but with residual lymphoid-primed multipotent progenitors (LMMPs) and granulocyte–monocyte progenitors (GMPs). WES led to the diagnosis of congenital amegakaryocytic thrombocytopenia (CAMT-MPL), with the identification of a novel homozygous germline missense variant (NM_032638.5:c.941T > G, p.Phe314Cys), located in exon 6 of the MPL gene. This variant affects a highly conserved phenylalanine in the extracellular domain of the thrombopoietin receptor. The patient underwent successful hematopoietic stem cell transplantation which is the only available curative treatment option for such patients.